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1
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Authors Younghoon Go1811
Ji Yun Jeong11011
Nam Ho Jeoung211
Jae-Han Jeon18
Bo-
Yoon Park19
Hyeon-Ji Kang19
Chae-Myeong Ha19
Young-Keun Choi1 Sun Joo Lee
1 Hye
Jin Ham8 Byung-Gyu Kim
8 Keun-Gyu Park
18 So Young Park
3 Chul-Ho Lee
4 Cheol Soo
Choi5 Tae-Sik Park
5 W N Paul Lee
6 Robert A Harris
712 and In-Kyu Lee
189 12
1Department of Internal Medicine Kyungpook National University School of Medicine
Daegu South Korea
2Department of Pharmaceutical Science and Technology Catholic University of Daegu
Gyeongsan South Korea
3Department of Physiology College of Medicine Yeungnam University Daegu South Korea
4Disease Model Research Center Korea Research Institute of Bioscience amp Biotechnology
Daejeon South Korea
5Lee Gil Ya Cancer and Diabetes Institute Gachon University of Medicine and Science
Inchon South Korea
6Department of Pediatrics Harbor-UCLA Medical Center 1000 West Carson Street
Torrance CA 90502 USA
7Roudebush VA Medical Center and the Department of Biochemistry and Molecular Biology
Indiana University School of Medicine Indianapolis IN 46202 USA
8Leading-edge Research Center for Drug Discovery and Development for Diabetes and
Metabolic Disease Kyungpook National University Daegu South Korea
9BK21 plus KNU Biomedical Convergence Programs at Kyungpook National University
Page 1 of 45 Diabetes
Diabetes Publish Ahead of Print published online July 6 2016
2
Daegu South Korea
Contact
In-Kyu Lee MD PhD
Division of Endocrinology and Metabolism
Kyungpook National University School of Medicine
50 Samduck-2Ga Jung-Gu Daegu South Korea 700-721
Tel +82-53-420-5564 Fax +82-53-426-2046 E-mail leeiknuackr
Robert A Harris PhD
Roudebush VA Medical Center and the Department of Biochemistry and Molecular Biology
Indiana University School of Medicine Indianapolis IN 46202
Tel + 327-440-8941 Fax + 317-988-3180 E-mail raharrisiuedu
Additional Footnotes
10Present address Department of Internal Medicine Soonchunhyang University Gumi
Hospital Gumi South Korea
11Co-first author
12Co-corresponding author
Page 2 of 45Diabetes
3
ABSTRACT
Hepatic steatosis is associated with increased insulin resistance and tricarboxylic acid
(TCA) cycle flux but decreased ketogenesis and pyruvate dehydrogenase complex (PDC)
flux This study examined whether hepatic PDC activation by inhibition of pyruvate
dehydrogenase kinase 2 (PDK2) ameliorates these metabolic abnormalities
Wild-type (WT) mice fed a HFD exhibited hepatic steatosis insulin resistance and
increased levels of pyruvate TCA cycle intermediates and malonyl-CoA but reduced
ketogenesis and PDC activity due to PDK2 induction Hepatic PDC activation by PDK2
inhibition attenuated hepatic steatosis improved hepatic insulin sensitivity reduced hepatic
glucose production increased capacity for β-oxidation and ketogenesis and decreased the
capacity for lipogenesis These results were attributed to altered enzymatic capacities and a
reduction in TCA anaplerosis that limited the availability of oxaloacetate (OAA) for the TCA
cycle which promoted ketogenesis
The present study reports that increasing hepatic PDC activity by inhibition of PDK2
ameliorates hepatic steatosis and insulin sensitivity by regulating TCA cycle anaplerosis and
ketogenesis The findings suggest PDK2 is a potential therapeutic target for non-alcoholic
fatty liver disease (NAFLD)
INTRODUCTION
Hepatic steatosis is induced by a number of lipid metabolic abnormalities including
increased de novo lipogenesis inhibited triacylglyceol (TG) release enhanced FA influx from
adipose tissue and reduced hepatic FA oxidation and ketogenesis (1) Recently it was
proposed that dysregulation of ketone body metabolism could potentially contribute to the
pathogenesis of non-alcoholic fatty liver disease (NAFLD) Reduced ketogenesis exacerbates
Page 3 of 45 Diabetes
4
diet-induced hepatic steatosis and hyperglycemia (2) and a ketotic diet reduces the body
weight of mice as much as a calorie-restricted diet (3) However the precise mechanism by
which impaired ketogenesis contributes to hepatic steatosis is still unclear
In the well-fed state acetyl-CoA produced from glucose is converted to FAs for storage
of excess energy or oxidized to CO2 in the TCA cycle to generate ATP by oxidative
phosphorylation In the fasting state two-thirds of the free FAs entering the liver are
converted to acetyl-CoA by β-oxidation and then further processed to ketone bodies which
act as an energy source for peripheral tissues The other one-third of FAs is utilized by the
TCA cycle to generate ATP to meet hepatic energy demands (4) Whether acetyl-CoA
produced by β-oxidation forms ketone bodies or enters the TCA cycle is determined by
anaplerotic influx of TCA cycle intermediates The conversion of pyruvate to OAA by
pyruvate carboxylase (PC) is one of the most important sources of anaplerosis in the liver
Pyruvate can also be converted to acetyl-CoA by oxidative decarboxylation mediated by
PDC PDC activity is inhibited via phosphorylation of the pyruvate dehydrogenase E1α
subunit (PDHE1α) which is mediated by increased expression of PDKs during fasting or in
an insulin-resistant state (5-7) Four isoforms of PDK (PDK1-4) are expressed in a tissue-
specific manner with unique expression profiles in response to different physiological
conditions (8) Among these isoforms PDK2 is the major PDK responsible for regulation of
PDC activity in the liver (8-10)
Recently it was reported that decreased PDC activity and enhanced pyruvate
carboxylation due to hepatic insulin resistance contribute to increased hepatic
gluconeogenesis in obese subjects with hepatic steatosis (1112) Because of competition for
pyruvate the balance between PDC and PC activity may play a critical role in metabolic
dysfunction caused by obesity and insulin resistance In this study we examined the
Page 4 of 45Diabetes
5
possibility that activation of PDC by PDK2 inhibition reduces the anaplerotic flux of
pyruvate into the TCA cycle which may increase ketogenesis and prevent hepatic steatosis
induced by a HFD Indeed we found that PDK2 inhibition ameliorates hepatic steatosis and
insulin resistance suggesting that PDK2 is a potential therapeutic target for NAFLD
RESERCH DESIGN AND METHODS
Animal Experiments
All experiments were approved by the Institutional Animal Care and Use Committee of
Kyungpook National University For the NAFLD mice model 8-week-old male WT
(C57BL6J) and PDK2 KO mice (13) were fed a HFD in which 20 of the calories were
derived from carbohydrates and 60 from fat (Research Diets D12492 pellets) Control WT
and PDK2 KO mice were fed an isocaloric LFD in which 70 of the calories were derived
from carbohydrates and 10 from fat (Research Diets D12450B pellets) The mice were
housed and maintained on a 12 h lightdark cycle at 22 plusmn 2degC After the mice were sacrificed
tissues were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature
of liquid nitrogen and stored at -80degC for analysis
Construction of PDK2 DN Recombinant Adenovirus and Its Infection In Vivo
pCMV6-KanNeo mouse PDK2 plasmid was purchased from OriGene The PDK2
dominant-negative (DN) mutant R157A (1415) was generated by site-directed mutagenesis
with the QuickChange II site-directed mutagenesis kit (Stratagene) using the following
primers forward 5rsquo-CCAGTACTTCCTGGACGCCTTCTACCTCAGC-3rsquo and reverse 5rsquo-
GCTGAGGTAGAAGGCGTCCAGGAAGTACTGG-3rsquo Recombinant adenovirus for PDK2
DN mutant was generated using pAd-Track-CMV shuttle vector as described previously (16)
Page 5 of 45 Diabetes
6
Recombinant PDK2 DN and GFP adenovirus were amplified in AD-293 cells and purified by
CsCl gradient centrifugation Adenovirus titers were determined using Adeno-X rapid titer
(BD Bioscience) according to the manufacturerrsquos instructions After 12 weeks of HFD
feeding in WT mice recombinant adenoviruses (10 times 109 plaque-forming units) expressing
PDK2 DN and GFP constructs were delivered by tail vein injection On day 3 after
adenovirus infection glucose tolerance tests were performed in overnight-fasted mice by
intraperitoneal (ip) injection of glucose (1 gkg body weight) On day 7 after adenovirus
infection mice were sacrificed after overnight fasting After the mice were sacrificed tissues
were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature of
liquid nitrogen
Measurement of PDC Activity
PDC activity was spectrophotometrically measured in a 96-well plate reader by coupling
to the reaction catalyzed by arylamineacetyltransferase as described previously (7) One unit
of PDC activity corresponds to the acetylation of 1 micromol of p-(p-aminophenyl azo)
benzenesulfonate per min at 30degC
Statistical Analysis
Statistical significance was determined by the unpaired Studentrsquos t-test when two groups
were compared Values are presented as the means plusmn SEM of the indicated number of
independent samples P values less than 005 were considered statistically significant
See the Supplementary materials and methods section for additional details
Page 6 of 45Diabetes
7
RESULTS
Up-Regulation of PDK2 Reduces Hepatic PDC Activity in HFD-Fed Mice
PDK2 expression is increased in the livers of obese animals (917) its role in the
development of hepatic metabolic diseases remains unclear We examined PDC activity and
the expression of PDK isoenzymes in the livers of WT mice fed a HFD As shown in Figure
1A PDC activity was significantly lower in the livers of mice fed a HFD than in those of
mice fed a LFD The reduction in hepatic PDC activity was associated with significant
increases in the phosphorylation of PDHE1α (Fig 1B) PDK2 mRNA levels were
significantly higher in the livers of HFD-fed mice compared to LFD-fed mice (Fig 1C)
Conversely the level of the PDK4 which is induced by a HFD in skeletal muscle and
heart(5) was markedly decreased in the livers of HFD-fed mice (Fig 1C) The protein levels
of PDK2 and PDK4 showed the same pattern as their respective mRNAs (Fig 1D) To
confirm that PDK2 regulates hepatic PDC activity in HFD-fed mice PDC activity and
phosphorylation of PDH E1α were measured in the livers of WT and PDK2 KO mice As
expected from the data shown in Figure 1A the HFD reduced hepatic PDC activity in WT
mice but not in PDK2 KO mice (Fig 1E) Consistent with this finding PDHE1α
phosphorylation was decreased in PDK2 KO mice compared to WT mice (Fig 1F) To
examine PDC activity change by PDK2 deficiency in other tissue we measured the
phosphorylation state of PDHE1α in muscle heart and kidney Phosphorylation of PDHE1α
was similar between HFD-fed PDK2 KO mice and HFD-fed WT mice in these tissues
(supplementary Fig 1) The serum levels of pyruvate lactate and FFA were not different
between WT and PDK2 KO mice after HFD feeding (Supplementary Fig 2) These findings
indicate that PDK2 is a major regulator of PDC activity in the liver of HFD-fed mice
Page 7 of 45 Diabetes
8
PDK2 Deficiency Prevents HFD-Induced Hepatic Steatosis
To elucidate the role of PDK2 in the development of hepatic steatosis WT and PDK2 KO
mice were fed a HFD to induce NAFLD After 16 weeks of HFD feeding body weight gain
in HFD-fed PDK2 KO mice was significantly lower than that of HFD-fed WT mice (Fig 2A)
Even though the body weights of HFD-fed PDK2 KO mice were lower than those of WT
mice there were no differences in food consumption or physical activity (Supplementary Fig
3A and 3B) Relative to HFD-fed WT mice HFD-fed PDK2 KO mice exhibited greater
energy expenditure (Supplementary Fig 3C)
The liverbody weight ratio of PDK2 KO mice fed a HFD was significantly lower than
that of WT mice (Fig 2B) The number and the size of the lipid droplets were greatly
decreased in livers of PDK2 KO mice fed a HFD (Fig 2C) However no difference in
phenotype in epididymal adipose tissue (EAT) was observed between WT and PDK2 KO
mice on either diet (Fig 2C) Consistent with the histological analysis the hepatic TG
content was significantly lower in PDK2 KO mice than in WT mice after HFD feeding (Fig
2D) The level of hepatic glycogen was also significantly reduced in PDK2 KO mice fed a
HFD compare to WT mice fed a HFD (Fig 2E) Serum activities of AST and ALT markers of
liver injury were also significantly elevated in WT mice fed a HFD but not in PDK2 KO
mice (Fig 2F and 2G) The expression of genes involved in inflammation including tumor
necrosis factor alpha (TNFα) monocyte chemoattractant protein-1 (MCP-1) and
plasminogen activator inhibitor-1 (PAI-1) were significantly lower in PDK2 KO mice than in
WT mice fed a HFD (Supplementary Fig 4A) These data indicate that PDK2 deficiency
could ameliorate HFD-induced hepatic steatosis injury and inflammation
Page 8 of 45Diabetes
9
PDK2 Deficiency Ameliorates Hepatic Insulin Resistance and Reduces Hepatic Glucose
Production
In HFD-fed mice PDK2 deficiency lowered fasting blood glucose and serum insulin
levels by 15 and 63 respectively relative to WT mice (Fig 3A and 3B) HFD-fed PDK2
KO mice processed glucose more efficiently than HFD-fed WT mice as evidenced by a 30
decrease in the area under the curve (AUC) during GTT (Fig 3C) However with LFD-fed
mice the elimination of glucose was similar between PDK2 KO mice and WT mice (Fig 3C)
which is well agreed with previous data (18) These observations suggest that PDK2
deficiency prevents impaired glucose homeostasis induced by a HFD
To better delineate the mechanism responsible for improved hepatic glucose homeostasis
in HFD-fed PDK2 KO mice we performed a hyperinsulinemic-euglycemic clamp study In
HFD-fed WT mice the glucose infusion rate (GIR) was lower hepatic glucose production
(HGP) was higher during the clamp and whole body glucose turnover was lower (Fig 3D)
than in LFD-fed WT mice However in HFD-fed PDK2 KO mice the GIR was increased
two-fold compared to HFD-fed WT mice (Fig 3D) Furthermore both basal and clamped
HGP were significantly lower in HFD-fed PDK2 KO mice than in HFD-fed WT mice (Fig
3D) Nevertheless whole body glucose turnover was not different between HFD-fed PDK2
KO mice and WT mice (Fig 3D) HGP measured by the pyruvate tolerance test (PTT) was
lower in HFD-fed PDK2 KO mice than in HFD-fed WT (Fig 3E) To evaluate insulin
signaling we examined the phosphorylation levels of AKT and FoxO1 in the liver and
muscle of HFD-fed WT and PDK2 KO mice after insulin injection Insulin-stimulated
phosphorylation of AKT and FoxO1 were significantly increased in the livers of PDK2 KO
mice compared to those of WT mice (Fig 3F) Interestingly in muscle the levels of AKT
and FoxO1 phosphorylation did not differ between HFD-fed PDK2 KO mice and HFD-fed
Page 9 of 45 Diabetes
10
WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by
PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be
caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation
of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine
whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we
measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The
amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-
fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the
phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2
KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of
hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by
a HFD
PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice
Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic
steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2
deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative
decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates
thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA
cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed
PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A
and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free
[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)
Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in
Page 10 of 45Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase
isoforms 2 and 4 by fatty acids and thyroid hormone Biochem J 2003369687-695
11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease Cell Metab
201114804-810
13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
contractions in PDH kinase 2 knockout mice effect of PDH kinase 1 compensation Am J
Physiol - Regul Integ Comp Physiol 2011300R1487-1493
14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate
dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-
13582
15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
kinase-4 structures reveal a metastable open conformation fostering robust core-free basal
activity J Biol Chem 200828325305-25315
Page 19 of 45 Diabetes
20
16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
alcoholic fatty liver disease J Biol Chem 200427932345-32353
20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
2012531080-1092
21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
Invest 20121221958-1959
22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
201233741-43
23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387
24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
383
26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
Page 20 of 45Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
2
Daegu South Korea
Contact
In-Kyu Lee MD PhD
Division of Endocrinology and Metabolism
Kyungpook National University School of Medicine
50 Samduck-2Ga Jung-Gu Daegu South Korea 700-721
Tel +82-53-420-5564 Fax +82-53-426-2046 E-mail leeiknuackr
Robert A Harris PhD
Roudebush VA Medical Center and the Department of Biochemistry and Molecular Biology
Indiana University School of Medicine Indianapolis IN 46202
Tel + 327-440-8941 Fax + 317-988-3180 E-mail raharrisiuedu
Additional Footnotes
10Present address Department of Internal Medicine Soonchunhyang University Gumi
Hospital Gumi South Korea
11Co-first author
12Co-corresponding author
Page 2 of 45Diabetes
3
ABSTRACT
Hepatic steatosis is associated with increased insulin resistance and tricarboxylic acid
(TCA) cycle flux but decreased ketogenesis and pyruvate dehydrogenase complex (PDC)
flux This study examined whether hepatic PDC activation by inhibition of pyruvate
dehydrogenase kinase 2 (PDK2) ameliorates these metabolic abnormalities
Wild-type (WT) mice fed a HFD exhibited hepatic steatosis insulin resistance and
increased levels of pyruvate TCA cycle intermediates and malonyl-CoA but reduced
ketogenesis and PDC activity due to PDK2 induction Hepatic PDC activation by PDK2
inhibition attenuated hepatic steatosis improved hepatic insulin sensitivity reduced hepatic
glucose production increased capacity for β-oxidation and ketogenesis and decreased the
capacity for lipogenesis These results were attributed to altered enzymatic capacities and a
reduction in TCA anaplerosis that limited the availability of oxaloacetate (OAA) for the TCA
cycle which promoted ketogenesis
The present study reports that increasing hepatic PDC activity by inhibition of PDK2
ameliorates hepatic steatosis and insulin sensitivity by regulating TCA cycle anaplerosis and
ketogenesis The findings suggest PDK2 is a potential therapeutic target for non-alcoholic
fatty liver disease (NAFLD)
INTRODUCTION
Hepatic steatosis is induced by a number of lipid metabolic abnormalities including
increased de novo lipogenesis inhibited triacylglyceol (TG) release enhanced FA influx from
adipose tissue and reduced hepatic FA oxidation and ketogenesis (1) Recently it was
proposed that dysregulation of ketone body metabolism could potentially contribute to the
pathogenesis of non-alcoholic fatty liver disease (NAFLD) Reduced ketogenesis exacerbates
Page 3 of 45 Diabetes
4
diet-induced hepatic steatosis and hyperglycemia (2) and a ketotic diet reduces the body
weight of mice as much as a calorie-restricted diet (3) However the precise mechanism by
which impaired ketogenesis contributes to hepatic steatosis is still unclear
In the well-fed state acetyl-CoA produced from glucose is converted to FAs for storage
of excess energy or oxidized to CO2 in the TCA cycle to generate ATP by oxidative
phosphorylation In the fasting state two-thirds of the free FAs entering the liver are
converted to acetyl-CoA by β-oxidation and then further processed to ketone bodies which
act as an energy source for peripheral tissues The other one-third of FAs is utilized by the
TCA cycle to generate ATP to meet hepatic energy demands (4) Whether acetyl-CoA
produced by β-oxidation forms ketone bodies or enters the TCA cycle is determined by
anaplerotic influx of TCA cycle intermediates The conversion of pyruvate to OAA by
pyruvate carboxylase (PC) is one of the most important sources of anaplerosis in the liver
Pyruvate can also be converted to acetyl-CoA by oxidative decarboxylation mediated by
PDC PDC activity is inhibited via phosphorylation of the pyruvate dehydrogenase E1α
subunit (PDHE1α) which is mediated by increased expression of PDKs during fasting or in
an insulin-resistant state (5-7) Four isoforms of PDK (PDK1-4) are expressed in a tissue-
specific manner with unique expression profiles in response to different physiological
conditions (8) Among these isoforms PDK2 is the major PDK responsible for regulation of
PDC activity in the liver (8-10)
Recently it was reported that decreased PDC activity and enhanced pyruvate
carboxylation due to hepatic insulin resistance contribute to increased hepatic
gluconeogenesis in obese subjects with hepatic steatosis (1112) Because of competition for
pyruvate the balance between PDC and PC activity may play a critical role in metabolic
dysfunction caused by obesity and insulin resistance In this study we examined the
Page 4 of 45Diabetes
5
possibility that activation of PDC by PDK2 inhibition reduces the anaplerotic flux of
pyruvate into the TCA cycle which may increase ketogenesis and prevent hepatic steatosis
induced by a HFD Indeed we found that PDK2 inhibition ameliorates hepatic steatosis and
insulin resistance suggesting that PDK2 is a potential therapeutic target for NAFLD
RESERCH DESIGN AND METHODS
Animal Experiments
All experiments were approved by the Institutional Animal Care and Use Committee of
Kyungpook National University For the NAFLD mice model 8-week-old male WT
(C57BL6J) and PDK2 KO mice (13) were fed a HFD in which 20 of the calories were
derived from carbohydrates and 60 from fat (Research Diets D12492 pellets) Control WT
and PDK2 KO mice were fed an isocaloric LFD in which 70 of the calories were derived
from carbohydrates and 10 from fat (Research Diets D12450B pellets) The mice were
housed and maintained on a 12 h lightdark cycle at 22 plusmn 2degC After the mice were sacrificed
tissues were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature
of liquid nitrogen and stored at -80degC for analysis
Construction of PDK2 DN Recombinant Adenovirus and Its Infection In Vivo
pCMV6-KanNeo mouse PDK2 plasmid was purchased from OriGene The PDK2
dominant-negative (DN) mutant R157A (1415) was generated by site-directed mutagenesis
with the QuickChange II site-directed mutagenesis kit (Stratagene) using the following
primers forward 5rsquo-CCAGTACTTCCTGGACGCCTTCTACCTCAGC-3rsquo and reverse 5rsquo-
GCTGAGGTAGAAGGCGTCCAGGAAGTACTGG-3rsquo Recombinant adenovirus for PDK2
DN mutant was generated using pAd-Track-CMV shuttle vector as described previously (16)
Page 5 of 45 Diabetes
6
Recombinant PDK2 DN and GFP adenovirus were amplified in AD-293 cells and purified by
CsCl gradient centrifugation Adenovirus titers were determined using Adeno-X rapid titer
(BD Bioscience) according to the manufacturerrsquos instructions After 12 weeks of HFD
feeding in WT mice recombinant adenoviruses (10 times 109 plaque-forming units) expressing
PDK2 DN and GFP constructs were delivered by tail vein injection On day 3 after
adenovirus infection glucose tolerance tests were performed in overnight-fasted mice by
intraperitoneal (ip) injection of glucose (1 gkg body weight) On day 7 after adenovirus
infection mice were sacrificed after overnight fasting After the mice were sacrificed tissues
were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature of
liquid nitrogen
Measurement of PDC Activity
PDC activity was spectrophotometrically measured in a 96-well plate reader by coupling
to the reaction catalyzed by arylamineacetyltransferase as described previously (7) One unit
of PDC activity corresponds to the acetylation of 1 micromol of p-(p-aminophenyl azo)
benzenesulfonate per min at 30degC
Statistical Analysis
Statistical significance was determined by the unpaired Studentrsquos t-test when two groups
were compared Values are presented as the means plusmn SEM of the indicated number of
independent samples P values less than 005 were considered statistically significant
See the Supplementary materials and methods section for additional details
Page 6 of 45Diabetes
7
RESULTS
Up-Regulation of PDK2 Reduces Hepatic PDC Activity in HFD-Fed Mice
PDK2 expression is increased in the livers of obese animals (917) its role in the
development of hepatic metabolic diseases remains unclear We examined PDC activity and
the expression of PDK isoenzymes in the livers of WT mice fed a HFD As shown in Figure
1A PDC activity was significantly lower in the livers of mice fed a HFD than in those of
mice fed a LFD The reduction in hepatic PDC activity was associated with significant
increases in the phosphorylation of PDHE1α (Fig 1B) PDK2 mRNA levels were
significantly higher in the livers of HFD-fed mice compared to LFD-fed mice (Fig 1C)
Conversely the level of the PDK4 which is induced by a HFD in skeletal muscle and
heart(5) was markedly decreased in the livers of HFD-fed mice (Fig 1C) The protein levels
of PDK2 and PDK4 showed the same pattern as their respective mRNAs (Fig 1D) To
confirm that PDK2 regulates hepatic PDC activity in HFD-fed mice PDC activity and
phosphorylation of PDH E1α were measured in the livers of WT and PDK2 KO mice As
expected from the data shown in Figure 1A the HFD reduced hepatic PDC activity in WT
mice but not in PDK2 KO mice (Fig 1E) Consistent with this finding PDHE1α
phosphorylation was decreased in PDK2 KO mice compared to WT mice (Fig 1F) To
examine PDC activity change by PDK2 deficiency in other tissue we measured the
phosphorylation state of PDHE1α in muscle heart and kidney Phosphorylation of PDHE1α
was similar between HFD-fed PDK2 KO mice and HFD-fed WT mice in these tissues
(supplementary Fig 1) The serum levels of pyruvate lactate and FFA were not different
between WT and PDK2 KO mice after HFD feeding (Supplementary Fig 2) These findings
indicate that PDK2 is a major regulator of PDC activity in the liver of HFD-fed mice
Page 7 of 45 Diabetes
8
PDK2 Deficiency Prevents HFD-Induced Hepatic Steatosis
To elucidate the role of PDK2 in the development of hepatic steatosis WT and PDK2 KO
mice were fed a HFD to induce NAFLD After 16 weeks of HFD feeding body weight gain
in HFD-fed PDK2 KO mice was significantly lower than that of HFD-fed WT mice (Fig 2A)
Even though the body weights of HFD-fed PDK2 KO mice were lower than those of WT
mice there were no differences in food consumption or physical activity (Supplementary Fig
3A and 3B) Relative to HFD-fed WT mice HFD-fed PDK2 KO mice exhibited greater
energy expenditure (Supplementary Fig 3C)
The liverbody weight ratio of PDK2 KO mice fed a HFD was significantly lower than
that of WT mice (Fig 2B) The number and the size of the lipid droplets were greatly
decreased in livers of PDK2 KO mice fed a HFD (Fig 2C) However no difference in
phenotype in epididymal adipose tissue (EAT) was observed between WT and PDK2 KO
mice on either diet (Fig 2C) Consistent with the histological analysis the hepatic TG
content was significantly lower in PDK2 KO mice than in WT mice after HFD feeding (Fig
2D) The level of hepatic glycogen was also significantly reduced in PDK2 KO mice fed a
HFD compare to WT mice fed a HFD (Fig 2E) Serum activities of AST and ALT markers of
liver injury were also significantly elevated in WT mice fed a HFD but not in PDK2 KO
mice (Fig 2F and 2G) The expression of genes involved in inflammation including tumor
necrosis factor alpha (TNFα) monocyte chemoattractant protein-1 (MCP-1) and
plasminogen activator inhibitor-1 (PAI-1) were significantly lower in PDK2 KO mice than in
WT mice fed a HFD (Supplementary Fig 4A) These data indicate that PDK2 deficiency
could ameliorate HFD-induced hepatic steatosis injury and inflammation
Page 8 of 45Diabetes
9
PDK2 Deficiency Ameliorates Hepatic Insulin Resistance and Reduces Hepatic Glucose
Production
In HFD-fed mice PDK2 deficiency lowered fasting blood glucose and serum insulin
levels by 15 and 63 respectively relative to WT mice (Fig 3A and 3B) HFD-fed PDK2
KO mice processed glucose more efficiently than HFD-fed WT mice as evidenced by a 30
decrease in the area under the curve (AUC) during GTT (Fig 3C) However with LFD-fed
mice the elimination of glucose was similar between PDK2 KO mice and WT mice (Fig 3C)
which is well agreed with previous data (18) These observations suggest that PDK2
deficiency prevents impaired glucose homeostasis induced by a HFD
To better delineate the mechanism responsible for improved hepatic glucose homeostasis
in HFD-fed PDK2 KO mice we performed a hyperinsulinemic-euglycemic clamp study In
HFD-fed WT mice the glucose infusion rate (GIR) was lower hepatic glucose production
(HGP) was higher during the clamp and whole body glucose turnover was lower (Fig 3D)
than in LFD-fed WT mice However in HFD-fed PDK2 KO mice the GIR was increased
two-fold compared to HFD-fed WT mice (Fig 3D) Furthermore both basal and clamped
HGP were significantly lower in HFD-fed PDK2 KO mice than in HFD-fed WT mice (Fig
3D) Nevertheless whole body glucose turnover was not different between HFD-fed PDK2
KO mice and WT mice (Fig 3D) HGP measured by the pyruvate tolerance test (PTT) was
lower in HFD-fed PDK2 KO mice than in HFD-fed WT (Fig 3E) To evaluate insulin
signaling we examined the phosphorylation levels of AKT and FoxO1 in the liver and
muscle of HFD-fed WT and PDK2 KO mice after insulin injection Insulin-stimulated
phosphorylation of AKT and FoxO1 were significantly increased in the livers of PDK2 KO
mice compared to those of WT mice (Fig 3F) Interestingly in muscle the levels of AKT
and FoxO1 phosphorylation did not differ between HFD-fed PDK2 KO mice and HFD-fed
Page 9 of 45 Diabetes
10
WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by
PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be
caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation
of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine
whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we
measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The
amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-
fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the
phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2
KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of
hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by
a HFD
PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice
Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic
steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2
deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative
decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates
thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA
cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed
PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A
and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free
[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)
Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in
Page 10 of 45Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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Page 19 of 45 Diabetes
20
16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
alcoholic fatty liver disease J Biol Chem 200427932345-32353
20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
2012531080-1092
21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
Invest 20121221958-1959
22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
201233741-43
23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387
24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
383
26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
Page 20 of 45Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
3
ABSTRACT
Hepatic steatosis is associated with increased insulin resistance and tricarboxylic acid
(TCA) cycle flux but decreased ketogenesis and pyruvate dehydrogenase complex (PDC)
flux This study examined whether hepatic PDC activation by inhibition of pyruvate
dehydrogenase kinase 2 (PDK2) ameliorates these metabolic abnormalities
Wild-type (WT) mice fed a HFD exhibited hepatic steatosis insulin resistance and
increased levels of pyruvate TCA cycle intermediates and malonyl-CoA but reduced
ketogenesis and PDC activity due to PDK2 induction Hepatic PDC activation by PDK2
inhibition attenuated hepatic steatosis improved hepatic insulin sensitivity reduced hepatic
glucose production increased capacity for β-oxidation and ketogenesis and decreased the
capacity for lipogenesis These results were attributed to altered enzymatic capacities and a
reduction in TCA anaplerosis that limited the availability of oxaloacetate (OAA) for the TCA
cycle which promoted ketogenesis
The present study reports that increasing hepatic PDC activity by inhibition of PDK2
ameliorates hepatic steatosis and insulin sensitivity by regulating TCA cycle anaplerosis and
ketogenesis The findings suggest PDK2 is a potential therapeutic target for non-alcoholic
fatty liver disease (NAFLD)
INTRODUCTION
Hepatic steatosis is induced by a number of lipid metabolic abnormalities including
increased de novo lipogenesis inhibited triacylglyceol (TG) release enhanced FA influx from
adipose tissue and reduced hepatic FA oxidation and ketogenesis (1) Recently it was
proposed that dysregulation of ketone body metabolism could potentially contribute to the
pathogenesis of non-alcoholic fatty liver disease (NAFLD) Reduced ketogenesis exacerbates
Page 3 of 45 Diabetes
4
diet-induced hepatic steatosis and hyperglycemia (2) and a ketotic diet reduces the body
weight of mice as much as a calorie-restricted diet (3) However the precise mechanism by
which impaired ketogenesis contributes to hepatic steatosis is still unclear
In the well-fed state acetyl-CoA produced from glucose is converted to FAs for storage
of excess energy or oxidized to CO2 in the TCA cycle to generate ATP by oxidative
phosphorylation In the fasting state two-thirds of the free FAs entering the liver are
converted to acetyl-CoA by β-oxidation and then further processed to ketone bodies which
act as an energy source for peripheral tissues The other one-third of FAs is utilized by the
TCA cycle to generate ATP to meet hepatic energy demands (4) Whether acetyl-CoA
produced by β-oxidation forms ketone bodies or enters the TCA cycle is determined by
anaplerotic influx of TCA cycle intermediates The conversion of pyruvate to OAA by
pyruvate carboxylase (PC) is one of the most important sources of anaplerosis in the liver
Pyruvate can also be converted to acetyl-CoA by oxidative decarboxylation mediated by
PDC PDC activity is inhibited via phosphorylation of the pyruvate dehydrogenase E1α
subunit (PDHE1α) which is mediated by increased expression of PDKs during fasting or in
an insulin-resistant state (5-7) Four isoforms of PDK (PDK1-4) are expressed in a tissue-
specific manner with unique expression profiles in response to different physiological
conditions (8) Among these isoforms PDK2 is the major PDK responsible for regulation of
PDC activity in the liver (8-10)
Recently it was reported that decreased PDC activity and enhanced pyruvate
carboxylation due to hepatic insulin resistance contribute to increased hepatic
gluconeogenesis in obese subjects with hepatic steatosis (1112) Because of competition for
pyruvate the balance between PDC and PC activity may play a critical role in metabolic
dysfunction caused by obesity and insulin resistance In this study we examined the
Page 4 of 45Diabetes
5
possibility that activation of PDC by PDK2 inhibition reduces the anaplerotic flux of
pyruvate into the TCA cycle which may increase ketogenesis and prevent hepatic steatosis
induced by a HFD Indeed we found that PDK2 inhibition ameliorates hepatic steatosis and
insulin resistance suggesting that PDK2 is a potential therapeutic target for NAFLD
RESERCH DESIGN AND METHODS
Animal Experiments
All experiments were approved by the Institutional Animal Care and Use Committee of
Kyungpook National University For the NAFLD mice model 8-week-old male WT
(C57BL6J) and PDK2 KO mice (13) were fed a HFD in which 20 of the calories were
derived from carbohydrates and 60 from fat (Research Diets D12492 pellets) Control WT
and PDK2 KO mice were fed an isocaloric LFD in which 70 of the calories were derived
from carbohydrates and 10 from fat (Research Diets D12450B pellets) The mice were
housed and maintained on a 12 h lightdark cycle at 22 plusmn 2degC After the mice were sacrificed
tissues were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature
of liquid nitrogen and stored at -80degC for analysis
Construction of PDK2 DN Recombinant Adenovirus and Its Infection In Vivo
pCMV6-KanNeo mouse PDK2 plasmid was purchased from OriGene The PDK2
dominant-negative (DN) mutant R157A (1415) was generated by site-directed mutagenesis
with the QuickChange II site-directed mutagenesis kit (Stratagene) using the following
primers forward 5rsquo-CCAGTACTTCCTGGACGCCTTCTACCTCAGC-3rsquo and reverse 5rsquo-
GCTGAGGTAGAAGGCGTCCAGGAAGTACTGG-3rsquo Recombinant adenovirus for PDK2
DN mutant was generated using pAd-Track-CMV shuttle vector as described previously (16)
Page 5 of 45 Diabetes
6
Recombinant PDK2 DN and GFP adenovirus were amplified in AD-293 cells and purified by
CsCl gradient centrifugation Adenovirus titers were determined using Adeno-X rapid titer
(BD Bioscience) according to the manufacturerrsquos instructions After 12 weeks of HFD
feeding in WT mice recombinant adenoviruses (10 times 109 plaque-forming units) expressing
PDK2 DN and GFP constructs were delivered by tail vein injection On day 3 after
adenovirus infection glucose tolerance tests were performed in overnight-fasted mice by
intraperitoneal (ip) injection of glucose (1 gkg body weight) On day 7 after adenovirus
infection mice were sacrificed after overnight fasting After the mice were sacrificed tissues
were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature of
liquid nitrogen
Measurement of PDC Activity
PDC activity was spectrophotometrically measured in a 96-well plate reader by coupling
to the reaction catalyzed by arylamineacetyltransferase as described previously (7) One unit
of PDC activity corresponds to the acetylation of 1 micromol of p-(p-aminophenyl azo)
benzenesulfonate per min at 30degC
Statistical Analysis
Statistical significance was determined by the unpaired Studentrsquos t-test when two groups
were compared Values are presented as the means plusmn SEM of the indicated number of
independent samples P values less than 005 were considered statistically significant
See the Supplementary materials and methods section for additional details
Page 6 of 45Diabetes
7
RESULTS
Up-Regulation of PDK2 Reduces Hepatic PDC Activity in HFD-Fed Mice
PDK2 expression is increased in the livers of obese animals (917) its role in the
development of hepatic metabolic diseases remains unclear We examined PDC activity and
the expression of PDK isoenzymes in the livers of WT mice fed a HFD As shown in Figure
1A PDC activity was significantly lower in the livers of mice fed a HFD than in those of
mice fed a LFD The reduction in hepatic PDC activity was associated with significant
increases in the phosphorylation of PDHE1α (Fig 1B) PDK2 mRNA levels were
significantly higher in the livers of HFD-fed mice compared to LFD-fed mice (Fig 1C)
Conversely the level of the PDK4 which is induced by a HFD in skeletal muscle and
heart(5) was markedly decreased in the livers of HFD-fed mice (Fig 1C) The protein levels
of PDK2 and PDK4 showed the same pattern as their respective mRNAs (Fig 1D) To
confirm that PDK2 regulates hepatic PDC activity in HFD-fed mice PDC activity and
phosphorylation of PDH E1α were measured in the livers of WT and PDK2 KO mice As
expected from the data shown in Figure 1A the HFD reduced hepatic PDC activity in WT
mice but not in PDK2 KO mice (Fig 1E) Consistent with this finding PDHE1α
phosphorylation was decreased in PDK2 KO mice compared to WT mice (Fig 1F) To
examine PDC activity change by PDK2 deficiency in other tissue we measured the
phosphorylation state of PDHE1α in muscle heart and kidney Phosphorylation of PDHE1α
was similar between HFD-fed PDK2 KO mice and HFD-fed WT mice in these tissues
(supplementary Fig 1) The serum levels of pyruvate lactate and FFA were not different
between WT and PDK2 KO mice after HFD feeding (Supplementary Fig 2) These findings
indicate that PDK2 is a major regulator of PDC activity in the liver of HFD-fed mice
Page 7 of 45 Diabetes
8
PDK2 Deficiency Prevents HFD-Induced Hepatic Steatosis
To elucidate the role of PDK2 in the development of hepatic steatosis WT and PDK2 KO
mice were fed a HFD to induce NAFLD After 16 weeks of HFD feeding body weight gain
in HFD-fed PDK2 KO mice was significantly lower than that of HFD-fed WT mice (Fig 2A)
Even though the body weights of HFD-fed PDK2 KO mice were lower than those of WT
mice there were no differences in food consumption or physical activity (Supplementary Fig
3A and 3B) Relative to HFD-fed WT mice HFD-fed PDK2 KO mice exhibited greater
energy expenditure (Supplementary Fig 3C)
The liverbody weight ratio of PDK2 KO mice fed a HFD was significantly lower than
that of WT mice (Fig 2B) The number and the size of the lipid droplets were greatly
decreased in livers of PDK2 KO mice fed a HFD (Fig 2C) However no difference in
phenotype in epididymal adipose tissue (EAT) was observed between WT and PDK2 KO
mice on either diet (Fig 2C) Consistent with the histological analysis the hepatic TG
content was significantly lower in PDK2 KO mice than in WT mice after HFD feeding (Fig
2D) The level of hepatic glycogen was also significantly reduced in PDK2 KO mice fed a
HFD compare to WT mice fed a HFD (Fig 2E) Serum activities of AST and ALT markers of
liver injury were also significantly elevated in WT mice fed a HFD but not in PDK2 KO
mice (Fig 2F and 2G) The expression of genes involved in inflammation including tumor
necrosis factor alpha (TNFα) monocyte chemoattractant protein-1 (MCP-1) and
plasminogen activator inhibitor-1 (PAI-1) were significantly lower in PDK2 KO mice than in
WT mice fed a HFD (Supplementary Fig 4A) These data indicate that PDK2 deficiency
could ameliorate HFD-induced hepatic steatosis injury and inflammation
Page 8 of 45Diabetes
9
PDK2 Deficiency Ameliorates Hepatic Insulin Resistance and Reduces Hepatic Glucose
Production
In HFD-fed mice PDK2 deficiency lowered fasting blood glucose and serum insulin
levels by 15 and 63 respectively relative to WT mice (Fig 3A and 3B) HFD-fed PDK2
KO mice processed glucose more efficiently than HFD-fed WT mice as evidenced by a 30
decrease in the area under the curve (AUC) during GTT (Fig 3C) However with LFD-fed
mice the elimination of glucose was similar between PDK2 KO mice and WT mice (Fig 3C)
which is well agreed with previous data (18) These observations suggest that PDK2
deficiency prevents impaired glucose homeostasis induced by a HFD
To better delineate the mechanism responsible for improved hepatic glucose homeostasis
in HFD-fed PDK2 KO mice we performed a hyperinsulinemic-euglycemic clamp study In
HFD-fed WT mice the glucose infusion rate (GIR) was lower hepatic glucose production
(HGP) was higher during the clamp and whole body glucose turnover was lower (Fig 3D)
than in LFD-fed WT mice However in HFD-fed PDK2 KO mice the GIR was increased
two-fold compared to HFD-fed WT mice (Fig 3D) Furthermore both basal and clamped
HGP were significantly lower in HFD-fed PDK2 KO mice than in HFD-fed WT mice (Fig
3D) Nevertheless whole body glucose turnover was not different between HFD-fed PDK2
KO mice and WT mice (Fig 3D) HGP measured by the pyruvate tolerance test (PTT) was
lower in HFD-fed PDK2 KO mice than in HFD-fed WT (Fig 3E) To evaluate insulin
signaling we examined the phosphorylation levels of AKT and FoxO1 in the liver and
muscle of HFD-fed WT and PDK2 KO mice after insulin injection Insulin-stimulated
phosphorylation of AKT and FoxO1 were significantly increased in the livers of PDK2 KO
mice compared to those of WT mice (Fig 3F) Interestingly in muscle the levels of AKT
and FoxO1 phosphorylation did not differ between HFD-fed PDK2 KO mice and HFD-fed
Page 9 of 45 Diabetes
10
WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by
PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be
caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation
of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine
whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we
measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The
amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-
fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the
phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2
KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of
hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by
a HFD
PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice
Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic
steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2
deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative
decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates
thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA
cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed
PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A
and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free
[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)
Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in
Page 10 of 45Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
4
diet-induced hepatic steatosis and hyperglycemia (2) and a ketotic diet reduces the body
weight of mice as much as a calorie-restricted diet (3) However the precise mechanism by
which impaired ketogenesis contributes to hepatic steatosis is still unclear
In the well-fed state acetyl-CoA produced from glucose is converted to FAs for storage
of excess energy or oxidized to CO2 in the TCA cycle to generate ATP by oxidative
phosphorylation In the fasting state two-thirds of the free FAs entering the liver are
converted to acetyl-CoA by β-oxidation and then further processed to ketone bodies which
act as an energy source for peripheral tissues The other one-third of FAs is utilized by the
TCA cycle to generate ATP to meet hepatic energy demands (4) Whether acetyl-CoA
produced by β-oxidation forms ketone bodies or enters the TCA cycle is determined by
anaplerotic influx of TCA cycle intermediates The conversion of pyruvate to OAA by
pyruvate carboxylase (PC) is one of the most important sources of anaplerosis in the liver
Pyruvate can also be converted to acetyl-CoA by oxidative decarboxylation mediated by
PDC PDC activity is inhibited via phosphorylation of the pyruvate dehydrogenase E1α
subunit (PDHE1α) which is mediated by increased expression of PDKs during fasting or in
an insulin-resistant state (5-7) Four isoforms of PDK (PDK1-4) are expressed in a tissue-
specific manner with unique expression profiles in response to different physiological
conditions (8) Among these isoforms PDK2 is the major PDK responsible for regulation of
PDC activity in the liver (8-10)
Recently it was reported that decreased PDC activity and enhanced pyruvate
carboxylation due to hepatic insulin resistance contribute to increased hepatic
gluconeogenesis in obese subjects with hepatic steatosis (1112) Because of competition for
pyruvate the balance between PDC and PC activity may play a critical role in metabolic
dysfunction caused by obesity and insulin resistance In this study we examined the
Page 4 of 45Diabetes
5
possibility that activation of PDC by PDK2 inhibition reduces the anaplerotic flux of
pyruvate into the TCA cycle which may increase ketogenesis and prevent hepatic steatosis
induced by a HFD Indeed we found that PDK2 inhibition ameliorates hepatic steatosis and
insulin resistance suggesting that PDK2 is a potential therapeutic target for NAFLD
RESERCH DESIGN AND METHODS
Animal Experiments
All experiments were approved by the Institutional Animal Care and Use Committee of
Kyungpook National University For the NAFLD mice model 8-week-old male WT
(C57BL6J) and PDK2 KO mice (13) were fed a HFD in which 20 of the calories were
derived from carbohydrates and 60 from fat (Research Diets D12492 pellets) Control WT
and PDK2 KO mice were fed an isocaloric LFD in which 70 of the calories were derived
from carbohydrates and 10 from fat (Research Diets D12450B pellets) The mice were
housed and maintained on a 12 h lightdark cycle at 22 plusmn 2degC After the mice were sacrificed
tissues were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature
of liquid nitrogen and stored at -80degC for analysis
Construction of PDK2 DN Recombinant Adenovirus and Its Infection In Vivo
pCMV6-KanNeo mouse PDK2 plasmid was purchased from OriGene The PDK2
dominant-negative (DN) mutant R157A (1415) was generated by site-directed mutagenesis
with the QuickChange II site-directed mutagenesis kit (Stratagene) using the following
primers forward 5rsquo-CCAGTACTTCCTGGACGCCTTCTACCTCAGC-3rsquo and reverse 5rsquo-
GCTGAGGTAGAAGGCGTCCAGGAAGTACTGG-3rsquo Recombinant adenovirus for PDK2
DN mutant was generated using pAd-Track-CMV shuttle vector as described previously (16)
Page 5 of 45 Diabetes
6
Recombinant PDK2 DN and GFP adenovirus were amplified in AD-293 cells and purified by
CsCl gradient centrifugation Adenovirus titers were determined using Adeno-X rapid titer
(BD Bioscience) according to the manufacturerrsquos instructions After 12 weeks of HFD
feeding in WT mice recombinant adenoviruses (10 times 109 plaque-forming units) expressing
PDK2 DN and GFP constructs were delivered by tail vein injection On day 3 after
adenovirus infection glucose tolerance tests were performed in overnight-fasted mice by
intraperitoneal (ip) injection of glucose (1 gkg body weight) On day 7 after adenovirus
infection mice were sacrificed after overnight fasting After the mice were sacrificed tissues
were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature of
liquid nitrogen
Measurement of PDC Activity
PDC activity was spectrophotometrically measured in a 96-well plate reader by coupling
to the reaction catalyzed by arylamineacetyltransferase as described previously (7) One unit
of PDC activity corresponds to the acetylation of 1 micromol of p-(p-aminophenyl azo)
benzenesulfonate per min at 30degC
Statistical Analysis
Statistical significance was determined by the unpaired Studentrsquos t-test when two groups
were compared Values are presented as the means plusmn SEM of the indicated number of
independent samples P values less than 005 were considered statistically significant
See the Supplementary materials and methods section for additional details
Page 6 of 45Diabetes
7
RESULTS
Up-Regulation of PDK2 Reduces Hepatic PDC Activity in HFD-Fed Mice
PDK2 expression is increased in the livers of obese animals (917) its role in the
development of hepatic metabolic diseases remains unclear We examined PDC activity and
the expression of PDK isoenzymes in the livers of WT mice fed a HFD As shown in Figure
1A PDC activity was significantly lower in the livers of mice fed a HFD than in those of
mice fed a LFD The reduction in hepatic PDC activity was associated with significant
increases in the phosphorylation of PDHE1α (Fig 1B) PDK2 mRNA levels were
significantly higher in the livers of HFD-fed mice compared to LFD-fed mice (Fig 1C)
Conversely the level of the PDK4 which is induced by a HFD in skeletal muscle and
heart(5) was markedly decreased in the livers of HFD-fed mice (Fig 1C) The protein levels
of PDK2 and PDK4 showed the same pattern as their respective mRNAs (Fig 1D) To
confirm that PDK2 regulates hepatic PDC activity in HFD-fed mice PDC activity and
phosphorylation of PDH E1α were measured in the livers of WT and PDK2 KO mice As
expected from the data shown in Figure 1A the HFD reduced hepatic PDC activity in WT
mice but not in PDK2 KO mice (Fig 1E) Consistent with this finding PDHE1α
phosphorylation was decreased in PDK2 KO mice compared to WT mice (Fig 1F) To
examine PDC activity change by PDK2 deficiency in other tissue we measured the
phosphorylation state of PDHE1α in muscle heart and kidney Phosphorylation of PDHE1α
was similar between HFD-fed PDK2 KO mice and HFD-fed WT mice in these tissues
(supplementary Fig 1) The serum levels of pyruvate lactate and FFA were not different
between WT and PDK2 KO mice after HFD feeding (Supplementary Fig 2) These findings
indicate that PDK2 is a major regulator of PDC activity in the liver of HFD-fed mice
Page 7 of 45 Diabetes
8
PDK2 Deficiency Prevents HFD-Induced Hepatic Steatosis
To elucidate the role of PDK2 in the development of hepatic steatosis WT and PDK2 KO
mice were fed a HFD to induce NAFLD After 16 weeks of HFD feeding body weight gain
in HFD-fed PDK2 KO mice was significantly lower than that of HFD-fed WT mice (Fig 2A)
Even though the body weights of HFD-fed PDK2 KO mice were lower than those of WT
mice there were no differences in food consumption or physical activity (Supplementary Fig
3A and 3B) Relative to HFD-fed WT mice HFD-fed PDK2 KO mice exhibited greater
energy expenditure (Supplementary Fig 3C)
The liverbody weight ratio of PDK2 KO mice fed a HFD was significantly lower than
that of WT mice (Fig 2B) The number and the size of the lipid droplets were greatly
decreased in livers of PDK2 KO mice fed a HFD (Fig 2C) However no difference in
phenotype in epididymal adipose tissue (EAT) was observed between WT and PDK2 KO
mice on either diet (Fig 2C) Consistent with the histological analysis the hepatic TG
content was significantly lower in PDK2 KO mice than in WT mice after HFD feeding (Fig
2D) The level of hepatic glycogen was also significantly reduced in PDK2 KO mice fed a
HFD compare to WT mice fed a HFD (Fig 2E) Serum activities of AST and ALT markers of
liver injury were also significantly elevated in WT mice fed a HFD but not in PDK2 KO
mice (Fig 2F and 2G) The expression of genes involved in inflammation including tumor
necrosis factor alpha (TNFα) monocyte chemoattractant protein-1 (MCP-1) and
plasminogen activator inhibitor-1 (PAI-1) were significantly lower in PDK2 KO mice than in
WT mice fed a HFD (Supplementary Fig 4A) These data indicate that PDK2 deficiency
could ameliorate HFD-induced hepatic steatosis injury and inflammation
Page 8 of 45Diabetes
9
PDK2 Deficiency Ameliorates Hepatic Insulin Resistance and Reduces Hepatic Glucose
Production
In HFD-fed mice PDK2 deficiency lowered fasting blood glucose and serum insulin
levels by 15 and 63 respectively relative to WT mice (Fig 3A and 3B) HFD-fed PDK2
KO mice processed glucose more efficiently than HFD-fed WT mice as evidenced by a 30
decrease in the area under the curve (AUC) during GTT (Fig 3C) However with LFD-fed
mice the elimination of glucose was similar between PDK2 KO mice and WT mice (Fig 3C)
which is well agreed with previous data (18) These observations suggest that PDK2
deficiency prevents impaired glucose homeostasis induced by a HFD
To better delineate the mechanism responsible for improved hepatic glucose homeostasis
in HFD-fed PDK2 KO mice we performed a hyperinsulinemic-euglycemic clamp study In
HFD-fed WT mice the glucose infusion rate (GIR) was lower hepatic glucose production
(HGP) was higher during the clamp and whole body glucose turnover was lower (Fig 3D)
than in LFD-fed WT mice However in HFD-fed PDK2 KO mice the GIR was increased
two-fold compared to HFD-fed WT mice (Fig 3D) Furthermore both basal and clamped
HGP were significantly lower in HFD-fed PDK2 KO mice than in HFD-fed WT mice (Fig
3D) Nevertheless whole body glucose turnover was not different between HFD-fed PDK2
KO mice and WT mice (Fig 3D) HGP measured by the pyruvate tolerance test (PTT) was
lower in HFD-fed PDK2 KO mice than in HFD-fed WT (Fig 3E) To evaluate insulin
signaling we examined the phosphorylation levels of AKT and FoxO1 in the liver and
muscle of HFD-fed WT and PDK2 KO mice after insulin injection Insulin-stimulated
phosphorylation of AKT and FoxO1 were significantly increased in the livers of PDK2 KO
mice compared to those of WT mice (Fig 3F) Interestingly in muscle the levels of AKT
and FoxO1 phosphorylation did not differ between HFD-fed PDK2 KO mice and HFD-fed
Page 9 of 45 Diabetes
10
WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by
PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be
caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation
of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine
whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we
measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The
amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-
fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the
phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2
KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of
hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by
a HFD
PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice
Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic
steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2
deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative
decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates
thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA
cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed
PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A
and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free
[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)
Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in
Page 10 of 45Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
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10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
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11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
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12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
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15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
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17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
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18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
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26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
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21
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Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
5
possibility that activation of PDC by PDK2 inhibition reduces the anaplerotic flux of
pyruvate into the TCA cycle which may increase ketogenesis and prevent hepatic steatosis
induced by a HFD Indeed we found that PDK2 inhibition ameliorates hepatic steatosis and
insulin resistance suggesting that PDK2 is a potential therapeutic target for NAFLD
RESERCH DESIGN AND METHODS
Animal Experiments
All experiments were approved by the Institutional Animal Care and Use Committee of
Kyungpook National University For the NAFLD mice model 8-week-old male WT
(C57BL6J) and PDK2 KO mice (13) were fed a HFD in which 20 of the calories were
derived from carbohydrates and 60 from fat (Research Diets D12492 pellets) Control WT
and PDK2 KO mice were fed an isocaloric LFD in which 70 of the calories were derived
from carbohydrates and 10 from fat (Research Diets D12450B pellets) The mice were
housed and maintained on a 12 h lightdark cycle at 22 plusmn 2degC After the mice were sacrificed
tissues were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature
of liquid nitrogen and stored at -80degC for analysis
Construction of PDK2 DN Recombinant Adenovirus and Its Infection In Vivo
pCMV6-KanNeo mouse PDK2 plasmid was purchased from OriGene The PDK2
dominant-negative (DN) mutant R157A (1415) was generated by site-directed mutagenesis
with the QuickChange II site-directed mutagenesis kit (Stratagene) using the following
primers forward 5rsquo-CCAGTACTTCCTGGACGCCTTCTACCTCAGC-3rsquo and reverse 5rsquo-
GCTGAGGTAGAAGGCGTCCAGGAAGTACTGG-3rsquo Recombinant adenovirus for PDK2
DN mutant was generated using pAd-Track-CMV shuttle vector as described previously (16)
Page 5 of 45 Diabetes
6
Recombinant PDK2 DN and GFP adenovirus were amplified in AD-293 cells and purified by
CsCl gradient centrifugation Adenovirus titers were determined using Adeno-X rapid titer
(BD Bioscience) according to the manufacturerrsquos instructions After 12 weeks of HFD
feeding in WT mice recombinant adenoviruses (10 times 109 plaque-forming units) expressing
PDK2 DN and GFP constructs were delivered by tail vein injection On day 3 after
adenovirus infection glucose tolerance tests were performed in overnight-fasted mice by
intraperitoneal (ip) injection of glucose (1 gkg body weight) On day 7 after adenovirus
infection mice were sacrificed after overnight fasting After the mice were sacrificed tissues
were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature of
liquid nitrogen
Measurement of PDC Activity
PDC activity was spectrophotometrically measured in a 96-well plate reader by coupling
to the reaction catalyzed by arylamineacetyltransferase as described previously (7) One unit
of PDC activity corresponds to the acetylation of 1 micromol of p-(p-aminophenyl azo)
benzenesulfonate per min at 30degC
Statistical Analysis
Statistical significance was determined by the unpaired Studentrsquos t-test when two groups
were compared Values are presented as the means plusmn SEM of the indicated number of
independent samples P values less than 005 were considered statistically significant
See the Supplementary materials and methods section for additional details
Page 6 of 45Diabetes
7
RESULTS
Up-Regulation of PDK2 Reduces Hepatic PDC Activity in HFD-Fed Mice
PDK2 expression is increased in the livers of obese animals (917) its role in the
development of hepatic metabolic diseases remains unclear We examined PDC activity and
the expression of PDK isoenzymes in the livers of WT mice fed a HFD As shown in Figure
1A PDC activity was significantly lower in the livers of mice fed a HFD than in those of
mice fed a LFD The reduction in hepatic PDC activity was associated with significant
increases in the phosphorylation of PDHE1α (Fig 1B) PDK2 mRNA levels were
significantly higher in the livers of HFD-fed mice compared to LFD-fed mice (Fig 1C)
Conversely the level of the PDK4 which is induced by a HFD in skeletal muscle and
heart(5) was markedly decreased in the livers of HFD-fed mice (Fig 1C) The protein levels
of PDK2 and PDK4 showed the same pattern as their respective mRNAs (Fig 1D) To
confirm that PDK2 regulates hepatic PDC activity in HFD-fed mice PDC activity and
phosphorylation of PDH E1α were measured in the livers of WT and PDK2 KO mice As
expected from the data shown in Figure 1A the HFD reduced hepatic PDC activity in WT
mice but not in PDK2 KO mice (Fig 1E) Consistent with this finding PDHE1α
phosphorylation was decreased in PDK2 KO mice compared to WT mice (Fig 1F) To
examine PDC activity change by PDK2 deficiency in other tissue we measured the
phosphorylation state of PDHE1α in muscle heart and kidney Phosphorylation of PDHE1α
was similar between HFD-fed PDK2 KO mice and HFD-fed WT mice in these tissues
(supplementary Fig 1) The serum levels of pyruvate lactate and FFA were not different
between WT and PDK2 KO mice after HFD feeding (Supplementary Fig 2) These findings
indicate that PDK2 is a major regulator of PDC activity in the liver of HFD-fed mice
Page 7 of 45 Diabetes
8
PDK2 Deficiency Prevents HFD-Induced Hepatic Steatosis
To elucidate the role of PDK2 in the development of hepatic steatosis WT and PDK2 KO
mice were fed a HFD to induce NAFLD After 16 weeks of HFD feeding body weight gain
in HFD-fed PDK2 KO mice was significantly lower than that of HFD-fed WT mice (Fig 2A)
Even though the body weights of HFD-fed PDK2 KO mice were lower than those of WT
mice there were no differences in food consumption or physical activity (Supplementary Fig
3A and 3B) Relative to HFD-fed WT mice HFD-fed PDK2 KO mice exhibited greater
energy expenditure (Supplementary Fig 3C)
The liverbody weight ratio of PDK2 KO mice fed a HFD was significantly lower than
that of WT mice (Fig 2B) The number and the size of the lipid droplets were greatly
decreased in livers of PDK2 KO mice fed a HFD (Fig 2C) However no difference in
phenotype in epididymal adipose tissue (EAT) was observed between WT and PDK2 KO
mice on either diet (Fig 2C) Consistent with the histological analysis the hepatic TG
content was significantly lower in PDK2 KO mice than in WT mice after HFD feeding (Fig
2D) The level of hepatic glycogen was also significantly reduced in PDK2 KO mice fed a
HFD compare to WT mice fed a HFD (Fig 2E) Serum activities of AST and ALT markers of
liver injury were also significantly elevated in WT mice fed a HFD but not in PDK2 KO
mice (Fig 2F and 2G) The expression of genes involved in inflammation including tumor
necrosis factor alpha (TNFα) monocyte chemoattractant protein-1 (MCP-1) and
plasminogen activator inhibitor-1 (PAI-1) were significantly lower in PDK2 KO mice than in
WT mice fed a HFD (Supplementary Fig 4A) These data indicate that PDK2 deficiency
could ameliorate HFD-induced hepatic steatosis injury and inflammation
Page 8 of 45Diabetes
9
PDK2 Deficiency Ameliorates Hepatic Insulin Resistance and Reduces Hepatic Glucose
Production
In HFD-fed mice PDK2 deficiency lowered fasting blood glucose and serum insulin
levels by 15 and 63 respectively relative to WT mice (Fig 3A and 3B) HFD-fed PDK2
KO mice processed glucose more efficiently than HFD-fed WT mice as evidenced by a 30
decrease in the area under the curve (AUC) during GTT (Fig 3C) However with LFD-fed
mice the elimination of glucose was similar between PDK2 KO mice and WT mice (Fig 3C)
which is well agreed with previous data (18) These observations suggest that PDK2
deficiency prevents impaired glucose homeostasis induced by a HFD
To better delineate the mechanism responsible for improved hepatic glucose homeostasis
in HFD-fed PDK2 KO mice we performed a hyperinsulinemic-euglycemic clamp study In
HFD-fed WT mice the glucose infusion rate (GIR) was lower hepatic glucose production
(HGP) was higher during the clamp and whole body glucose turnover was lower (Fig 3D)
than in LFD-fed WT mice However in HFD-fed PDK2 KO mice the GIR was increased
two-fold compared to HFD-fed WT mice (Fig 3D) Furthermore both basal and clamped
HGP were significantly lower in HFD-fed PDK2 KO mice than in HFD-fed WT mice (Fig
3D) Nevertheless whole body glucose turnover was not different between HFD-fed PDK2
KO mice and WT mice (Fig 3D) HGP measured by the pyruvate tolerance test (PTT) was
lower in HFD-fed PDK2 KO mice than in HFD-fed WT (Fig 3E) To evaluate insulin
signaling we examined the phosphorylation levels of AKT and FoxO1 in the liver and
muscle of HFD-fed WT and PDK2 KO mice after insulin injection Insulin-stimulated
phosphorylation of AKT and FoxO1 were significantly increased in the livers of PDK2 KO
mice compared to those of WT mice (Fig 3F) Interestingly in muscle the levels of AKT
and FoxO1 phosphorylation did not differ between HFD-fed PDK2 KO mice and HFD-fed
Page 9 of 45 Diabetes
10
WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by
PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be
caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation
of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine
whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we
measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The
amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-
fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the
phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2
KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of
hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by
a HFD
PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice
Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic
steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2
deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative
decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates
thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA
cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed
PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A
and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free
[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)
Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in
Page 10 of 45Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
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10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
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12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
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13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
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15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
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18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
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19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
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20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
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26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
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28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
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Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
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rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
6
Recombinant PDK2 DN and GFP adenovirus were amplified in AD-293 cells and purified by
CsCl gradient centrifugation Adenovirus titers were determined using Adeno-X rapid titer
(BD Bioscience) according to the manufacturerrsquos instructions After 12 weeks of HFD
feeding in WT mice recombinant adenoviruses (10 times 109 plaque-forming units) expressing
PDK2 DN and GFP constructs were delivered by tail vein injection On day 3 after
adenovirus infection glucose tolerance tests were performed in overnight-fasted mice by
intraperitoneal (ip) injection of glucose (1 gkg body weight) On day 7 after adenovirus
infection mice were sacrificed after overnight fasting After the mice were sacrificed tissues
were rapidly collected and freeze-clamped with Wollenberger tongs at the temperature of
liquid nitrogen
Measurement of PDC Activity
PDC activity was spectrophotometrically measured in a 96-well plate reader by coupling
to the reaction catalyzed by arylamineacetyltransferase as described previously (7) One unit
of PDC activity corresponds to the acetylation of 1 micromol of p-(p-aminophenyl azo)
benzenesulfonate per min at 30degC
Statistical Analysis
Statistical significance was determined by the unpaired Studentrsquos t-test when two groups
were compared Values are presented as the means plusmn SEM of the indicated number of
independent samples P values less than 005 were considered statistically significant
See the Supplementary materials and methods section for additional details
Page 6 of 45Diabetes
7
RESULTS
Up-Regulation of PDK2 Reduces Hepatic PDC Activity in HFD-Fed Mice
PDK2 expression is increased in the livers of obese animals (917) its role in the
development of hepatic metabolic diseases remains unclear We examined PDC activity and
the expression of PDK isoenzymes in the livers of WT mice fed a HFD As shown in Figure
1A PDC activity was significantly lower in the livers of mice fed a HFD than in those of
mice fed a LFD The reduction in hepatic PDC activity was associated with significant
increases in the phosphorylation of PDHE1α (Fig 1B) PDK2 mRNA levels were
significantly higher in the livers of HFD-fed mice compared to LFD-fed mice (Fig 1C)
Conversely the level of the PDK4 which is induced by a HFD in skeletal muscle and
heart(5) was markedly decreased in the livers of HFD-fed mice (Fig 1C) The protein levels
of PDK2 and PDK4 showed the same pattern as their respective mRNAs (Fig 1D) To
confirm that PDK2 regulates hepatic PDC activity in HFD-fed mice PDC activity and
phosphorylation of PDH E1α were measured in the livers of WT and PDK2 KO mice As
expected from the data shown in Figure 1A the HFD reduced hepatic PDC activity in WT
mice but not in PDK2 KO mice (Fig 1E) Consistent with this finding PDHE1α
phosphorylation was decreased in PDK2 KO mice compared to WT mice (Fig 1F) To
examine PDC activity change by PDK2 deficiency in other tissue we measured the
phosphorylation state of PDHE1α in muscle heart and kidney Phosphorylation of PDHE1α
was similar between HFD-fed PDK2 KO mice and HFD-fed WT mice in these tissues
(supplementary Fig 1) The serum levels of pyruvate lactate and FFA were not different
between WT and PDK2 KO mice after HFD feeding (Supplementary Fig 2) These findings
indicate that PDK2 is a major regulator of PDC activity in the liver of HFD-fed mice
Page 7 of 45 Diabetes
8
PDK2 Deficiency Prevents HFD-Induced Hepatic Steatosis
To elucidate the role of PDK2 in the development of hepatic steatosis WT and PDK2 KO
mice were fed a HFD to induce NAFLD After 16 weeks of HFD feeding body weight gain
in HFD-fed PDK2 KO mice was significantly lower than that of HFD-fed WT mice (Fig 2A)
Even though the body weights of HFD-fed PDK2 KO mice were lower than those of WT
mice there were no differences in food consumption or physical activity (Supplementary Fig
3A and 3B) Relative to HFD-fed WT mice HFD-fed PDK2 KO mice exhibited greater
energy expenditure (Supplementary Fig 3C)
The liverbody weight ratio of PDK2 KO mice fed a HFD was significantly lower than
that of WT mice (Fig 2B) The number and the size of the lipid droplets were greatly
decreased in livers of PDK2 KO mice fed a HFD (Fig 2C) However no difference in
phenotype in epididymal adipose tissue (EAT) was observed between WT and PDK2 KO
mice on either diet (Fig 2C) Consistent with the histological analysis the hepatic TG
content was significantly lower in PDK2 KO mice than in WT mice after HFD feeding (Fig
2D) The level of hepatic glycogen was also significantly reduced in PDK2 KO mice fed a
HFD compare to WT mice fed a HFD (Fig 2E) Serum activities of AST and ALT markers of
liver injury were also significantly elevated in WT mice fed a HFD but not in PDK2 KO
mice (Fig 2F and 2G) The expression of genes involved in inflammation including tumor
necrosis factor alpha (TNFα) monocyte chemoattractant protein-1 (MCP-1) and
plasminogen activator inhibitor-1 (PAI-1) were significantly lower in PDK2 KO mice than in
WT mice fed a HFD (Supplementary Fig 4A) These data indicate that PDK2 deficiency
could ameliorate HFD-induced hepatic steatosis injury and inflammation
Page 8 of 45Diabetes
9
PDK2 Deficiency Ameliorates Hepatic Insulin Resistance and Reduces Hepatic Glucose
Production
In HFD-fed mice PDK2 deficiency lowered fasting blood glucose and serum insulin
levels by 15 and 63 respectively relative to WT mice (Fig 3A and 3B) HFD-fed PDK2
KO mice processed glucose more efficiently than HFD-fed WT mice as evidenced by a 30
decrease in the area under the curve (AUC) during GTT (Fig 3C) However with LFD-fed
mice the elimination of glucose was similar between PDK2 KO mice and WT mice (Fig 3C)
which is well agreed with previous data (18) These observations suggest that PDK2
deficiency prevents impaired glucose homeostasis induced by a HFD
To better delineate the mechanism responsible for improved hepatic glucose homeostasis
in HFD-fed PDK2 KO mice we performed a hyperinsulinemic-euglycemic clamp study In
HFD-fed WT mice the glucose infusion rate (GIR) was lower hepatic glucose production
(HGP) was higher during the clamp and whole body glucose turnover was lower (Fig 3D)
than in LFD-fed WT mice However in HFD-fed PDK2 KO mice the GIR was increased
two-fold compared to HFD-fed WT mice (Fig 3D) Furthermore both basal and clamped
HGP were significantly lower in HFD-fed PDK2 KO mice than in HFD-fed WT mice (Fig
3D) Nevertheless whole body glucose turnover was not different between HFD-fed PDK2
KO mice and WT mice (Fig 3D) HGP measured by the pyruvate tolerance test (PTT) was
lower in HFD-fed PDK2 KO mice than in HFD-fed WT (Fig 3E) To evaluate insulin
signaling we examined the phosphorylation levels of AKT and FoxO1 in the liver and
muscle of HFD-fed WT and PDK2 KO mice after insulin injection Insulin-stimulated
phosphorylation of AKT and FoxO1 were significantly increased in the livers of PDK2 KO
mice compared to those of WT mice (Fig 3F) Interestingly in muscle the levels of AKT
and FoxO1 phosphorylation did not differ between HFD-fed PDK2 KO mice and HFD-fed
Page 9 of 45 Diabetes
10
WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by
PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be
caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation
of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine
whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we
measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The
amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-
fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the
phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2
KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of
hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by
a HFD
PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice
Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic
steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2
deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative
decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates
thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA
cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed
PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A
and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free
[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)
Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in
Page 10 of 45Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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4 Williamson JR Scholz R Browning ET Control Mechanisms of Gluconeogenesis
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6 Sugden MC Holness MJ Recent advances in mechanisms regulating glucose
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7 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
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8 Wu P Blair PV Sato J Jaskiewicz J Popov KM Harris RA Starvation increases the
amount of pyruvate dehydrogenase kinase in several mammalian tissues Arch Biochem
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9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
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10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase
isoforms 2 and 4 by fatty acids and thyroid hormone Biochem J 2003369687-695
11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease Cell Metab
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13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
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14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate
dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-
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15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
kinase-4 structures reveal a metastable open conformation fostering robust core-free basal
activity J Biol Chem 200828325305-25315
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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
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20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
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21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
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22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
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23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
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24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
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26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
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related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
7
RESULTS
Up-Regulation of PDK2 Reduces Hepatic PDC Activity in HFD-Fed Mice
PDK2 expression is increased in the livers of obese animals (917) its role in the
development of hepatic metabolic diseases remains unclear We examined PDC activity and
the expression of PDK isoenzymes in the livers of WT mice fed a HFD As shown in Figure
1A PDC activity was significantly lower in the livers of mice fed a HFD than in those of
mice fed a LFD The reduction in hepatic PDC activity was associated with significant
increases in the phosphorylation of PDHE1α (Fig 1B) PDK2 mRNA levels were
significantly higher in the livers of HFD-fed mice compared to LFD-fed mice (Fig 1C)
Conversely the level of the PDK4 which is induced by a HFD in skeletal muscle and
heart(5) was markedly decreased in the livers of HFD-fed mice (Fig 1C) The protein levels
of PDK2 and PDK4 showed the same pattern as their respective mRNAs (Fig 1D) To
confirm that PDK2 regulates hepatic PDC activity in HFD-fed mice PDC activity and
phosphorylation of PDH E1α were measured in the livers of WT and PDK2 KO mice As
expected from the data shown in Figure 1A the HFD reduced hepatic PDC activity in WT
mice but not in PDK2 KO mice (Fig 1E) Consistent with this finding PDHE1α
phosphorylation was decreased in PDK2 KO mice compared to WT mice (Fig 1F) To
examine PDC activity change by PDK2 deficiency in other tissue we measured the
phosphorylation state of PDHE1α in muscle heart and kidney Phosphorylation of PDHE1α
was similar between HFD-fed PDK2 KO mice and HFD-fed WT mice in these tissues
(supplementary Fig 1) The serum levels of pyruvate lactate and FFA were not different
between WT and PDK2 KO mice after HFD feeding (Supplementary Fig 2) These findings
indicate that PDK2 is a major regulator of PDC activity in the liver of HFD-fed mice
Page 7 of 45 Diabetes
8
PDK2 Deficiency Prevents HFD-Induced Hepatic Steatosis
To elucidate the role of PDK2 in the development of hepatic steatosis WT and PDK2 KO
mice were fed a HFD to induce NAFLD After 16 weeks of HFD feeding body weight gain
in HFD-fed PDK2 KO mice was significantly lower than that of HFD-fed WT mice (Fig 2A)
Even though the body weights of HFD-fed PDK2 KO mice were lower than those of WT
mice there were no differences in food consumption or physical activity (Supplementary Fig
3A and 3B) Relative to HFD-fed WT mice HFD-fed PDK2 KO mice exhibited greater
energy expenditure (Supplementary Fig 3C)
The liverbody weight ratio of PDK2 KO mice fed a HFD was significantly lower than
that of WT mice (Fig 2B) The number and the size of the lipid droplets were greatly
decreased in livers of PDK2 KO mice fed a HFD (Fig 2C) However no difference in
phenotype in epididymal adipose tissue (EAT) was observed between WT and PDK2 KO
mice on either diet (Fig 2C) Consistent with the histological analysis the hepatic TG
content was significantly lower in PDK2 KO mice than in WT mice after HFD feeding (Fig
2D) The level of hepatic glycogen was also significantly reduced in PDK2 KO mice fed a
HFD compare to WT mice fed a HFD (Fig 2E) Serum activities of AST and ALT markers of
liver injury were also significantly elevated in WT mice fed a HFD but not in PDK2 KO
mice (Fig 2F and 2G) The expression of genes involved in inflammation including tumor
necrosis factor alpha (TNFα) monocyte chemoattractant protein-1 (MCP-1) and
plasminogen activator inhibitor-1 (PAI-1) were significantly lower in PDK2 KO mice than in
WT mice fed a HFD (Supplementary Fig 4A) These data indicate that PDK2 deficiency
could ameliorate HFD-induced hepatic steatosis injury and inflammation
Page 8 of 45Diabetes
9
PDK2 Deficiency Ameliorates Hepatic Insulin Resistance and Reduces Hepatic Glucose
Production
In HFD-fed mice PDK2 deficiency lowered fasting blood glucose and serum insulin
levels by 15 and 63 respectively relative to WT mice (Fig 3A and 3B) HFD-fed PDK2
KO mice processed glucose more efficiently than HFD-fed WT mice as evidenced by a 30
decrease in the area under the curve (AUC) during GTT (Fig 3C) However with LFD-fed
mice the elimination of glucose was similar between PDK2 KO mice and WT mice (Fig 3C)
which is well agreed with previous data (18) These observations suggest that PDK2
deficiency prevents impaired glucose homeostasis induced by a HFD
To better delineate the mechanism responsible for improved hepatic glucose homeostasis
in HFD-fed PDK2 KO mice we performed a hyperinsulinemic-euglycemic clamp study In
HFD-fed WT mice the glucose infusion rate (GIR) was lower hepatic glucose production
(HGP) was higher during the clamp and whole body glucose turnover was lower (Fig 3D)
than in LFD-fed WT mice However in HFD-fed PDK2 KO mice the GIR was increased
two-fold compared to HFD-fed WT mice (Fig 3D) Furthermore both basal and clamped
HGP were significantly lower in HFD-fed PDK2 KO mice than in HFD-fed WT mice (Fig
3D) Nevertheless whole body glucose turnover was not different between HFD-fed PDK2
KO mice and WT mice (Fig 3D) HGP measured by the pyruvate tolerance test (PTT) was
lower in HFD-fed PDK2 KO mice than in HFD-fed WT (Fig 3E) To evaluate insulin
signaling we examined the phosphorylation levels of AKT and FoxO1 in the liver and
muscle of HFD-fed WT and PDK2 KO mice after insulin injection Insulin-stimulated
phosphorylation of AKT and FoxO1 were significantly increased in the livers of PDK2 KO
mice compared to those of WT mice (Fig 3F) Interestingly in muscle the levels of AKT
and FoxO1 phosphorylation did not differ between HFD-fed PDK2 KO mice and HFD-fed
Page 9 of 45 Diabetes
10
WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by
PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be
caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation
of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine
whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we
measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The
amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-
fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the
phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2
KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of
hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by
a HFD
PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice
Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic
steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2
deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative
decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates
thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA
cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed
PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A
and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free
[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)
Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in
Page 10 of 45Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
REFERENCES
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Clin Invest 2004114147-152
2 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175
3 Kennedy AR Pissios P Otu H et al A high-fat ketogenic diet induces a unique
metabolic state in mice Am J Physiol Endocrinol Metabol 2007292E1724-E1739
4 Williamson JR Scholz R Browning ET Control Mechanisms of Gluconeogenesis
and Ketogenesis II Interactions between fatty acid oxidation and the citric acid cycle in
perfused rat liver J Biol Chem 19692444617-4627
5 Jeoung NH Harris RA Pyruvate dehydrogenase kinase-4 deficiency lowers blood
glucose and improves glucose tolerance in diet-induced obese mice Am J Physiol Endocrinol
Metab 2008295E46-54
6 Sugden MC Holness MJ Recent advances in mechanisms regulating glucose
oxidation at the level of the pyruvate dehydrogenase complex by PDKs Am J Physiol
Endocrinol Metab 2003284E855-862
7 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
Page 18 of 45Diabetes
19
8 Wu P Blair PV Sato J Jaskiewicz J Popov KM Harris RA Starvation increases the
amount of pyruvate dehydrogenase kinase in several mammalian tissues Arch Biochem
Biophys 20003811-7
9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
dehydrogenase kinases 2 and 4 in young and middle-aged Otsuka Long-Evans Tokushima
Fatty rats induction by elevated levels of free fatty acids Metabolism 200655317-323
10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase
isoforms 2 and 4 by fatty acids and thyroid hormone Biochem J 2003369687-695
11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease Cell Metab
201114804-810
13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
contractions in PDH kinase 2 knockout mice effect of PDH kinase 1 compensation Am J
Physiol - Regul Integ Comp Physiol 2011300R1487-1493
14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate
dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-
13582
15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
kinase-4 structures reveal a metastable open conformation fostering robust core-free basal
activity J Biol Chem 200828325305-25315
Page 19 of 45 Diabetes
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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
alcoholic fatty liver disease J Biol Chem 200427932345-32353
20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
2012531080-1092
21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
Invest 20121221958-1959
22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
201233741-43
23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387
24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
383
26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
Page 20 of 45Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
8
PDK2 Deficiency Prevents HFD-Induced Hepatic Steatosis
To elucidate the role of PDK2 in the development of hepatic steatosis WT and PDK2 KO
mice were fed a HFD to induce NAFLD After 16 weeks of HFD feeding body weight gain
in HFD-fed PDK2 KO mice was significantly lower than that of HFD-fed WT mice (Fig 2A)
Even though the body weights of HFD-fed PDK2 KO mice were lower than those of WT
mice there were no differences in food consumption or physical activity (Supplementary Fig
3A and 3B) Relative to HFD-fed WT mice HFD-fed PDK2 KO mice exhibited greater
energy expenditure (Supplementary Fig 3C)
The liverbody weight ratio of PDK2 KO mice fed a HFD was significantly lower than
that of WT mice (Fig 2B) The number and the size of the lipid droplets were greatly
decreased in livers of PDK2 KO mice fed a HFD (Fig 2C) However no difference in
phenotype in epididymal adipose tissue (EAT) was observed between WT and PDK2 KO
mice on either diet (Fig 2C) Consistent with the histological analysis the hepatic TG
content was significantly lower in PDK2 KO mice than in WT mice after HFD feeding (Fig
2D) The level of hepatic glycogen was also significantly reduced in PDK2 KO mice fed a
HFD compare to WT mice fed a HFD (Fig 2E) Serum activities of AST and ALT markers of
liver injury were also significantly elevated in WT mice fed a HFD but not in PDK2 KO
mice (Fig 2F and 2G) The expression of genes involved in inflammation including tumor
necrosis factor alpha (TNFα) monocyte chemoattractant protein-1 (MCP-1) and
plasminogen activator inhibitor-1 (PAI-1) were significantly lower in PDK2 KO mice than in
WT mice fed a HFD (Supplementary Fig 4A) These data indicate that PDK2 deficiency
could ameliorate HFD-induced hepatic steatosis injury and inflammation
Page 8 of 45Diabetes
9
PDK2 Deficiency Ameliorates Hepatic Insulin Resistance and Reduces Hepatic Glucose
Production
In HFD-fed mice PDK2 deficiency lowered fasting blood glucose and serum insulin
levels by 15 and 63 respectively relative to WT mice (Fig 3A and 3B) HFD-fed PDK2
KO mice processed glucose more efficiently than HFD-fed WT mice as evidenced by a 30
decrease in the area under the curve (AUC) during GTT (Fig 3C) However with LFD-fed
mice the elimination of glucose was similar between PDK2 KO mice and WT mice (Fig 3C)
which is well agreed with previous data (18) These observations suggest that PDK2
deficiency prevents impaired glucose homeostasis induced by a HFD
To better delineate the mechanism responsible for improved hepatic glucose homeostasis
in HFD-fed PDK2 KO mice we performed a hyperinsulinemic-euglycemic clamp study In
HFD-fed WT mice the glucose infusion rate (GIR) was lower hepatic glucose production
(HGP) was higher during the clamp and whole body glucose turnover was lower (Fig 3D)
than in LFD-fed WT mice However in HFD-fed PDK2 KO mice the GIR was increased
two-fold compared to HFD-fed WT mice (Fig 3D) Furthermore both basal and clamped
HGP were significantly lower in HFD-fed PDK2 KO mice than in HFD-fed WT mice (Fig
3D) Nevertheless whole body glucose turnover was not different between HFD-fed PDK2
KO mice and WT mice (Fig 3D) HGP measured by the pyruvate tolerance test (PTT) was
lower in HFD-fed PDK2 KO mice than in HFD-fed WT (Fig 3E) To evaluate insulin
signaling we examined the phosphorylation levels of AKT and FoxO1 in the liver and
muscle of HFD-fed WT and PDK2 KO mice after insulin injection Insulin-stimulated
phosphorylation of AKT and FoxO1 were significantly increased in the livers of PDK2 KO
mice compared to those of WT mice (Fig 3F) Interestingly in muscle the levels of AKT
and FoxO1 phosphorylation did not differ between HFD-fed PDK2 KO mice and HFD-fed
Page 9 of 45 Diabetes
10
WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by
PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be
caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation
of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine
whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we
measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The
amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-
fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the
phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2
KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of
hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by
a HFD
PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice
Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic
steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2
deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative
decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates
thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA
cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed
PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A
and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free
[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)
Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in
Page 10 of 45Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
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rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
9
PDK2 Deficiency Ameliorates Hepatic Insulin Resistance and Reduces Hepatic Glucose
Production
In HFD-fed mice PDK2 deficiency lowered fasting blood glucose and serum insulin
levels by 15 and 63 respectively relative to WT mice (Fig 3A and 3B) HFD-fed PDK2
KO mice processed glucose more efficiently than HFD-fed WT mice as evidenced by a 30
decrease in the area under the curve (AUC) during GTT (Fig 3C) However with LFD-fed
mice the elimination of glucose was similar between PDK2 KO mice and WT mice (Fig 3C)
which is well agreed with previous data (18) These observations suggest that PDK2
deficiency prevents impaired glucose homeostasis induced by a HFD
To better delineate the mechanism responsible for improved hepatic glucose homeostasis
in HFD-fed PDK2 KO mice we performed a hyperinsulinemic-euglycemic clamp study In
HFD-fed WT mice the glucose infusion rate (GIR) was lower hepatic glucose production
(HGP) was higher during the clamp and whole body glucose turnover was lower (Fig 3D)
than in LFD-fed WT mice However in HFD-fed PDK2 KO mice the GIR was increased
two-fold compared to HFD-fed WT mice (Fig 3D) Furthermore both basal and clamped
HGP were significantly lower in HFD-fed PDK2 KO mice than in HFD-fed WT mice (Fig
3D) Nevertheless whole body glucose turnover was not different between HFD-fed PDK2
KO mice and WT mice (Fig 3D) HGP measured by the pyruvate tolerance test (PTT) was
lower in HFD-fed PDK2 KO mice than in HFD-fed WT (Fig 3E) To evaluate insulin
signaling we examined the phosphorylation levels of AKT and FoxO1 in the liver and
muscle of HFD-fed WT and PDK2 KO mice after insulin injection Insulin-stimulated
phosphorylation of AKT and FoxO1 were significantly increased in the livers of PDK2 KO
mice compared to those of WT mice (Fig 3F) Interestingly in muscle the levels of AKT
and FoxO1 phosphorylation did not differ between HFD-fed PDK2 KO mice and HFD-fed
Page 9 of 45 Diabetes
10
WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by
PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be
caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation
of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine
whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we
measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The
amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-
fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the
phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2
KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of
hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by
a HFD
PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice
Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic
steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2
deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative
decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates
thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA
cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed
PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A
and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free
[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)
Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in
Page 10 of 45Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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4 Williamson JR Scholz R Browning ET Control Mechanisms of Gluconeogenesis
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6 Sugden MC Holness MJ Recent advances in mechanisms regulating glucose
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7 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
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8 Wu P Blair PV Sato J Jaskiewicz J Popov KM Harris RA Starvation increases the
amount of pyruvate dehydrogenase kinase in several mammalian tissues Arch Biochem
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9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
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10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase
isoforms 2 and 4 by fatty acids and thyroid hormone Biochem J 2003369687-695
11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease Cell Metab
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13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
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14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate
dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-
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15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
kinase-4 structures reveal a metastable open conformation fostering robust core-free basal
activity J Biol Chem 200828325305-25315
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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
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20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
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21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
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22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
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23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
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24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
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26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
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related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
10
WT mice (Supplementary Fig 5) suggesting that the liver is the major organ affected by
PDK2 deficiency in HFD-fed mice Hepatic insulin resistance caused by obesity may be
caused by increased intracellular DAG and long chain acyl-CoA esters leading to activation
of protein kinase Cε (PKCε) which negatively affects insulin signaling (19) To examine
whether PDK2 deficiency ameliorates hepatic insulin resistance induced by a HFD we
measured DAG levels in the livers of HFD-fed PDK2 KO mice and HFD-fed WT mice The
amounts of 160 181 160-181 and total DAG were markedly lower in the livers of HFD-
fed PDK2 KO mice than in those of HFD-fed WT mice (Fig 3G) Furthermore the
phosphorylation of PKCε at Ser729 was significantly reduced in the livers of HFD-fed PDK2
KO mice compared to HFD-fed WT mice (Fig 3H) These results suggest that activation of
hepatic PDC by PDK2 deficiency ameliorates HGP and hepatic insulin resistance induced by
a HFD
PDK2 Deficiency Reduces TCA cycle Intermediates and Malonyl-CoA in HFD-Fed Mice
Altered hepatic TCA cycle anaplerosis and ketogenesis are associated with hepatic
steatosis and insulin resistance (20) We hypothesized that hepatic PDC activation by PDK2
deficiency shifts the fate of pyruvate from TCA cycle anaplerosis by PC to oxidative
decarboxylation by PDC This shift would reduce the levels of TCA cycle intermediates
thereby reducing TCA cycle flux Thus we measured hepatic pyruvate lactate and TCA
cycle intermediates in WT and PDK2 KO mice Levels of lactate and pyruvate in HFD-fed
PDK2 KO mice were significantly lower than in HFD-fed WT mice (Supplementary Fig 6A
and 6B) However the [lactate][pyruvate] ratio an index of the cytosolic free
[NADH][NAD] ratio was not different between two groups (Supplementary Fig 6C)
Hepatic TCA cycle intermediates (OAA citrate and succinate) were significantly higher in
Page 10 of 45Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
11
HFD-fed WT mice than in LFD-fed WT mice (Fig 4A) By contrast the TCA cycle
intermediates in HFD-fed PDK2 KO mice were dramatically lower than in HFD-fed WT
mice (Fig 4A) Despite the increase in hepatic PDC activity caused by PDK2 deficiency
there were no differences in the levels of hepatic acetyl-CoA and ATP in PDK2 KO mice
compared to WT mice fed either diet (Fig 4B) Most importantly the increase in malonyl-
CoA levels in the livers of WT mice fed a HFD was reduced significantly in the livers of
HFD-fed PDK2 KO mice (Fig 4B) Since malonyl-CoA is a major negative regulator of β-
oxidation via inhibition of CPT1 (21) we measured hepatic β-HB levels as an index of
ketogenesis The levels of hepatic β-HB were significantly decreased in HFD-fed WT mice
compared to LFD-fed WT mice (Fig 4C) confirming that the reduction in ketogenesis is
associated with hepatic steatosis induced by a HFD However β-HB levels were completely
restored in HFD-fed PDK2 KO mice compared to HFD-fed WT mice (Fig 4C) We then
determined the rate of β-HB production in WT and PDK2 KO mice using a stable isotope
([13
U]β-HB) dilution method and found that the decreased rate of β-HB production in HFD-
fed WT mice was restored in HFD-fed PDK2 KO mice (Fig 4D)
Next we examined the expression of genes involved in β-oxidation ketogenesis and
lipogenesis in the livers of WT and PDK2 KO mice As expected the expression of genes
involved in β-oxidation and ketogenesis including peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC1α) peroxisome proliferator-activated receptor gamma
coactivator 1-beta (PGC1β) carnitine palmitoyltransferase 1-liver (CPT1-L) peroxisome
proliferator-activated receptor alpha (PPARα) and 3-hydroxy-3-methylglutaryl-CoA synthase
2 (HMGCS2) were markedly down-regulated in the livers of WT mice fed a HFD compared
to a LFD However the expression levels of these genes were either partially or completely
restored in the livers of PDK2 KO mice (Fig 4E) The expression of genes involved in
Page 11 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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4 Williamson JR Scholz R Browning ET Control Mechanisms of Gluconeogenesis
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8 Wu P Blair PV Sato J Jaskiewicz J Popov KM Harris RA Starvation increases the
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9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
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10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
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11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease Cell Metab
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13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
contractions in PDH kinase 2 knockout mice effect of PDH kinase 1 compensation Am J
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14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate
dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-
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15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
kinase-4 structures reveal a metastable open conformation fostering robust core-free basal
activity J Biol Chem 200828325305-25315
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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
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20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
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21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
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22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
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23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
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24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
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26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
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related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
12
lipogenesis such as sterol regulatory element-binding protein 1c (SREBP1c) fatty acid
synthase (FAS) acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1)
was reduced in HFD-fed PDK2 KO mice relative to HFD-fed WT mice (Supplementary Fig
7) These findings suggest that activation of hepatic PDC reduced TCA cycle activity by
decreasing the anaplerotic influx of OAA This resulted in reduction of malonyl-CoA in liver
of PDK2 KO mice fed a HFD
Alteration of Anaplerotic Flux into the TCA cycle by PDK2 Deficiency Promotes
Ketogenesis
In the previous results a decrease in TCA cycle intermediates and an increase in ketone
body were observed in the liver of PDK2 KO mice fed a HFD (Fig 4) These results suggest
that increased PDC activity by PDK2 deficiency might diminish hepatic PC flux and TCA
cycle flux To examine this hypothesis we employed stable isotopomer flux study with 13
C6-
glucose supplement with 2 mM octanoate as a FA substrate in primary hepatocytes isolated
from WT and PDK2 KO mice The PDC flux (measured by acetyl-CoA[M+2] enrichment) in
primary hepatocytes of PDK2 KO mice was significantly increased compared to that of WT
hepatocytes (Fig 5A) However PC flux (measured by aspartate[M+3] and citrate[M+3]) and
TCA flux (measured by citrate[M+2] glutamate[M+2] succinate[M+2] and aspartate[M+2])
in primary hepatocyte of PDK2 KO mice were significantly lower than that of WT mice (Fig
5A) These results indicate that TCA flux is decreased due to reduced anaplerotic PC flux
even though the PDC flux is increased which suggests that PDK2 deficiency leads to the
production of ketone bodies from FA rather than complete oxidation to CO2 by TCA cycle
In order to examine this hypothesis we measured the production of β-HB and CO2 with
octanoate in the primary hepatocytes obtained from WT mice and PDK2 KO mice Oxidation
Page 12 of 45Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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2 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
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4 Williamson JR Scholz R Browning ET Control Mechanisms of Gluconeogenesis
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5 Jeoung NH Harris RA Pyruvate dehydrogenase kinase-4 deficiency lowers blood
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6 Sugden MC Holness MJ Recent advances in mechanisms regulating glucose
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Endocrinol Metab 2003284E855-862
7 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
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8 Wu P Blair PV Sato J Jaskiewicz J Popov KM Harris RA Starvation increases the
amount of pyruvate dehydrogenase kinase in several mammalian tissues Arch Biochem
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9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
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10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
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isoforms 2 and 4 by fatty acids and thyroid hormone Biochem J 2003369687-695
11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
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13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
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15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
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18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
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19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
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20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
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23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
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24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
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25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
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26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
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21
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27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
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28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
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Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
13
of [14
C1]-octanoate to 14
CO2 was significantly reduced and the rate of β-HB production
increased in primary hepatocytes from PDK2 KO mice compared to WT mice (Fig 5B and
5C) On the contrary in the presence of glutamine another anaplerotic precursor of the TCA
cycle increased ketogenesis in PDK2-deficient hepatocytes was reduced to a level similar to
that of WT hepatocytes (Fig 5C) indicating that limited TCA anaplerotic influx is
responsible for induction of ketogenesis in PDK2 KO hepatocytes (Fig 5D)
Liver-Specific PDK2 Inhibition Prevents Hepatic Steatosis and Restores Glucose
Homeostasis in HFD-Fed Mice
It has been reported that a mutation in the DW motif in the carboxy-terminal tails of the
PDKs and the reciprocal DW motif (R157A) in the amino-terminal domain of the PDKs
inactivates their enzymatic activity showing these motifs are indispensable for kinase activity
(1415)
To determine the liver-specific effect of PDK2 inhibition HFD-fed WT mice were
infected with PDK2 R157A DN mutant adenovirus (Ad-PDK2 DN) or GFP adenovirus (Ad-
GFP) PDHE1α phosphorylation was significantly decreased by Ad-PDK2 DN (Fig 6A)
indicating that PDC is activated in the livers of HFD-fed WT mice (Fig 6A)
The number and the size of the lipid droplets and TG levels were reduced in the livers of
HFD-fed mice infected with Ad-PDK2 DN compared with those infected with Ad-GFP (Fig
6B and 6C) Similar to PDK2 KO mice hepatic TCA cycle intermediates (OAA citrate and
succinate) were also significantly decreased in HFD-fed mice infected with Ad-PDK2 DN
(Fig 6D) There were no differences in the levels of acetyl-CoA and ATP in the livers of the
two groups of mice (Fig 6F) Malonyl-CoA levels were reduced in Ad-PDK2 DN-treated
mice (Fig 6F) whereas the levels of hepatic β-HB were significantly increased (Fig 6E)
Page 13 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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2 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175
3 Kennedy AR Pissios P Otu H et al A high-fat ketogenic diet induces a unique
metabolic state in mice Am J Physiol Endocrinol Metabol 2007292E1724-E1739
4 Williamson JR Scholz R Browning ET Control Mechanisms of Gluconeogenesis
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Metab 2008295E46-54
6 Sugden MC Holness MJ Recent advances in mechanisms regulating glucose
oxidation at the level of the pyruvate dehydrogenase complex by PDKs Am J Physiol
Endocrinol Metab 2003284E855-862
7 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
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8 Wu P Blair PV Sato J Jaskiewicz J Popov KM Harris RA Starvation increases the
amount of pyruvate dehydrogenase kinase in several mammalian tissues Arch Biochem
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9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
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Fatty rats induction by elevated levels of free fatty acids Metabolism 200655317-323
10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase
isoforms 2 and 4 by fatty acids and thyroid hormone Biochem J 2003369687-695
11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease Cell Metab
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13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
contractions in PDH kinase 2 knockout mice effect of PDH kinase 1 compensation Am J
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14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate
dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-
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15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
kinase-4 structures reveal a metastable open conformation fostering robust core-free basal
activity J Biol Chem 200828325305-25315
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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
alcoholic fatty liver disease J Biol Chem 200427932345-32353
20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
2012531080-1092
21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
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22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
201233741-43
23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387
24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
383
26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
Page 20 of 45Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
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fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
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3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
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hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
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enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
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transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
14
These results suggest that hepatic PDC activation by Ad-PDK2 DN prevents hepatic steatosis
by decreasing TCA cycle anaplerosis and increasing ketogenesis
To examine whether hepatic PDC activation by Ad-PDK2 DN ameliorates dysregulation
of glucose metabolism induced by a HFD fasting blood glucose and serum insulin were
measured and a GTT was performed In HFD-fed mice liver-specific PDK2 inhibition
improved hyperglycemia hyperinsulinemia and glucose intolerance (Fig 6Gndash6I
respectively) These results indicate that inhibition of PDK2 in the liver is able to attenuate
the impaired glucose homeostasis induced by a HFD
DISCISSION
As the link between glycolysis and the TCA cycle and a precursor for gluconeogenesis
and lipogenesis pyruvate plays a critical role in both anabolic and catabolic metabolism
depending upon the tissue and the physiological condition of the organism (22) In the insulin
resistant condition pyruvate is utilized for gluconeogenesis as well as de novo fat synthesis
rather than ATP generation in the TCA cycle resulting in the induction of hyperglycemia and
hepatic steatosis (23) Although abnormal regulation of pyruvate metabolism has long been
recognized in diabetes and metabolic disease (24-26) much remains to be elucidated about
the molecular mechanisms that are involved The fate of pyruvate is decided by the relative
activity of PC to PDC In this study we provide evidence that decreased PDC activity by
increased PDK2 expression contributes to the development of hepatic steatosis in HFD-fed
mice Conversely inhibition of PDK2 prevents the development of HFD-induced hepatic
steatosis via increased ketogenesis due to decreased anaplerotic influx Furthermore HFD-
fed PDK2 KO mice are resistant to impaired glucose metabolism and weight gain compared
to HFD-fed WT mice
Page 14 of 45Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
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17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
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18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
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19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
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20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
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25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
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26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
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28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
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Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
15
The large decrease in TCA cycle intermediates in the livers of PDK2 KO mice may
promote β-oxidation and ketogenesis The reduction in TCA intermediates is presumably
caused by decreased availability of OAA due to reduced anaplerotic influx from pyruvate
which would reduce the rate of ATP production in the TCA cycle Complete oxidation of
palmitate to CO2 and water generates 129 ATPs whereas only 16 ATPs are produced when
palmitate is converted into β-HB Surprisingly hepatic ATP levels were not different between
PDK2 KO mice and WT mice To compensate for reduced ATP production by the TCA cycle
FA consumption via ketogenesis must be greatly enhanced in the liver of HFD-PDK2 KO
mice leading to less fat accumulation Consistent with this the enzymatic capacity for β-
oxidation and ketogenesis was greater in HFD-PDK2 KO mice than in HFD-WT mice The
observed reduction in malonyl-CoA levels and increase of β-HB production rate also supports
the promotion of β-oxidation and ketogenesis in PDK2-deficient mice
Recently it was reported that increased anaplerotic flux into the TCA cycle (from
pyruvate to OAA by PC) is correlated with NAFLD in human and rodents (1112)
Furthermore the relative PDC to TCA flux (VPDHVTCA) is diminished in the livers of rats
with chronic lipid-induced hepatic insulin resistance (11) The reduction in VPDHVTCA is
likely due to increased PDK2 activity in HFD feeding PDK2 deficiency may increase PDC
activity and thereby restore VPDHVTCA To examine this hypothesis we measured PC PDC
and TCA cycle flux using 13
C6-glucose isotopomer in primary hepatocytes obtained from WT
and PDK2 KO mice As shown in Fig 5 increased PDC flux by PDK2 deficiency
significantly reduced PC flux and TCA cycle flux PC activity is allosterically regulated by
acetyl-CoA But the allosteric effect of acetyl-CoA on PC activity may not be different
between WT mice and PDK2 KO mice since hepatic acetyl-CoA levels of WT mice and
PDK2 KO mice were comparable to each other Instead the lowered PC flux may be due to
Page 15 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
REFERENCES
1 Browning JD Horton JD Molecular mediators of hepatic steatosis and liver injury J
Clin Invest 2004114147-152
2 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175
3 Kennedy AR Pissios P Otu H et al A high-fat ketogenic diet induces a unique
metabolic state in mice Am J Physiol Endocrinol Metabol 2007292E1724-E1739
4 Williamson JR Scholz R Browning ET Control Mechanisms of Gluconeogenesis
and Ketogenesis II Interactions between fatty acid oxidation and the citric acid cycle in
perfused rat liver J Biol Chem 19692444617-4627
5 Jeoung NH Harris RA Pyruvate dehydrogenase kinase-4 deficiency lowers blood
glucose and improves glucose tolerance in diet-induced obese mice Am J Physiol Endocrinol
Metab 2008295E46-54
6 Sugden MC Holness MJ Recent advances in mechanisms regulating glucose
oxidation at the level of the pyruvate dehydrogenase complex by PDKs Am J Physiol
Endocrinol Metab 2003284E855-862
7 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
Page 18 of 45Diabetes
19
8 Wu P Blair PV Sato J Jaskiewicz J Popov KM Harris RA Starvation increases the
amount of pyruvate dehydrogenase kinase in several mammalian tissues Arch Biochem
Biophys 20003811-7
9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
dehydrogenase kinases 2 and 4 in young and middle-aged Otsuka Long-Evans Tokushima
Fatty rats induction by elevated levels of free fatty acids Metabolism 200655317-323
10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase
isoforms 2 and 4 by fatty acids and thyroid hormone Biochem J 2003369687-695
11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease Cell Metab
201114804-810
13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
contractions in PDH kinase 2 knockout mice effect of PDH kinase 1 compensation Am J
Physiol - Regul Integ Comp Physiol 2011300R1487-1493
14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate
dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-
13582
15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
kinase-4 structures reveal a metastable open conformation fostering robust core-free basal
activity J Biol Chem 200828325305-25315
Page 19 of 45 Diabetes
20
16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
alcoholic fatty liver disease J Biol Chem 200427932345-32353
20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
2012531080-1092
21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
Invest 20121221958-1959
22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
201233741-43
23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387
24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
383
26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
Page 20 of 45Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
16
pyruvate limitation induced by increased PDC flux caused by PDK2 deficiency In addition
the reduced PC flux by PDK2 deficiency decreased the availability of OAA which affect the
decreased TCA cycle flux in PDK2 KO hepatocytes Recently Satapaei et al also showed
that induction of the TCA cycle promotes hepatic oxidative stress and inflammation (20)
Together with our results showing reduced expression of inflammatory markers in the livers
of PDK2 KO mice these findings are in agreement with the role of oxidative TCA cycle flux
as a major source of electrons for oxidative stress in cells Although testing the effect of
PDK2 deletion on oxidative stress under conditions of nutritional overburden was beyond the
limits of the present study decreased oxidative metabolism by the TCA cycle is a likely
mechanism for the protective effect of PDK2 deficiency
Importantly PDK2 deficiency decreased HGP and improved glucose tolerance in the
HFD condition as assessed by the GTT PTT and the hyperinsulinemic-euglycemic clamp
As OAA is a main precursor for gluconeogenesis in the liver it is not surprising that
activation of PDC causes a decrease in gluconeogenesis In agreement with our data it has
been demonstrated that hepatic PC activity correlates with gluconeogenesis in vivo and ex
vivo in lipid-induced obesity models (2027) In addition we found that hepatic levels of
pyruvate and OAA in PDK2 KO mice were lower than in WT mice after HFD challenge
which may reduce HGP as well as lipogenesis in PDK2 KO mice
DAG-mediated activation of PKCε can cause hepatic steatosis-associated insulin resistance
(2829) Consistent with these findings mice lacking PDK2 were resistant to HFD-induced
hepatic DAG accumulation and PKCε activation and therefore had significantly improved
hepatic insulin signaling This improvement was apparent in vivo as evidenced by decreased
serum fasting insulin levels and decreased HGP in the HFD-PDK2 KO mice
In conclusion we confirmed that hepatic PDC activation by inhibition of PDK2 could
Page 16 of 45Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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2 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175
3 Kennedy AR Pissios P Otu H et al A high-fat ketogenic diet induces a unique
metabolic state in mice Am J Physiol Endocrinol Metabol 2007292E1724-E1739
4 Williamson JR Scholz R Browning ET Control Mechanisms of Gluconeogenesis
and Ketogenesis II Interactions between fatty acid oxidation and the citric acid cycle in
perfused rat liver J Biol Chem 19692444617-4627
5 Jeoung NH Harris RA Pyruvate dehydrogenase kinase-4 deficiency lowers blood
glucose and improves glucose tolerance in diet-induced obese mice Am J Physiol Endocrinol
Metab 2008295E46-54
6 Sugden MC Holness MJ Recent advances in mechanisms regulating glucose
oxidation at the level of the pyruvate dehydrogenase complex by PDKs Am J Physiol
Endocrinol Metab 2003284E855-862
7 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
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19
8 Wu P Blair PV Sato J Jaskiewicz J Popov KM Harris RA Starvation increases the
amount of pyruvate dehydrogenase kinase in several mammalian tissues Arch Biochem
Biophys 20003811-7
9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
dehydrogenase kinases 2 and 4 in young and middle-aged Otsuka Long-Evans Tokushima
Fatty rats induction by elevated levels of free fatty acids Metabolism 200655317-323
10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase
isoforms 2 and 4 by fatty acids and thyroid hormone Biochem J 2003369687-695
11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease Cell Metab
201114804-810
13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
contractions in PDH kinase 2 knockout mice effect of PDH kinase 1 compensation Am J
Physiol - Regul Integ Comp Physiol 2011300R1487-1493
14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate
dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-
13582
15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
kinase-4 structures reveal a metastable open conformation fostering robust core-free basal
activity J Biol Chem 200828325305-25315
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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
alcoholic fatty liver disease J Biol Chem 200427932345-32353
20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
2012531080-1092
21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
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22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
201233741-43
23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387
24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
383
26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
Page 20 of 45Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
17
prevent obesity-induced hepatic steatosis and improve hepatic insulin sensitivity and glucose
homeostasis most likely by augmentation of FA consumption associated with reduction of
TCA cycle anaplerosis resulting in a reduction in OAA availability and induction of
ketogenesis in the livers of HFD-mice (Fig 7) These findings may open up a new avenue of
treatment for NAFLD and its complications
ARTICLE INFORMATION
Acknowledgments
We thank Dr Youngmin Hong and Dr Ji-Young Choi (Dong-il SHIMADZU Corp Technical
Research Center South Korea) for help with the analysis of hepatic metabolites using LC-
MSMS
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant
awarded by the Korean Ministry of Education (NRF-2012R1A2A1A03670452) and the
Korea Health Technology RampD Project through the Korea Health Industry Development
Institute (KHIDI) funded by the Ministry of Health amp Welfare Republic of Korea
(HI16C1501)
Duality of Interest
No potential conflicts of interest relevant to this article were reported
Author contributions
Page 17 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
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7 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
Page 18 of 45Diabetes
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9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
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19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
alcoholic fatty liver disease J Biol Chem 200427932345-32353
20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
2012531080-1092
21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
Invest 20121221958-1959
22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
201233741-43
23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387
24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
383
26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
Page 20 of 45Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
18
YG JYJ NHJ RAH and I-KL generated the hypothesis designed the experiments and
wrote the manuscript B-YP H-JK C-MH Y-KC and SJL performed the
experiments J-HJ HJH B-KK K-GP SYP C-HL CSC T-SP and WNPL
analyzed and discussed the data
REFERENCES
1 Browning JD Horton JD Molecular mediators of hepatic steatosis and liver injury J
Clin Invest 2004114147-152
2 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175
3 Kennedy AR Pissios P Otu H et al A high-fat ketogenic diet induces a unique
metabolic state in mice Am J Physiol Endocrinol Metabol 2007292E1724-E1739
4 Williamson JR Scholz R Browning ET Control Mechanisms of Gluconeogenesis
and Ketogenesis II Interactions between fatty acid oxidation and the citric acid cycle in
perfused rat liver J Biol Chem 19692444617-4627
5 Jeoung NH Harris RA Pyruvate dehydrogenase kinase-4 deficiency lowers blood
glucose and improves glucose tolerance in diet-induced obese mice Am J Physiol Endocrinol
Metab 2008295E46-54
6 Sugden MC Holness MJ Recent advances in mechanisms regulating glucose
oxidation at the level of the pyruvate dehydrogenase complex by PDKs Am J Physiol
Endocrinol Metab 2003284E855-862
7 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
Page 18 of 45Diabetes
19
8 Wu P Blair PV Sato J Jaskiewicz J Popov KM Harris RA Starvation increases the
amount of pyruvate dehydrogenase kinase in several mammalian tissues Arch Biochem
Biophys 20003811-7
9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
dehydrogenase kinases 2 and 4 in young and middle-aged Otsuka Long-Evans Tokushima
Fatty rats induction by elevated levels of free fatty acids Metabolism 200655317-323
10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase
isoforms 2 and 4 by fatty acids and thyroid hormone Biochem J 2003369687-695
11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease Cell Metab
201114804-810
13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
contractions in PDH kinase 2 knockout mice effect of PDH kinase 1 compensation Am J
Physiol - Regul Integ Comp Physiol 2011300R1487-1493
14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate
dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-
13582
15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
kinase-4 structures reveal a metastable open conformation fostering robust core-free basal
activity J Biol Chem 200828325305-25315
Page 19 of 45 Diabetes
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16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
alcoholic fatty liver disease J Biol Chem 200427932345-32353
20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
2012531080-1092
21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
Invest 20121221958-1959
22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
201233741-43
23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387
24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
383
26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
Page 20 of 45Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
19
8 Wu P Blair PV Sato J Jaskiewicz J Popov KM Harris RA Starvation increases the
amount of pyruvate dehydrogenase kinase in several mammalian tissues Arch Biochem
Biophys 20003811-7
9 Bajotto G Murakami T Nagasaki M et al Increased expression of hepatic pyruvate
dehydrogenase kinases 2 and 4 in young and middle-aged Otsuka Long-Evans Tokushima
Fatty rats induction by elevated levels of free fatty acids Metabolism 200655317-323
10 Holness MJ Bulmer K Smith ND Sugden MC Investigation of potential
mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase
isoforms 2 and 4 by fatty acids and thyroid hormone Biochem J 2003369687-695
11 Alves TC Befroy DE Kibbey RG et al Regulation of hepatic fat and glucose
oxidation in rats with lipid-induced hepatic insulin resistance Hepatology (Baltimore Md)
2011531175-1181
12 Sunny NE Parks EJ Browning JD Burgess SC Excessive hepatic mitochondrial
TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease Cell Metab
201114804-810
13 Dunford EC Herbst EA Jeoung NH et al PDH activation during in vitro muscle
contractions in PDH kinase 2 knockout mice effect of PDH kinase 1 compensation Am J
Physiol - Regul Integ Comp Physiol 2011300R1487-1493
14 Klyuyeva A Tuganova A Popov KM The carboxy-terminal tail of pyruvate
dehydrogenase kinase 2 is required for the kinase activity Biochemistry 20054413573-
13582
15 Wynn RM Kato M Chuang JL Tso S-C Li J Chuang DT Pyruvate dehydrogenase
kinase-4 structures reveal a metastable open conformation fostering robust core-free basal
activity J Biol Chem 200828325305-25315
Page 19 of 45 Diabetes
20
16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
alcoholic fatty liver disease J Biol Chem 200427932345-32353
20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
2012531080-1092
21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
Invest 20121221958-1959
22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
201233741-43
23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387
24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
383
26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
Page 20 of 45Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
20
16 He T-C Zhou S Da Costa LT Yu J Kinzler KW Vogelstein B A simplified system
for generating recombinant adenoviruses Proc Natl Acad Sci USA 1998952509-2514
17 Hwang B Wu P Harris RA Additive effects of clofibric acid and pyruvate
dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high
saturated fat diet FEBS J 20122791883-1893
18 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
19 Samuel VT Liu ZX Qu X et al Mechanism of hepatic insulin resistance in non-
alcoholic fatty liver disease J Biol Chem 200427932345-32353
20 Satapati S Sunny NE Kucejova B et al Elevated TCA cycle function in the
pathology of diet-induced hepatic insulin resistance and fatty liver J Lipid Res
2012531080-1092
21 Foster DW Malonyl-CoA the regulator of fatty acid synthesis and oxidation J Clin
Invest 20121221958-1959
22 Divakaruni AS Murphy AN A Mitochondrial Mystery Solved Science
201233741-43
23 Jitrapakdee S St Maurice M Rayment I Cleland W Wallace J Attwood P Structure
mechanism and regulation of pyruvate carboxylase Biochem J 2008413369-387
24 Cotter DG Ercal B Huang X et al Ketogenesis prevents diet-induced fatty liver
injury and hyperglycemia J Clin Invest 20141245175-5190
25 Garland P Newsholme E Randle P Effect of fatty acids ketone bodies diabetes and
starvation on pyruvate metabolism in rat heart and diaphragm muscle Nature 1962195381-
383
26 Jeoung NH Harris CR Harris RA Regulation of pyruvate metabolism in metabolic-
Page 20 of 45Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
21
related diseases Rev Endo Metabol 20141599-110
27 Lee P Leong W Tan T Lim M Han W Radda GK In Vivo hyperpolarized carbon-
13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an
insulin-resistant mouse model Hepatology (Baltimore Md) 201357515-524
28 Samuel VT Liu ZX Wang A et al Inhibition of protein kinase Cepsilon prevents
hepatic insulin resistance in nonalcoholic fatty liver disease J Clin Invest 2007117739-745
29 Kumashiro N Erion DM Zhang D et al Cellular mechanism of insulin resistance in
nonalcoholic fatty liver disease Proc Natl Acad Sci U S A 201110816381-16385
Figure Legends
Figure 1 HFD Reduces Hepatic PDC Activity by Up-Regulation of PDK2
(A) Hepatic PDC activity in overnight-fasted WT mice fed a LFD or a HFD for 16 weeks (n
= 5) (B) Phosphorylation state of PDHE1α in the livers obtained from the mice in (A) The
bar graphs on the right show quantification of PDHE1α phosphorylation (C and D) Hepatic
expression of mRNA (C) and protein (D) of PDK isoenzymes in HFD-fed mice (n = 5) (E)
Hepatic PDC activity in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16
weeks (n = 5) (F) Phosphorylation of PDHE1α and expression of PDK isoforms in the liver
of HFD-fed WT and HFD-fed PDK2 KO mice (E) The bar graphs on the right show
quantification of PDHE1α phosphorylation Data are presented as the mean plusmn SEM p lt 005
and
p lt 001
Figure 2 PDK2 Deficiency Ameliorates Hepatic Steatosis in Mice Fed a HFD
(A and B) Body weight gain (n = 12) (A) liverbody weight ratio (n = 6) (B) and
representative images of the livers (B) of WT and PDK2 KO mice fed a LFD or a HFD (C)
Page 21 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
22
Representative histological appearance of HampE-stained liver and epididymal fat tissue (EAT)
obtained from the mice in (A) Scale bar 100 microm (D-F) Level of hepatic triacylglycerol (TG)
(D) hepatic glycogen (E) serum AST (E) and ALT (F) activities in overnight-fasted WT and
PDK2KO mice (n = 6) Date are presented as the mean plusmn SEM p lt 005
p lt 001 and
p
lt 0001
Figure 3 PDK2 Deficiency Improves Glucose Homeostasis and Ameliorates Hepatic
Glucose Production and Insulin Resistance Induced by a HFD
(A and B) Blood glucose (A) and serum insulin (B) levels of overnight-fasted WT mice and
PDK2 KO mice fed a HFD or LFD (n = 6 per) (C-E) Glucose tolerance test (C)
hyperinsulinemic-euglycemic clamp study (D) and pyruvate tolerance test (
p lt 001 WT-
HFD vs PDK2 KO-HFD) (E) with overnight-fasted WT and PDK2 KO mice fed a LFD or a
HFD (n = 6) (F) Hepatic insulin signaling in WT and PDK2 KO mice fed a HFD (n = 5) (G
and H) Levels of hepatic diacylglycerol (DAG)(G) and PKCε phosphorylation (H) in WT and
PDK2 KO mice fed a HFD for 16 weeks (n = 5) The bar graphs on the right show
quantification of PKCε phosphorylation Data are presented as the mean plusmn SEM p lt 005
p lt 001 and
p lt 0001
Figure 4 Enhanced Hepatic PDC Activity Reduces TCA Cycle Intermediates and
Malonyl-CoA and Increases Ketogenesis
(A) Levels of hepatic TCA cycle intermediates such as oxaloacetate (OAA) citrate and
succinate in overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n =
6) (B and C) Level of hepatic metabolites such as acetyl-CoA (B) ATP (B) malonyl-CoA
(B) and β-hydroxybutyrate (β-HB) (C) obtained from the mice in (A) (D) β-HB production
Page 22 of 45Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
23
rates in WT and PDK2 KO mice after 18 h fasting was measured using constant infusion of
[U-13
C4]sodium DL-3-hydroxyburyrate (n = 6) (E) Hepatic gene expression of β-
oxidationketogenic enzymes obtained from the mice in (A) Data are presented as the mean
plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 5 PDK2 Deficiency Enhances Ketogenesis by Decreasing TCA Cycle Anaplerosis
in Mouse Primary Hepatocytes
(A) The incorporation of 13
C atoms from 13
C6-glucose to acetyl-CoA[M+2] (PDC flux red)
aspartate[M+3] citrate[M+3] (PC flux green) citrate[M+2] glutamate[M+2]
succinate[M+2] and aspartate[M+2] (TCA cycle flux) WT and PDK2 KO primary
hepatocytes were treated with 25 mM 13
C6-glucose and 2 mM sodium octanoate for 2 h (n =
5) The enrichment of 13
C isotopomer were determined by LC-MS (B) Oxidation of [1-14
C]
sodium octanoate to 14
CO2 in WT and PDK2 KO primary hepatocytes (n = 6) (C) WT and
PDK2 KO primary hepatocytes were treated in control or ketogenic media (2 mM octanoate)
with or without 4 mM glutamine for 4 h Levels of β-HB in the culture media were
determined enzymatic methods (n = 6) (D) Schematic models for induction of ketogenesis in
the primary hepatocytes of PDK2 KO mice All data are presented as the mean plusmn SEM
p lt
001
p lt 0001
Figure 6 Liver-Specific PDC Activation by Ad-PDK2 dominant negative (DN) Prevents
Hepatic Steatosis and Improves Glucose Homeostasis in HFD-Fed Mice
(A and B) Representative hepatic PDHE1α phosphorylation (A) and histological appearance
of liver tissue (B) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (C-F) The levels
of hepatic TG (C) TCA cycle intermediates (D) β-HB (E) acetyl-CoA (F) ATP (F) and
Page 23 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
24
malonyl-CoA (F) in HFD-fed mice infected with Ad-GFP or Ad-PDK2 DN (n = 5) (G and
H) Blood glucose (G) and serum insulin (H) levels in HFD-fed mice infected with Ad-GFP or
Ad-PDK2 DN (n = 5) (I) GTT in HFD-fed mice infected with Ad GFP or Ad-PDK2 DN (n =
5) All data are presented as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Figure 7 Schematic Model of Changes in Hepatic Glucose and Lipid Metabolism Via
PDC Activation Induced by PDK2 Inhibition in Liver of HFD-Fed Mice
Page 24 of 45Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
A B
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
LFD HFD
C
Rel
ativ
e m
RN
A l
eve
ls
PDK1 PDK2 PDK3 PDK4
mouse liver
D E
FWT-HFD PDK2 KO-HFD
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK1
PDK2
PDK3
PDK4
-tubulin
mouse liver
Hep
atic
PD
C a
ctiv
ity
(Ug
wei
gh
t)
LFD HFD
PDK1
LFD HFD
PDK2
PDK3
PDK4
-tubulin
mouse liver
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S29
3)P
DH
E1
pP
DH
E1
(S30
0)P
DH
E1
Figure 1
Page 25 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Figure 2
A B
Bo
dy
wei
gh
t (g
)
Weeks
p lt 005
WT PDK2 KO WT PDK2KO
LFD HFD
Liv
erb
od
y w
eig
ht
()
E
LFD HFD
D
Ser
um
AS
T (
UL
)
LFD HFD
LFD HFD
Liver
EAT
WT PDK2 KOWT PDK2 KO
LFD HFD
Hep
atic
TG
(m
g
g t
issu
e)
Ser
um
AL
T (
UL
)
LFD HFD
F
C
G
Hep
atic
gly
cog
en
(mg
g t
issu
e)
LFD HFD
Page 26 of 45Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Figure 3
D
E F
Time (min)
pA
KT
AK
T
pF
oxO
1F
ox
O1
WT-HFD PDK2 KO-HFD
pFoxO1 (S256)
FoxO1
pAKT (S473)
AKT
Insulin ‐ ‐ ‐ + + + ‐ ‐ ‐ + + +
A B C
Blo
od
glu
cose
(m
gd
L)
GIR
(m
gk
gm
in)
Bas
al H
GP
(m
gk
gm
in)
Cla
mp
HG
P (
mg
kg
min
)
Glu
cose
tu
rno
ver
(mg
kg
min
)
Insulinstimulated
Insulinstimulated
DA
G (
nm
olg
tis
sue)
pPKC (S729)
PKC
-tubulin
WT-HFD PDK2 KO-HFD
mouse liver
mouse liver
Blo
od
glu
cose
(m
gd
L)
LFD HFD
Ser
um
in
sulin
(n
gm
L)
LFD HFD
Blo
od
glu
cose
(m
gd
L)
Time (min)
AU
C (
mg
dL
x m
in)
LFD HFD
LFD HFD LFD HFD LFD HFD LFD HFD
G H
pP
KC(
S72
9)P
KC
Page 27 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Figure 4
B
A
C
Hep
atic
-H
B
(m
olg
tis
sue)
-H
B p
rod
uct
ion
rat
e(m
gm
ink
g)
Hep
atic
OA
A
(m
olg
tis
sue)
LFD HFD
Hep
atic
cit
rate
(
mo
lg t
issu
e)
LFD HFD
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
LFD HFD
LFD HFD
Hep
atic
ace
tyl-
Co
A
(AU
)
LFD HFD
Hep
atic
AT
P (
AU
)
LFD HFD
Hep
atic
mal
on
yl-C
oA
(A
U)
LFD HFD
D
LFD HFD
Rel
ativ
e m
RN
A l
eve
ls
PPAR HMGCS2
-oxidationketogenesis
E
PGC1 PGC1 CPT1-L
Page 28 of 45Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Figure 5
A
C
Control Octanoate OctanoateGlutamine
-H
B p
rod
uct
ion
rat
e(
mo
lmin
mg
pro
tein
)
14C
O2
pro
du
ctio
n
(dp
m1
5 x
106
cells
)
B
D
E
nri
chm
ent
PDC flux PC flux
E
nri
chm
ent
E
nri
chm
ent
TCA cycle flux
Page 29 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Figure 6
D
Hep
atic
OA
A
(m
olg
tis
sue)
Hep
atic
cit
rate
(
mo
lg t
issu
e)
Hep
atic
su
ccin
ate
(m
olg
tis
sue)
E
Hep
atic
-H
B
(m
olg
tis
sue)
F
Hep
atic
ace
tyl-
Co
A
(AU
)
Hep
atic
A
TP
(A
U)
Hep
atic
m
alo
nyl
-Co
A(A
U)
GB
loo
d g
luco
se (
mg
dL
)
H
Ser
um
in
sulin
(n
gm
l)
I
Blo
od
glu
cose
(m
gd
L)
AU
C (
mg
dL
x m
in)
pPDHE1 (S293)
pPDHE1 (S300)
PDHE1
PDK2
-tubulin
mouse liver
B C
Hep
atic
TG
(m
gg
tis
sue)
A
Ad-GFP Ad-PDK2 DN
Page 30 of 45Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Figure 7
Induced pathwayReduced pathway
Page 31 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Inhibition of pyruvate dehydrogenase kinase 2 protects against hepatic steatosis
through modulation of TCA cycle anaplerosis and ketogenesis
Younghoon Go Ji Yun Jeong Nam Ho Jeoung Jae-Han Jeon Bo-Yoon Park Hyeon-Ji Kang
Chae-Myeong Ha Young-Keun Choi Sun Joo Lee Hye Jin Ham Byung-Gyu Kim Keun-
Gyu Park So Young Park Chul-Ho Lee Cheol Soo Choi Tae-Sik Park W N Paul Lee
Robert A Harris and In-Kyu Lee
SUPPLEMENTARY MATERIALS
Primary Hepatocyte Culture
Primary mouse hepatocytes were prepared from 8ndash10-week-old WT and PDK2 KO mice
by the collagenase perfusion method described previously (1) To measure 14
CO2 production
primary hepatocytes were cultured in T-25 collagen coated plates containing 4 ml of Krebs-
Henseleit bicarbonate buffer (pH 74) supplemented with 30 mM glucose containing 200
mCimmol [1-14
C]sodium octanoate (American Radiolabeled Chemicals) 14
CO2 was
collected after incubation for 2 h For stimulation of ketone body production cells were
incubated in media containing 2 mM sodium octanoate for 4 h (2)
13C-Isotopomer labeling studies
Primary hepatocytes of WT and PDK2 KO mice were pre-incubated in serum-free DMEM
medium (25 mM glucose) for 6 h to reach metabolic steady state Then they were washed
with glucose-free DMEM medium (Sigma-Aldrich) supplemented with 1 mM sodium lactate
(Sigma-Aldrich) and 01 mM sodium pyruvate (Sigma-Aldrich) and incubated with the tracer
Page 32 of 45Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
25 mM of [U-13
C6] glucose (Sigma-Aldrich) in DMEM medium with 2 mM sodium
octanoate for 2 h Cells were washed with 3 ml of ice-cold 09 NaCl (two times) and then
collected in Eppendorf tube (3) Cells were resuspended with 200 l of ice-cold metabolite
extraction solution (chloroformmethanolwater (131 vv)) and then sonicated After
incubation on ice for 1 h metabolite sample were collected by centrifugation at 13000 rpm
for 5 min All the samples were lysophilized and resuspended in 300 l of water containing
01 formic acid prior to LC-MSMS analysis Analytes were separated on a
pentafluorophenyl column (100mmtimes21mm 3microm) by gradient elution using an HPLC Nexera
coupled with an LCMS-8060 mass spectrometer (Shimadzu Japan) The mobile phase
consisted of water-acetonitrile (01 formic acid) at a flow rate of 02mLmin Q3 selected
ion monitoring (SIM) scan mode was used to obtain target metabolite isotopomer information
and raw spectrum intensity data of which was extracted respectively from within a retention
time range of each multiple reaction monitoring (MRM) scan performed simultaneously
Subsequently 13
C-MIDs (mass isotopomer distributions) were determined and corrected for
natural isotope abundance from the SIM scan data of target metabolite isotopomer
Western Blot Analysis
Tissue powder prepared under liquid nitrogen was homogenized with a lysis buffer (20
mmolL Tris pH74 10 mmolL Na4P2OH 100 mmolL NaF 2 mmolL Na3VO4 5 mmolL
EDTA pH80 01 mmolL PMSF 1 NP-40) containing proteinase inhibitors and
phosphatase inhibitors Protein concentration was measured by the BCA Protein Assay
Reagent (Thermo scientific) Proteins were separated on 6-15 SDS-polyacrylamide gel and
transferred to a PVDF membrane (Millipore) Membranes were immunoblotted with primary
antibodies recognizing PDK2 phospho-PKCε(Ser729) PKCε (Santa Cruz Biotechnology)
phospho-AKT (Ser473) AKT phospho-FoxO1 (S272) FoxO1 (Cell signaling Technology)
Page 33 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
PDK1 (Assay designs) PDK3 antiserum (4) PDK4 antiserum (5) phospho-PDHE1α
(Ser293) phospho-PDHE1α (Ser300) (EMD Chemicals) and β-tubulin (Sigma-Aldrich)
Band intensities were quantified with ImageJ software (v 142q NIH Bethesda MD USA)
Quantitative Real-Time RT-PCR
Total RNA was isolated from tissue extracts using TRIzol reagent (Invitrogen) according
to the manufacturerrsquos instructions cDNA was synthesized from 2 microg of total RNA using a
cDNA synthesis kit (Fermentas) Quantitative Real-Time RT-PCR was carried out using the
Power SYBR Green PCR Master Mix Kit (Applied Biosystems) in ViiA 7 Real Time PCR
system (Applied Biosystems) 36B4 was used for normalization The primer sequences are
given in the Supplementary Table 1
Measurement of Metabolites in Liver
Hepatic metabolites were deproteinized with 7 perchloric acid followed by neutralization
with 20 KOH and precipitation of KClO4 from tissues of overnight-fasted mice Hepatic
pyruvate (6) β-HB (7) OAA (8) and citrate (9) were measured by enzymatic methods
Hepatic succinate was measured with a Succinic Acid Assay kit (Megazyme) Hepatic ATP
acetyl-CoA and malonyl-CoA levels were measured using a liquid chromatography-tandem
mass spectrometry (LC-MSMS) (LCMS-8040 Shimadzu Japan) with an internal standard
such as L-methionine sulfone or 2-morpholinoethanesulfonic acid (10) Hepatic TG levels
were determined with a TG Quantification kit (BioVision) DAGs from liver tissue were
assessed by LC-MSMS as described previously (11) with a bench-top tandem mass
spectrometer
Page 34 of 45Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Glucose and Pyruvate Tolerance Tests
Glucose and pyruvate tolerance tests were performed in overnight-fasted mice by ip
injection of glucose (1 gkg body weight) or pyruvate (2 gkg body weight) Tail blood
glucose was measured with an Accu-Chek glucometer (Roche)
Measurement of Serum Parameters
Serum activities of ALT and AST were measured with a Hitachi 7020 biochemical
analyzer Insulin levels were measured with a Milliplex Map assay kit (Millipore)
Measurement of Energy Expenditure Using an Indirect Calorimetry System
Food intake physical activity CO2 production rates O2 consumption rates and energy
expenditure (EE) were measured with a TSE indirect calorimetry system (Lab Master TSE
systems) for 48 h Mice were individually housed in metabolic cages with 12 h lightdark
cycles at 22 plusmn 2degC
Hyperinsulinemic-Euglycemic Clamp Study
Four days prior to the experiments silicone catheters (Helix Medical San Mateo CA
USA) were inserted into the right internal jugular veins of anesthetized mice After overnight
fasting mice were placed in a rat-size restrainer and a three-way valve was attached to the
jugular vein catheter to deliver glucose or insulin A 2 h hyperinsulinemic-euglycemic clamp
was carried out as described previously (12) Briefly insulin was infused at 10 pmol kg-1
min-1
and 20 glucose was infused at a variable rate to maintain euglycemia To determine
insulin-stimulated whole body glucose uptake and hepatic glucose production (HGP) [3-
3H]glucose (PerkinElmer Life and Analytical Sciences) was continuously infused for 2 h
before (005 microCimin) and throughout the clamp (01 microCimin) Blood samples were collected
Page 35 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
via the tail vein and plasma glucose was measured with a Beckman Glucose Analyzer2
(Beckman) Insulin-stimulated whole body glucose uptake was determined as the ratio of the
[3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of
the clamp Hepatic glucose production during the clamps was determined by subtracting the
glucose infusion rate from the whole body glucose uptake rate
References
1 Koo S-H Flechner L Qi L et al The CREB coactivator TORC2 is a key regulator of
fasting glucose metabolism Nature 20054371109-1111
2 Sengupta S Peterson TR Laplante M Oh S Sabatini DM mTORC1 controls
fasting-induced ketogenesis and its modulation by ageing Nature 20104681100-1104
3 Sapcariu SC Kanashova T Weindl D Ghelfi J Dittmar G Hiller K Simultaneous
extraction of proteins and metabolites from cells in culture MethodsX 2014174-80
4 Jeoung NH Rahimi Y Wu P Lee WNP Harris RA Fasting induces ketoacidosis and
hypothermia in PDHK2PDHK4-double-knockout mice Biochem J 2012443829-839
5 Jeoung N Wu P Joshi M Jaskiewicz J Bock C Depaoli-Roach A Harris R Role of
pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during
starvation Biochem J 2006397417-425
6 Czok R Lamprecht W Pyruvate phosphoenolpyruvate and D-glycerate-2-phosphate
In Methods of enzymatic analysis Bergmeyer HU (ed) 1974 p 1446-145
7 Williamson DH Mellanby J Krebs HA Enzymic determination of d(minus)-β-
hydroxybutyric acid and acetoacetic acid in blood Biochem J 19628290
8 Wahlefeld A Oxaloacetate UV spectrophotometric determination In Methods of
Page 36 of 45Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
enzymatic analysis in Bergmeyer HU (ed) 1974 p 1604-1608
9 Dagley S Citrate UV spectrophotometric determination In Methods of enzymatic
analysis in Bergmeyer HU(ed) 1974 p1562-1565
10 Kubo A Ohmura M Wakui M et al Semi-quantitative analyses of metabolic
systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG
mice Anal Bioanal Chem 20114001895-1904
11 Kim D-K Kim JR Koh M et al Estrogen-related receptor γ (ERRγ) is a novel
transcriptional regulator of phosphatidic acid phosphatase LIPIN1 and inhibits hepatic
insulin signaling J Biol Chem 201128638035-38042
12 Sung HK Kim Y-W Choi SJ et al COMP-angiopoietin-1 enhances skeletal muscle
blood flow and insulin sensitivity in mice Am J Physiol Endocrinol Metab 2009297E402-
E409
Page 37 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Supplementary Table 1 Primer sequences used for real-time RT-PCR
Gene Forward primer (5-3) Reverse primer (5-3)
PDK1
PDK2
PDK3
CACCACGCGGACAAAGG
CCCCGTCCCCGTTGTC
GGAGCAATCCCAGCAGTGAA
GCCCAGCGTGACGTGAA
TCGCAGGCATTGCTGGAT
TGATCTTGTCCTGTTTAGCCTTGT
PDK4
TNFa
MCP-1
PAI-1
CCATGAGAAGAGCCCAGAAGA
GACGTGGAACTGGCAGAAGAG
ACCTGGATCGGAACCAAATG
CACCCCTTCCAGAGTCCCATA
GAACTTTGACCAGCGTGTCTACAA
CCGCCTGGAGTTCTGGAA
CCTTAGGGCAGATGCAGTTTTAA
GCTGAAACACTTTTACTCCGAAGTT
SREBP1c AGAGGGTGAGCCTGACAA CCTCTGCAATTTCCAGATCT
FAS
ACC1
ACCTGGTAGACCACTGCATTGAC
CGCTCAGGTCACCAAAAAGAAT
CCTGATGAAACGACACATTCTCA
GGGTCCCGGCCACATAA
SCD1 CTGCCCCTGCGGATCTT GCCCATTCGTACACGTCATTCT
PGC1α TGCGGGATGATGGAGACA GCGAAAGCGTCACAGGTGTA
PGC1β TCGAAATCTCTCCAGTGACATGA GCACTCTACAATCTCACCGAACA
CPT1-l TGACCCAAAACAGTATCCCAATC CGCCACGGGACCAAAG
PPARα GAACAAAGACGGGATGCTC ACAGAACGGCTTCCTCAGGTT
HMGCS2 CTTAGCAGCAAGTTTCTTTTCATTCC GATGGACACACTAGACACCAGTTTCT
36B4 ACCTCCTTCTTCCGGCTTT CTCCAGTCTTTATCAGCTGC
Page 38 of 45Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Supplementary Fig 1
Supplementary Fig 1 Immunoblots of PDHE1αααα phosphorylation and PDK isoforms of
heart muscle and kidney in WT mice and PDK2 KO mice fed a HFD
Page 39 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Supplementary Fig 2
Supplementary Fig 2 Blood metabolic parameters of overnight-fasted WT and PDK2
KO mice Serum lactate pyruvate and free fatty acid (FFA) were determined in overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD for 16 weeks (n = 6) Data are presented
as the mean plusmn SEM p lt 005
Page 40 of 45Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Supplementary Fig 3
Supplementary Fig 3 Indirect calorimetry analysis of WT and PDK2 KO mice fed a
HFD Food intake (A) physical activity (B) and energy expenditure (C) were measured in
WT and PDK2 KO mice fed a HFD (n = 6) The data are presented as the mean plusmn SEM p lt
005 p lt 001
Page 41 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Supplementary Fig 4
Supplementary Fig 4 mRNA expression of inflammatory genes from the livers of WT
and PDK2 KO mice Hepatic mRNA Expression of TNFα MCP-1 and PAI-1on overnight-
fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented as the
mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 42 of 45Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Supplementary Fig 5
Supplementary Fig 5 Insulin signaling in muscle of WT and PDK2 KO mice fed a HFD
Immunoblot analysis of the insulin signaling pathway components in the muscle of WT and
PDK2 KO mice fed a HFD (n = 5) The bar graphs on the right show quantification of the
protein bands detected by immunoblot analysis
Page 43 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Supplementary Fig 6
Supplementary Fig 6 Hepatic metabolites of WT and PDK2 KO mice Level of hepatic
lactate (A) pyruvate (B) and lactatepyruvate ratio in overnight-fasted WT and PDK2 KO
mice fed a LFD or a HFD (n = 6) The data are presented as the mean plusmn SEM p lt 0001
Page 44 of 45Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes
Supplementary Fig 7
Supplementary Fig 7 mRNA expression of lipogenic genes from the livers of WT and
PDK2 KO mice Hepatic mRNA expression of SREBP1c FAS ACC1 and SCD1 on
overnight-fasted WT and PDK2 KO mice fed a LFD or a HFD (n = 6) The data are presented
as the mean plusmn SEM p lt 005
p lt 001
p lt 0001
Page 45 of 45 Diabetes