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Metabolic Crosstalk: molecular links between glycogen and lipid metabolism in
obesity
Binbin Lu1, Dave Bridges
1, Yemen Yang
1, Kaleigh Fisher
1, Alan Cheng
1, Louise Chang
1,
Zhuoxian Meng1, Jiandie Lin
1, Michael Downes
2, Ruth T. Yu
2, Christopher Liddle
2,3,
Ronald M. Evans2, and Alan R. Saltiel
1
1Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA
2Salk Institute for Biological Sciences, La Jolla, CA 92037, USA
3Storr Liver Unit, Westmead Millennium Institute and University of Sydney, Westmead
Hospital, Westmead, NSW 2145, Australia
Address correspondence to:
A.R. Saltiel, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109,
USA. Phone: (734) 615-9787; Fax: (734) 763-6492; E-mail: [email protected].
Running title: PTG-Dependent Glycogen and Lipid Accumulation
Abstract word count: 185
Total word Count: 4450
Tables: 0
Figures: 8
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Diabetes Publish Ahead of Print, published online April 10, 2014
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ABSTRACT
Glycogen and lipid are major storage forms of energy that are tightly regulated by
hormones and metabolic signals. Here, we demonstrate that feeding mice a high fat diet
(HFD) increased hepatic glycogen, due to increased expression of the glycogenic
scaffolding protein PTG/R5. PTG promoter activity was increased and glycogen levels
were augmented in mice and cells after activation of mechanistic target of rapamycin
complex 1 (mTORC1) and its downstream target sterol regulatory element binding
protein 1 (SREBP1). Deletion of the PTG gene in mice prevented HFD-induced hepatic
glycogen accumulation. Surprisingly, PTG deletion also blocked hepatic steatosis in
HFD-fed mice, and reduced the expression of numerous lipogenic genes. Additionally,
PTG deletion reduced fasting glucose and insulin levels in obese mice, while improving
insulin sensitivity, a result of reduced hepatic glucose output. This metabolic crosstalk
was due to decreased mTORC1 and SREBP activity in PTG knockout mice or
knockdown cells, suggesting a positive feedback loop in which once accumulated,
glycogen stimulates the mTORC1/SREBP1 pathway to shift energy storage to
lipogenesis. Together, these data reveal a previously unappreciated broad role for
glycogen in the control of energy homeostasis.
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INTRODUCTION
Glycogen represents the first choice for energy storage and utilization. Its metabolism is
tightly regulated by both hormones and nutritional status, which control the activities of
glycogen synthase (GS) and phosphorylase (GP) through a variety of pathways (1). GS
and GP activities are coordinated by glycogen-targeting subunits that serve as “molecular
scaffolds”, bringing together these enzymes with phosphatases and kinases in a
macromolecular complex, and in the process promoting activation of GS and inactivation
of GP. Six different proteins target protein phosphatase 1 (PP1) to glycogen (2). In liver,
PTG and GL are expressed at approximately equivalent levels (3), and together facilitate
the mobilization and storage of hepatic glycogen. PTG overexpression dramatically
increases glycogen content, due to a redistribution of PP1 and GS to glycogen particles,
and a corresponding marked increase in GS activity and glycogen synthesis (4–7).
Despite its profound effects, how PTG controls glycogen metabolism remains uncertain.
One potential mode of regulation may be transcriptional. Both noradrenaline and
adenosine up-regulate PTG mRNA levels in astrocytes and hepatocytes, concomitant
with increased glycogen synthesis (Allaman et al., 2000). The FoxA2 forkhead class
transcription factor directly binds to and transactivates the PTG promoter in vitro (10).
Moreover, two SREBP/USF binding units in the PTG promoter also suggest potential
transcriptional regulation by SREBP.
These indications that PTG may be under transcriptional regulation prompted us to study
its expression during different dietary conditions, and assess its importance in energy
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homeostasis. We show here that HFD produces an increase in hepatic glycogen via a
process that involves induction of PTG mRNA and protein levels, which occurs
downstream of mTORC1 activation and SREBP1 induction. Surprisingly, PTG knockout
mice on HFD not only exhibited decreased glycogen levels in liver, but also improved
insulin sensitivity as well as decreased hepatic steatosis, both of which can be attributed
to reduced activation of mTORC1. Taken together, these data indicate that upon its
accumulation in liver, glycogen elicits a positive feedback loop involving mTORC1,
resulting in increased expression of lipogenic genes, and thus a coordinated synthesis of
glycogen and lipid.
RESEARCH DESIGN AND METHODS
Animals. Generation of PTG KO mice has been described previously (11). These mice
had been maintained on a mixed genetic background, but for these studies were crossed
ten times with C57BL/6J mice (Jackson Laboratories). Wild-type littermates were used as
control. Male mice between 8-10 weeks were given a high fat diet (45% fat, D12451;
Research Diets Inc.) or continued on a normal diet (4.5% fat, 5L0D; Lab Diet) for the
same duration. All mice were maintained in temperature and humidity controlled
conditions with a 12hr light/dark cycle, and had free access to food and water. All
protocols were approved by the University of Michigan Animal Care and Use Committee.
Antibodies. The following antibodies were used: GS, pGS (S641), S6K1, pS6K1 (T389),
S6, pS6 (S235/236) (all from Cell Signaling), SREBP1 (a gift from Dr. Jay Horton). PTG
was detected after AMPD using a rabbit polyclonal antibody raised against the murine
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PTG sequence as described previously (11). A mouse monoclonal antibody raised against
RalA (from BD sciences) and rabbit polyclonal antibody raised against CREB (from Cell
Signaling) was used as a loading control for whole cell lysate and nuclear extracts,
respectively. For detection, Western Lightning Enhanced Chemiluminescence kit (Perkin
Elmer Life Sciences) was used. Individual protein bands were quantified by Image J.
Glycogen Measurement and Glucose conversion into Glycogen Assay. Glycogen was
measured as previously published (12). Glucose conversion into glycogen assay was
performed as described in (13).
Triglyceride Measurement. Frozen tissues were homogenized in buffer containing 50
mM Tris, 5 mM EDTA, 30 mM Mannitol. Crude lysate was mixed with KOH and then
extracted with chloroform: methanol (2:1). After vigorous vortexing and short incubation,
samples were spun down at 13,000 rpm for 10 min. The bottom layer was dried and
resuspended in butanol mixture (3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol)
before being assayed for triglyceride content using a Triglyceride Assay Kit from Sigma-
Aldrich.
Quantitative Real-Time PCR. Total RNA was isolated using Ambion RNA isolation kit
(Life Technologies) and subjected to quantitative real-time PCR as described (14).
Primers are listed in Supplementary Table 1.
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Western blotting and Amylose Pull-down Assay. Whole cell lysates and nuclear
extracts were prepared as described (11,15). Amylose pull-down (AMPD) assays were
performed for analyzing PTG and glycogen-bound GS protein levels. Briefly, 1 mg of
total proteins from crude lysates were incubated with amylose resin (New England
Biolabs) for 2hrs at 4°C before spun down at 3,000 rpm for 2 min. Proteins bound to the
resin were washed and released by boiling in the presence of SDS sample buffer, and
subjected to western blotting.
Luciferase Constructs and Luciferase Reporter Assay. PTG promoter fragments were
generated as described previously (10). The -493 to +36 fragment (T3) was used as a
template to produce mutations in the putative SREBP binding sites. The following primer
sequences were used in site-directed mutagenesis: M1, 5′-
GATTGGTCGGAGGGACCGTGGCTTTGATAAGCTGCC -3′ and 5′-
GGCAGCTTATCAAAGCCACGGTCCCTCCGACCAATC-3′; M2, 5'
GATTGGTCGGAGGGACCGGTTGTCGTGATCTGGCTTTGATAAG-3'and 5'-
CTTATCAAAGCCAGATCACGACAACCGGTCCCTCCGACCAATC-3'; M3, 5'-
GTCGGAGGGACCGGTTGTCGACATCTGGCTTTGATAAGCTGC-3' and 5'-
GCAGCTTATCAAAGCCAGATGTCGACAACCGGTCCCTCCGAC-3'. HEK-293A
and TSC2 MEF cells were cultured in DMEM containing 10% FBS. TSC2 null MEF’s
were obtained from Dr. David Kwiatkowski (Brigham and Women’s Hospital) (16).
Luciferase reporter assay was conducted as described (10).
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Chromatin Immunoprecipitation (ChIP) Assay. TSC2 MEF cells were treated with 1%
formaldehyde for 10 min and processed with a ChIP assay kit as described in (10). An
aliquot of DNA was used for quantitative real-time PCR using the following primers
corresponding to the mouse PTG genomic sequences. P1, 5’-
CTGGAGTTCCAGGCTGTGTA-3’ and 5’-GAGAACGGCTCAGAGGGTAG-3’; P2,
5’-GGGAAACACACACACACACA-3’ and 5’-GAGAAGTTGGCTGGCTCTTC-3’; P3,
5’-TCACGTGATCTGGCTTTGAT-3’ and 5’-CTGAGCAACTTCGCACTCAG-3’; P4,
5’-TTTGATTGGTCGGAGGGA-3’ and 5’-AACCGGGAGGCAGCTTAT-3’.
Blood Glucose and Insulin Measurements. Blood glucose was measured by OneTouch
Ultra Glucometer. Plasma insulin concentrations were measured by Ultra Sensitive
Mouse Insulin ELISA kit (Crystal Chem Inc.).
Glucose, Insulin and Pyruvate Tolerance Tests. For glucose tolerance test (GTT), mice
were fasted 6hrs before an intraperitoneal (IP) injection of 1.5g/kg of body weight of
glucose. For insulin tolerance tests (ITT), mice were fasted 3hrs before an IP injection of
0.75U/kg (ND) or 1.5 U/kg (HFD) of body weight of Humulin R (Eli Lilly). For pyruvate
tolerance test (PTT), mice were fasted overnight before an IP injection of 1.5g/kg of body
weight of sodium pyruvate. Blood glucose concentrations were measured before and after
the injection at the indicated time points using the One Touch Ultra glucometer
(Lifescan).
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shRNA Knockdown Experiments. Mouse pGIPZ lentiviral-shRNA clones targeting
SREBP1and PTG were provided by the University of Michigan Biomedical Research
Core Facilities. Lentiviral production was done as described (17).
RNA-seq and Data Analysis. Total RNA was isolated from livers of biological
triplicates using Trizol (Invitrogen) and the RNeasy mini kit (Qiagen). Libraries were
prepared from 100-500ng total RNA (TrueSeq v2, Illumina) and sequenced on the
Illumina HiSeq 2000 using bar-coded multiplexing and a 100bp read length. Data
analysis was accomplished according to (16) utilizing UCSC mm9 as a reference
sequence. Heatmaps were visualized using Java Treeview 1.1.6r2
(http://jtreeview.sourceforge.net). RNA-seq data are available from the Gene Expression
Omnibus (http://www.ncbi.nlm.nih.gov/geo/) as accession GSE45319.
RESULTS
High Fat Diet Increases Hepatic Glycogen in Fasted Mice.
To understand better the role of glycogen metabolism in the overall control of energy
disposition, C57BL/6J mice were put on high fat diet (HFD) for 12 weeks and became
obese, glucose intolerant and insulin resistant (Supplementary Figure 1). Mice were
subjected to 16hrs fasting followed by 6hrs refeeding before sacrifice. Hepatic glycogen
levels were almost completely depleted by fasting and replenished after refeeding (Figure
1A). Interestingly, while levels were similar in the refed state, HFD produced a 3-fold
increase in hepatic glycogen levels in fasted mice (Figure 1A). Hepatic PTG (Ppp1r3c)
mRNA levels were approximately 2-fold increased by HFD in both the fasted and refed
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states, concomitant with similar induction in SREBP1 (Srebf1) mRNA levels (Figure 1B).
Phosphorylation of the mTORC1 downstream target S6K and its substrate S6 were
dramatically increased by HFD in fasted mice (Figure 1C). Fasting reduced SREBP1
processing; however, we still detected an increase in the active, mature form of SREBP1
protein after HFD (Figure 1C), consistent with previous reports (17,18). PTG protein
levels corresponded well with the HFD-dependent increase in mTORC1 activity and
SREBP1 induction (Figure 1C), suggesting that the mTORC1/SREBP1 pathway may
contribute to the elevated glycogenesis observed in obesity in the fasted state.
Glycogen phosphorylase (GP) activity did not change, ruling out the possibility that
increased glycogen content in obesity results from reduced GP activity (Figure 1D).
While total GS protein levels decreased during refeeding, the mRNA levels of the
predominant GS isoform GYS2 in liver were not changed, suggesting a post-
transcriptional modification of GS in response to refeeding (Figure 1B). Since the GS
antibody might not efficiently recognize the dephosphorylated form of the enzyme, we
measured GS activity (-/+ G6P ratio) in liver lysate, and observed increased GS activity
in response to refeeding in liver from ND but not HFD mice (Figure 1E).
The PTG Promoter is Regulated by SREBP1 and mTORC1
The coordinated increases in hepatic SREBP1 and PTG protein and transcript levels in
response to nutritional status, and the correlation of these findings with increased
glycogen levels, suggested an SREBP-dependent mechanism for the regulation of PTG
transcription. Therefore, we generated mutations of SREBP binding sites on the PTG
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promoter (10) and assessed their activity by a luciferase-based promoter assay.
Expression of active SREBP-1c led to an approximately 2-fold increase in the activity of
the wild-type PTG promoter, an effect significantly blunted in the mutants (Figure 2A),
suggesting that SREBP1 modulates PTG promoter activity through SRE elements
proximal to the transcriptional start site.
To determine whether mTORC1 can regulate PTG transcript levels, we examined TSC2-/-
murine embryo fibroblasts (MEFs) that have high mTORC1 activity (19). PTG promoter
activity was elevated in TSC2-/-
cells, and was reduced by rapamycin treatment (Figure
2B). However, this activation was not seen in the PTG promoter mutant M1, in which the
only available SRE was deleted. Chromatin Immunoprecipitation (ChIP) assay confirmed
the direct interaction of SREBP1 with the PTG promoter, and this interaction was
enhanced at the SREBP1 binding site by constitutive activation of mTORC1 (Figure 2C).
Similarly, overexpression of the constitutively active form of Rheb (20) activated the
PTG promoter in a rapamycin-sensitive manner, and this effect was abolished by deletion
of the proximal SRE in the PTG promoter (Figure 2D).
Transcriptional Regulation of PTG expression by SREBP1 is Required for the
Increased Glycogen Produced by mTORC1 Activation
TSC2-/-
MEFs have increased protein levels of PTG and SREBP1, and higher steady state
levels of glycogen and a greater rate of glucose conversion into glycogen compared to
wild type MEFs (Figure 3A, C and D, control), suggesting that mTORC1 activation is
sufficient for increased glycogen content in vitro. Because PTG binds to and localizes
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PP1 and its substrate GS to glycogen (4), we performed amylose pull-down (AMPD)
assays to measure PTG and GS bound to a glycogen-like matrix. Amounts of glycogen-
bound GS increased with PTG (Figure 3A, control), explaining the elevated glycogen
content in TSC2-/-
cells. These data reinforce the notion that PTG plays a dual
scaffolding/dephosphorylation role by recruiting GS to the glycogen pellet and promoting
its dephosphorylation.
In order to test if the stimulation of glycogen synthesis by mTORC1 is dependent on
SREBP1, we performed lentiviral-mediated shRNA knockdown of SREBP1. The levels
of PTG protein and mRNA were reduced by SREBP1 knockdown, as was the recruitment
of GS to the amylose precipitate (Figure 3A and B). Glycogen levels (Figure 3C) and the
conversion of glucose into glycogen (Figure 3D) were similarly reduced. Collectively,
these data suggest a role for mTORC1 in the regulation of PTG gene expression, via
SREBP binding to an SRE in the PTG promoter.
PTG Levels Control Glycogen Synthesis and Contribute to Feedback Regulation of
mTORC1 and SREBP1.
Lentiviral-mediated shRNA knockdown of PTG produced a 60-70% reduction in both
protein and transcript levels of PTG in TSC2-/-
cells (Figure 4A and B). PTG knockdown
did not affect other glycogen-targeting subunits such as GL, GM and the catalytic subunit
PP1 (Supplementary Figure 2A and B and Figure 4A). Interestingly, PTG knockdown
also reduced SREBP1 protein and mRNA levels, suggesting a positive feedback
mechanism in which PTG regulates SREBP1.
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PTG knockdown produced a 60-70% reduction in both steady state glycogen content and
the rate of glycogen synthesis in TSC-/-
but not TSC+/+
cells (Figure 4C and D), consistent
with the knockdown efficiency of PTG in these cells. The reduced glycogen synthesis
was not due to reduced glucose transport, as glucose uptake was not affected by PTG
knockdown (Figure 4E).
We next examined regulation of mTORC1 activity and glycogen levels by PTG in
HEK293A cells in which we stably overexpressed Flag-GFP-tagged PTG. Glycogen
levels in these cells were 50 fold higher compared to control cells (Figure 4F) due to the
constitutive activation of PTG proteins (Figure 4G). Moreover, phosphorylation of S6K
was 2-fold increased in cells overexpressing PTG (Figure 4G), suggesting that the
scaffolding protein also regulates mTORC1 signaling through a positive feedback
mechanism. Taken together, these results provide evidence that regulation of PTG
expression is directly responsible for the increased glycogen in the context of activated
mTORC1.
In HEK293A cells and the liver cell line Hepa1c, we also observed similar regulation of
glycogen synthesis through PTG. Transient overexpression of WT PTG in these cells
resulted in a 2-3 fold increase in glycogen content. More importantly, these effects were
blocked by overexpression of PTG mutants that are incompetent to bind to GS or GP,
suggesting that PTG is critical in the recruitment of GS and GP to glycogen particles
(Supplementary Figure 2C).
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Homozygous Deletion of PTG Reduces Glycogen and Lipid Levels in vivo.
Our previous studies on a PTG heterozygous KO mouse suggested that PTG acts as a
PP1 scaffolding protein and plays a vital role in glycogen metabolism (11). These mice
had been maintained on a mixed background in which homozygous deletion of the PTG
gene was embryonic lethal. Subsequent backcrosses of these mice into a C57BL/6J
background produced viable homozygous PTG KO mice (Supplementary Figure 3A).
The genotyping of more than 200 female and male homozygous PTG KO pups derived
from heterozygous breeding pairs coming from the first backcross accounted for only ~10%
of the total births, a number significantly lower than the expected Mendelian inheritance
ratio of 25% (Supplementary Figure 3B). However, sequential backcross of the
heterozygous PTG KO mice with C57BL/6J mice produced both female and male
homozygous PTG KO mice born at the expected frequency (Supplementary Figure 3B).
While the mechanism underlying the effect of genetic background on survival of PTG
KO mice remains unknown, all the experiments described in this study were performed
on male PTG KO mice derived from heterozygous breeding pairs backcrossed ten times
with C57BL/6J mice, and littermates without PTG gene deletion (WT) were used as
controls.
To study the role of PTG in glucose homeostasis in vivo, we fed PTG KO mice HFD. No
obvious differences in weight gain or food intake were observed between WT and KO,
fed either ND or HFD, demonstrating that the PTG KO mouse exhibits a normal growth
rate and eating behavior (Supplementary Figure 3C and D).
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Both hepatic glycogen and triglyceride levels were greatly reduced in PTG KO mice fed
HFD for 12 weeks (Supplementary Figure 3E and F). Consistent with the increased
hepatic glycogen levels observed in HFD WT mice, a 2-fold increase in PTG and
SREBP1 mRNA was detected (Supplementary Figure 3G). The mRNA levels of the
glycogen targeting subunit GL were also increased by about 2-fold under HFD conditions
(Supplementary Figure 3G). However, glucose and insulin tolerance tests were
indistinguishable between PTG WT and KO mice, either on ND or HFD (Supplementary
Figure 3H and I). Interestingly, neither GS nor GP activity was changed in the liver
lysates from KO mice (Supplementary Figure 4), reinforcing the hypothesis that PTG is a
critical scaffold to recruit GS or GP to the glycogen particles. In this regard, it is possible
that the phosphorylation status or activity of non-glycogen targeted GS or GP (i.e. GS or
GP in whole cell lysate) is largely irrelevant to glycogen accumulation.
We next explored glycogen synthesis more broadly by directly examining glucose
conversion into glycogen in vitro in isolated primary hepatocytes (Figure 5 A-C) or in
vivo during a hyperinsulinemic-euglycemic clamp study (Figure 5D and E). In primary
hepatocytes isolated from ND mice, we observed a 60% reduction in 14C-glucose
incorporation into glycogen in PTG KO, but no obvious difference in lipid or CO2
incorporation, consistent with the mild reduction in TG observed in ND PTG KO mice.
Both 3H-glucose incorporation into glycogen and lipids were 40-50% reduced in PTG
KO mice fed HFD for 8-10 weeks, as measured in a hyperinsulinemic-euglycemic clamp
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study, suggesting that both glycogen and lipid synthesis in HFD mice are reduced when
PTG is depleted.
PTG Ablation Reduces Hepatic Steatosis after Long-term HFD.
Because of the profound effects of PTG deletion on lipid accumulation in liver, we also
studied mice fed HFD for 24 weeks. Similar to what was observed with shorter-term
HFD feeding, the 24-week HFD-induced elevation in glycogen was prevented in PTG
KO mice (Figure 6A). Triglyceride content was 40% decreased in the liver from PTG KO
mice fed HFD compared to WT littermates, corresponding to decreased glycogen levels
(Figure 6B). Moreover, HFD-induced liver steatosis was significantly attenuated by PTG
ablation, consistent with the marked reduction in triglycerides (Figure 6C).
To further investigate alleviation of the hepatosteatotic phenotype, we measured the
expression of key metabolic and inflammatory genes in the livers. PTG KO mice on HFD
had lower expression of Srebf1 and its downstream target gene fatty acid synthase (Fasn)
(Figure 6D). Gluconeogenic gene expression was modestly higher in HFD mice and was
not different with PTG ablation (Figure 6E). However, glycolytic genes such as pyruvate
kinase isozymes R/L (Pklr) and glucokinase (Gck), which were more highly expressed in
HFD relative to ND mice, were expressed at lower levels in PTG KO mice (Figure 6E).
In addition, HFD-induced expression of several inflammatory genes and markers of
macrophage infiltration, including F4/80 (Emr1), CD11C (Itgax), Rantes (Ccl5), and
MCP-1 (Ccl2), were reduced in livers from PTG KO mice (Figure 6F).
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PTG Ablation Improves Glucose Tolerance and Insulin Sensitivity after Long-term
HFD.
These changes in glycogen and lipid metabolism prompted us to investigate the role of
PTG in the overall control of carbohydrate metabolism. After 24 weeks of HFD, both
hyperglycemia and hyperinsulinemia were attenuated in PTG KO mice (Figure 7A). We
also performed oral glucose and intraperitoneal (IP) insulin and pyruvate tolerance tests.
PTG ablation did not affect insulin sensitivity or glucose tolerance in ND mice. However,
PTG KO mice displayed much improved glucose tolerance (Figure 7B), as well as
improved sensitivity to IP injection of insulin (Figure 7C) and pyruvate (Figure 7D).
AMPD assays from liver lysates revealed that levels of glycogen-bound GS protein were
increased by HFD, consistent with the HFD-induced elevation in PTG protein levels
(Figure 7E). Moreover, the increase in glycogen-bound GS protein was suppressed in
PTG KO mice, likely due to the lack of recruitment of GS to glycogen in the absence of
PTG. These data demonstrate that the recruitment of GS to glycogen is elevated in HFD
mice due at least in part to increased PTG protein, causing increased hepatic glycogen
content. Interestingly, both mTORC1 activity and SREBP1 levels were greatly
attenuated in PTG KO mice, confirming in vivo the feedback regulation of the
mTORC1/SREBP1 pathway by PTG and glycogen.
Taken together, these results suggest that in short term HFD-fed PTG KO mice, the
reduction in hepatic glycogen likely contributes to lower glucose output, in the process
mildly reducing serum glucose and insulin levels. Over time, this modulation of
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metabolic activity also leads to dramatically improved insulin sensitivity in the KO mice,
perhaps through reductions in mTORC1 activity known to negatively regulate insulin
receptor signaling (21,22).
A PTG-SREBP1/mTORC1 Feedback Mechanism Regulates Lipogenesis.
Although ablation of PTG did not have a major impact on systemic glucose tolerance and
insulin sensitivity after short term HFD, there were dramatic effects on hepatic lipid
metabolism (Figure 5F), which might reflect a crosstalk between pools of glycogen and
upstream nutrient-sensing pathways. In an effort to determine the mechanism, we
conducted quantitative real-time PCR for Srebf1 and its target genes Fasn, Ldlr, and
Hmgcr. We observed a trend toward decreases in the mRNA levels of these genes in the
PTG KO mice on HFD, although these changes were not statistically significant (Figure
8A).
To thoroughly examine the entire transcriptional landscape of PTG WT and KO livers,
we performed unbiased RNA-Seq and gene set enrichment analysis. We found that sterol,
steroid, cholesterol, and lipid biosynthetic/metabolic processes were differentially
regulated by PTG ablation in HFD mice (Figure 8B). More specifically, the transcript
levels of Srebf1 and its hepatic target genes such as Fasn, Ldlr, Mvk, Hmgcr were
significantly reduced in the PTG KO (Figure 8C and Supplementary Table 2), consistent
with quantitative real-time PCR data. Collectively, these data reveal a novel crosstalk
mechanism in which the mTORC1/SREBP pathway can sense elevated levels of
glycogen, and then initiate the transcriptional changes that increase lipid synthesis.
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DISCUSSION
Despite its well-established role as the first choice for energy storage and mobilization,
the coordination of glycogen metabolism with that of lipid synthesis and breakdown
remains poorly understood, particularly in the liver. Data presented here reveals that upon
reaching threshold levels during nutrient excess, glycogen may trigger increased
expression of lipogenic genes, thus shifting energy storage from glycogen to lipid. This
shift in the mode of energy storage may result from a positive feedback loop involving
mTORC1 and its transcriptional factor target SREBP1.
The activation of glycogen synthase by insulin requires the localization of PP1 to
glycogen particles through glycogen-targeting subunits, which act as molecular scaffolds
to unite PP1 with GS and GP for regulation (23). Studies reported here confirm the
crucial role of PTG in controlling glycogen synthesis (4,5). PTG KO mice have markedly
reduced glycogen levels in liver. Moreover, biochemical evaluation of GS localization in
PTG KO mice demonstrates conclusively that PTG is essential to direct GS to glycogen
particles in liver.
Experiments described here also reveal the surprising finding that the regulation of
glycogen synthesis by fasting/refeeding and HFD can be largely attributed to changes in
the expression of PTG. The increase in glycogen levels found in liver from HFD mice are
mirrored by increased expression of PTG, but attenuated in PTG KO mice. Both in vivo
and in vitro studies revealed an important role for mTORC1 in PTG expression,
consistent with findings by ourselves and others that glycogen synthesis is sensitive to
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inhibition by rapamycin (24–26). We propose that mTORC1 may regulate glycogen
metabolism through a transcriptional mTORC1-SREBP-PTG pathway, which in turn
modifies the phosphorylation status of GS in the glycogen compartment (Figure 8D).
This idea is supported by studies in both embryonic fibroblasts and mice, in which there
was a clear causal relationship between mTORC1 function, SREBP and PTG levels and
glycogen accumulation. The regulation of SREBP processing by mTORC1 has been
widely studied in the context of lipid homeostasis (27,28). Our data suggest that dietary
modulations in mTORC1 activity may affect not only lipid storage, but also carbohydrate
storage through regulation of glycogen synthesis.
Also surprising was the effect of PTG ablation on glucose metabolism. PTG mice on the
original mixed background were homozygous lethal, so we studied the heterozygous
knockouts and reported that they have decreased hepatic glycogen levels and a worsened
metabolic phenotype after 10 months or longer with ND feeding (11). However, we were
surprised to find after several years that the homozygous PTG KO mice were viable by
backcrossing heterozygotes against C57BL6/J mice. After short term HFD, the new
homozygous PTG KO mice showed reduced hepatic glucose output compared to WT
littermates, but little effect on systemic insulin sensitivity, likely reflecting reduced
glycogen that serves as a source of glucose. However, after longer term HFD, PTG KO
mice exhibited profoundly reduced fasting glucose and insulin levels, along with
improved glucose, pyruvate and insulin tolerance, reflecting increased systemic insulin
sensitivity, and accompanied by changes in expression of genes involved in lipogenesis,
glycolysis, inflammation and macrophage infiltration. Thus, although GS is increased
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while GP is decreased by insulin action, and both enzymes have been considered as
targets for the treatment of type 2 diabetes, reducing glycogen synthesis may
paradoxically improve insulin resistance in obesity.
The effect of PTG deletion on hepatic steatosis was particularly intriguing. Because PTG
does not interact with lipid-metabolizing enzymes, it is likely that this observation
reflects crosstalk between pools of glycogen and upstream nutrient-sensing pathways.
These ideas are consistent with observations using a glycogen phosphorylase inhibitor, in
which administration of the drug resulted in a short-term reduction in blood sugar by
prevention of hepatic glycogen breakdown, but was accompanied by steatosis after longer
term treatment due to increased lipid synthesis (29). Glycogen levels are increased in the
liver of HFD mice under fasting conditions, while the mTORC1 pathway is constitutively
elevated, along with increased levels of PTG, suggesting that diet-induced activation of
mTORC1 may increase glycogen stores in the fasted state by increasing the levels of
PTG. This occurs even in the face of reduced insulin signaling in HFD, and is similar to
what is observed for obesity-induced hepatic triglyceride accumulation (reviewed in (30)).
Conversely, PTG KO mice, which have reduced levels of hepatic glycogen, have reduced
hepatic lipids and reduced mTORC1/SREBP. Our transcriptional landscape analysis by
RNA-seq revealed a significant down-regulation of SREBP1 and numerous target genes
involved in lipogenesis. Although we cannot rule out other mechanisms explaining
reduced hepatic lipid levels, such as reduced glucose and insulin, these transcriptional
changes most likely underlie the alterations in lipid levels in the PTG KO livers, which
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21
perhaps reflect an interesting feedback regulation of PTG on mTORC1/SREBP and
lipogenesis to mediate crosstalk between pools of glycogen and lipids.
How might mTORC1 sense levels of glycogen? We propose a molecular sensor
upstream of the mTORC1 complex, similar to what might be involved in the sensing of
amino acids (31). One possible candidate is AMP-activated kinase, a negative regulator
of mTORC1 that is thought to be directly attenuated by the binding of glycogen to its β
subunit (32–34). Although we did not observe a decrease in AMPK or ACC
phosphorylation in liver from PTG mice on short term HFD, there were reductions in
muscle tissue (data not shown), suggesting that this enzyme may contribute to the overall
regulation of mTORC1. However, it also remains possible that there are other glycogen
sensors in the mTOR pathway. Future studies will focus on the regulation of this
pathway by glycogen, with the hope of developing new therapeutic targets for the
treatment of insulin resistance and nonalcoholic fatty liver disease.
ACKNOWLEDGEMENTS
We thank the University of Michigan Vector Core and the Comprehensive Cancer Center
Tissue Core for shRNA clones and histology service. We thank Dr. Jay Horton for
SREBP1 antibodies and Dr. David Kwiatkowski for the TSC2 knockout fibroblasts. We
also thank Dr. Ken Inoki and the members of the Saltiel laboratory for helpful
discussions.
FUNDING
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22
This work was supported by NIH grants R01DK060591 to ARS and R24DK090962 to
ARS and RME, and a mentor-based postdoctoral fellowship from the American Diabetes
Association to BL. This work was also funded by grants from the National Institutes of
Health (DK057978), the Leona M. and Harry B. Helmsley Charitable Trust, the Glenn
Foundation for Medical Research, and the Ellison Medical Foundation to RME. RME is
an investigator of the Howard Hughes Medical Institute and March of Dimes Chair in
Molecular and Developmental Biology at the Salk Institute.
DUALITY OF INTEREST
No potential conflicts of interest relevant to this article were reported.
AUTHOR CONTRIBUTIONS
BL and ARS developed the overall research plan. BL generated the figures and wrote the
manuscript. BL, DB, AC, LC, ZM conducted experiments, researched data, performed
analysis and interpretation. YY and KF carried out experiments. DB and ARS contributed
to discussion, interpretation and reviewed/edited manuscript. MD, RTY, CL, RME
performed RNA-Seq and data analysis. ZM and JL performed the primary hepatocyte
experiment. ARS is the guarantor of this work and, as such, had full access to all the data
in the study and takes responsibility for the integrity of the data and the accuracy of the
data analysis.
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Figure Legends
Figure 1. High Fat Diet Increases Hepatic Glycogen in Fasted Mice
(A) Glycogen content in the liver of C57BL/6J mice on HFD for 12 weeks and sacrificed
after 16hrs fasting followed by 6hrs refeeding (n=8 each group).
(B) Quantitative real-time PCR showing relative mRNA level of PTG, SREBP1 and
GYS2 from the liver of mice in (A).
(C) Representative western blots from the liver of mice in (A). WCL, whole cell lysate;
N.E., nuclear extracts; AMPD, after an amylose pull-down assay.
(D) Hepatic GP activity and (E) GS activity from ND and HFD mice sacrificed after
16hrs fasting followed by 6hrs refeeding (n=6-8 mice each group).
Values are expressed as mean ± SE.*indicates statistical significance by student t-test
(*P<0.05).
Figure 2. The PTG Promoter is Regulated by SREBP1 and mTORC1
(A) HEK293 cells were cotransfected with nuclear active form of SREBP-1c (1-471) and
the indicated reporter constructs of the PTG promoter. Cells were harvested 24 hrs after
transfection and assayed for luciferase activity. Results were normalized (Renilla
luciferase) and expressed as fold increase over the basal value of each construct.
(B) TSC2 MEF cells were transfected with the indicated luciferase reporter constructs of
the PTG promoter and treated with Rapamycin (100nM) or DMSO before harvest.
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(C) A schematic representation of the PTG promoter region (top) and Chromatin
immunoprecipitation (CHIP) experiment showing increased SREBP1 activity in TSC2-/-
cells (bottom). Cross-linked chromatin was immunoprecipitated with SREBP1 antibody
and extensively washed before elution and precipitation of DNA. Quantitative PCR was
performed with the indicated primer pairs. Primer sequences were listed in the Research
Design and Methods.
(D) Effect of Rheb and rapamycin on the PTG promoter activity. HEK293 cells were
cotransfected with Rheb or vector and the indicated luciferase reporter construct of the
PTG promoter harboring the deletion of the putative SREBP1 binding site. Cells were
subsequently treated with DMSO or rapamycin (100nM) for 24hrs before harvest and
assayed for luciferase activity. Results were normalized (Renilla luciferase) and
expressed as fold increase over the basal value of each construct.
All the Values are expressed as mean ± SE (n=3). *indicates statistical significance by
student t-test (*P<0.05).
Figure 3. Transcriptional Regulation of PTG expression by SREBP1 is Required for
the Increased Glycogen Produced by mTORC1 Activation
(A) Western blots showing SREBP1 knockdown in TSC2 MEFs. Cells were transfected
with lentiviral mediated control shRNA or shRNAs targeting SREBP1 and selected for
cells stably expressing shRNAs. Stable cells were lysed and subjected to western blotting
against indicated antibodies.
(B) Quantitative real-time PCR showing relative mRNA level of SREBP1 and PTG from
cells in (A).
(C) Glycogen content of cells in (A).
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(D) 14C-Glucose conversion into glycogen of cells in (A).
All the Values are expressed as mean ± SE (n=3). *indicates statistical significance by
student t-test (*P<0.05).
Figure 4. PTG Levels Control Glycogen Synthesis and Contribute to Feedback
Regulation of mTORC1 and SREBP1.
(A) Western blots showing PTG knockdown in TSC2 MEFs. Cells were transfected with
lentiviral mediated control shRNA or shRNAs targeting PTG and selected for cells stably
expressing shRNAs. Stable cells were lysed and subjected to western blotting against
indicated antibodies.
(B) Quantitative real-time PCR showing relative mRNA level of PTG and SREBP1 from
cells in (A).
(C) Glycogen content of cells in (A).
(D) 14C-Glucose conversion into glycogen of cells in (A).
(E) 2-DG uptake of cells in (A).
(F) Glycogen levels of HEK293A cells stably overexpressing Flag-tagged PTG.
(G) Representative western blots showing activation of mTORC1 activity by
overexpressing PTG. Numbers above blots indicate relative band intensity as quantified
by Image J software.
Values are expressed as mean ± SE (n=3 experiments). *indicates statistical significance
by student t-test (*P<0.05) n.s. indicates not significant by student t-test.
Figure 5. PTG Ablation Results in Decreased Glucose Incorporation into Glycogen
and Lipids.
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14C-glucose incorporation into glycogen (A), lipids (B) and CO2 (C) in primary
hepatocytes isolated from 6-8 week old PTG WT or KO mice on ND. Values are
expressed as mean ± SE (n=3 triplicate experiments). * indicates statistical significance
by student t-test (*P<0.05)
3H-glucose incorporation into glycogen (D) and lipids (E) during a hyperinsulinemic-
euglycemic clamp study using PTG WT or KO mice on HFD for 8-10 weeks.
Values are expressed as mean ± SE (n=10-12 mice each group). * indicates statistical
significance by student t-test (*P<0.05)
Figure 6. PTG Ablation Reduces Hepatic Steatosis in vivo after Long Term HFD.
(A) Glycogen content and (B) TG levels in the liver of WT and PTG KO mice on HFD
for 24 weeks and fasted overnight (n=8-10 mice each group).
(C) Representative H&E staining of liver sections of mice in (A).
(D) Quantitative real-time PCR showing relative mRNA level of SREBP1 and FAS from
mice in (A).
Quantitative real-time PCR showing relative mRNA level of glucose metabolism genes
(E) and inflammatory genes and macrophage markers (F) from mice in (A).
Figure 7. PTG Ablation Improves Glucose Tolerance and Insulin Sensitivity in vivo
after Long Term HFD.
(A) Fasting blood glucose and insulin levels of WT and PTG KO mice on HFD for 24
weeks and fasted overnight.
(B) Oral glucose tolerance test, (C) Insulin tolerance test and (D) Pyruvate tolerance test
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of WT and PTG KO mice on HFD for 24 weeks.
(E) Representative blot (Left) and quantification (right) from the liver of mice in (A).
All the Values are expressed as mean ± SE (n=8-10). *indicates statistical significance by
student t-test (*P<0.05).
Figure 8. A PTG-SREBP1/mTORC1 Feedback Mechanism Regulates Lipogenesis
(A) Quantitative real-time PCR showing relative mRNA level of SREBP1 and its target
genes from the liver of mice on HFD for 12 weeks and fasted overnight (n=8-10 mice
each group).
(B) GO term enrichment analysis of RNA-seq data from the livers of mice in (A).
(C) Unbiased hierarchical clustering analysis of RNA-Seq data from the livers of mice in
(A) showing down-regulation of SREBP1 hepatic target genes in PTG KO mice on HFD .
Genes shown on the heatmap are listed in Supplementary Table 2.
(D) A schematic model showing the feedback regulation of PTG and glycogen on
mTORC1/SREBP and lipogenesis.
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A
pGSS6K
pS6K
PTG
S6
m-SREBP1
GS
pS6RalA
Fasted Refed Fasted RefedND HFD
B
WCL
AMPD
Figure1. High Fat Diet Increases Hepatic Glycogen in Fasted Mice.
ND
HFD
G
lyco
gen
cont
ent
(μm
ol g
luco
se/g
tiss
ue)
120
100
80
60
40
20
0Fasted Refed
0.0
0.5
1.0
4.5
Fasted Refed
Rel
ativ
e m
RN
A
Ppp1r3c
0
2
4
6
8
10
12
14
16
Fasted Refed
Rel
ativ
e m
RN
A
Srebf1
ND HFD
ND HFD 4.0
3.5
3.0
2.5
2.0
1.5
*
*
*
*
N.E. CREB
C
0 2 4 6 8
10 12 14
Fasted Refed
pS6K
/S6K
0 2 4 6 8
10 12 14 16 ND
HFD
pS6/
S6
Fasted Refed
ND HFD
* *
0 1 2 3 4 5 6
PTG
Fasted Refed
ND HFD *
*
0
5
10
15
20
Fasted Refed
m-S
RE
BP
1 ND HFD
*
0
0.4
0.8
1.2
1.6
Rel
ativ
e m
RN
A
Gys2
Fasted Refed
0.2
0.6
1.0
1.4
0
0.2
0.4
0.6
0.8
1.0
GS
Act
ivity
ratio
(-/+
G6P
)
0
0.2
0.4
0.6
0.8
1.0
Fasted
Refe
d
*
ND HFD
0
GP
activ
ity (n
mol
/min
/mg
prot
ein) 25
20
15
10
5
0
-AMP+AMP
GP
activ
ity ra
tio(-
/+A
MP
)
ND-Fas
ted
HFD-Refe
d
HFD-Fas
ted
ND-Refe
d
ND HFD
GS
act
ivity
(nm
ol/m
in/m
g pr
otei
n) 14
12
10
8
6
0
4
2
-G6P+G6P
ND-Fas
ted
HFD-Refe
d
HFD-Fas
ted
ND-Refe
d
Fasted
Refe
d
1.2*
ND HFD
ED
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WT -70 agggaccgGTTCACGTGATC tggctttgat -41
M1 -70 agggaccg------------------------- tggctttgat -41M2 -70 agggaccgGTTGTCGTGATCtggctttgat -41M3 -70 agggaccgGTTGTCGACATCtggctttgat -41
0
0.5
1.0
1.5
2.0
2.5
3.0
WT M1 M2
Rel
ativ
e Lu
c A
ctiv
ity
(fold
incr
ease
)
VectorSREBP-1c
AB
+/++/+-/- -/-
TSC2 DMSOTSC2 RapaTSC2 DMSOTSC2 Rapa
WT M1M3
* * *
Rel
ativ
e Lu
c A
ctiv
ity(F
old
incr
ease
)
WT M1
Vector DMSOVector RapaRheb DMSORheb Rapa
Rel
ativ
e Lu
cife
rase
Act
ivity
(F
old
chan
ge)
* *
0
0.5
1.0
1.5
2.0
2.5
DC
TSS
-2015
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Pre
cipi
tatio
n re
lativ
e to
inpu
t (%
)
TSC2+/+ TSC2-/-
SREBP binding sitePrimer pairs
1 2 4 3
1 2 3 4
*
*
0
0.5
1.0
1.5
2.0
2.5
Figure 2. The PTG promoter is regulated by SREBP1 and mTORC1.
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(µmol
/mg
prot
ein)
Contro
l
SREBP1-sh2
Contro
l
SREBP1-sh2
0.6
0
40
60
80
100
120
(fmol
/mg
prot
ein/
60m
in)
Gly
coge
n sy
nthe
sis
Gly
coge
n co
nten
t
TSC2
m-SREBP1
PTG
GS
RalA
GS
pGS
TSC2 TSC2
WCL
AMPD
+/+ -/-A
C
Figure 3. Transcriptional Regulation of PTG expression by SREBP1 is Required for the Increased Glycogen Produced by mTORC1 Activation
0.5
0.4
0.3
0.2
0.1
0
TSC2+/+TSC2-/-
CREBN.E.
Con
trol
SR
EB
P1-
sh1
SR
EB
P1-
sh2
Con
trol
SR
EB
P1-
sh1
SR
EB
P1-
sh2
B
Rel
ativ
e m
RN
A
2.0
1.6
1.2
0.8
0.4
0
Contro
l
SREBP1-sh1
SREBP1-sh2
0
0.5
1.0
1.5
2.0
2.5
Srebf1 Ppp1r3c
* * *
* *
**
* *
Contro
l
SREBP1-sh1
SREBP1-sh2
* *
D
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D
TSC2
PTG
GS
RalA
m-SREBP1
GS
pGS
TSC2 TSC2
WCL
AMPD
+/+ -/-A
C
0
0.1
0.2
0.3
0.4
0.5
(µmol
/mg
prot
ein)
0.6
Gly
coge
n co
nten
t
0
0.5
1.0
1.5
2.0
2.5
(nm
ol/m
g pr
otei
n/60
min
)G
lyco
gen
synt
hesi
s
TSC2+/+TSC2-/-
Contro
l
PTG-sh
1
PTG-sh
2
Contro
l
PTG-sh
1
PTG-sh
2
* * *
CREBN.E.
Con
trol
PTG
-sh1
PTG
-sh2
Con
trol
PTG
-sh1
PTG
-sh2
Figure 4. PTG Levels Control Glycogen Synthesis and Contribute to Feedback Regulation of mTORC1 and SREBP1.
B
0
0.5
1.0
1.5
2.0
2.5
3.0
Ppp1r3c
0
0.5
1.0
1.5
2.0
2.5
Srebf1
Rel
ativ
e m
RN
A
Contro
l
PTG-sh
1
PTG-sh
2Con
trol
PTG-sh
1
PTG-sh
2
** *
*
* *
0
10
20
30
40
50
60
70
80
2-D
G u
ptak
e (µ
mol
/g p
rote
in/5
min
)
Contro
l
PTG-sh
1
PTG-sh
2
n.sE
S6K
RalA
Flag-PTG
pS6K
S6K
pS6K
WCL
IP:
PTG stable 293A
PTG stab
le29
3A
20
15
10
5
0
mm
ol g
luco
se/m
g pr
otei
n
F
IgG S6K IgG S6K
IB
G
1.3 2.0 2.5 1.8 1.0 1.1 1.3 1.4
1.5 2.0 2.3 1.1 1.3 1.0
PP1
PP1
AMPD endo-PTGOX-PTG
1.5 1.8 1.6 1.4
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0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
WT KO 14C
glu
cose
inco
rpor
atio
n in
to g
lyco
gen
0
0.2
0.4
0.6
0.8
1.0
1.2
WT KO 0
0.2
0.4
0.6
0.8
1.0
1.2
WT KO
14C
glu
cose
inco
rpor
atio
n in
to li
pids
14C
glu
cose
inco
rpor
atio
n in
to C
O2
0
0.2
0.4
0.6
0.8
1.0
1.2
WT KO
3H g
luco
se in
corp
orat
ion
into
gly
coge
n
0
0.2
0.4
0.6
0.8
1.0
1.2
WT KO
3H g
luco
se in
corp
orat
ion
into
lipi
ds
A B C
D E
Figure 5. PTG Ablation Results in Decreased Glucose Incorporation into Glycogen and Lipids.
* *
*
Page 35 of 49
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0
5
10
15
20
25
30
35
ND HFD
AG
lyco
gen
cont
ent
(μm
ol g
luco
se/g
tiss
ue) WT
KO
0
40
80
120
160
200
ND HFDTG
(μ
mol
gly
cero
l/g t
issu
e) **** * WT
KO
WT KO
ND
HFD
CB* *
*
WTKO
WTKO
Srebf1 Fasn
2.5
2.0
1.5
1.0
0.5
0.0
2.5
2.0
1.5
1.0
0.5
0.0ND HFD ND HFD
Rel
ativ
e ge
ne e
xpre
ssio
n
Rel
ativ
e ge
ne e
xpre
ssio
n
#
*
D
0
1
2
3
4
5
6
G6pc Pck1 Pdk4 Pklr Gck
ND-WT
HFD-WT
HFD-KOR
elat
ive
gene
exp
ress
ion
glucose metabolism genes
#
#
#
*
E
Tnf Emr1 Itgax Ccl5 Ccl2
ND-WT
HFD-WT
HFD-KO
0
1
2
3
4
5
Rel
ativ
e ge
ne e
xpre
ssio
n
inflammatory genes and macrophage markers
*
#
##
#
#
*
*
*
F
Figure 6. PTG Ablation Reduces Hepatic Steatosis in vivo after Long Term HFD.
Page 36 of 49
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PTG
RalA
pGSS6K
S6pS6
pS6K
GS
m-SREBP1
WT KOWT KOND HFD
GS
WCL
AMPD
CREBN.E.
A
Figure 7. PTG Ablation Improves Glucose Tolerance and Insulin Sensitivity in vivo afterLong Term HFD.
0
1
2
3
4
5
6
Fast
ing
insu
lin (n
g/m
l)
020406080
100120140160180200
ND HFD ND HFD
Fast
ing
gluc
ose
(mg/
dL)
% o
f bas
al b
lood
glu
cose
0
50
100
150
200
250
300
0 30 60 90 1200 30 60 90 120 0 30 60 90 120
HFD-WTHFD-KO
ND-WTND-KO
HFD-WTHFD-KO
% o
f bas
al b
lood
glu
cose 120
100
80
60
40
20
0
120
100
80
60
40
20
0
Time after injection (min) Time after injection (min) Time after injection (min)B
lood
glu
cose
(mg/
dL)
PTTITT ITT
B
C D
E *G
S in
AMPD
PTG
in A
MP
D **
WTKO
*
pS6K
/S6K
m-S
RE
BP
1
0
1
2
3
0
1.0
2.0 * *
0
1
2
3
4
ND HFD
* *
0
1
2
3 * *
ND HFD
0.5
1.5
050
100150200250300350400
0 30 60 90 1200
100
200
300
400
500
600
ND-WTND-KO
HFD-WTHFD-KOB
lood
glu
cose
(mg/
dL)
Time after injection (min)
Blo
od g
luco
se (m
g/dL
)
0 30 60 90 120Time after injection (min)
OGTT OGTT* * * *
** * *
**
* *
* * * * * *
WTKO
*
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C
WT KO
mTORC1
HFD
Glycogenesis
FASNSCD1
LDLRHMGCR
SREBP1
Lipogenesis
Glycogen
PTG
TG
GS
0
5
10
15
20
25
30
sterol
meta
bolic
proc
ess
steroi
d meta
bolic
proc
ess
chole
sterol
meta
bolic
proc
ess
sterol
bios
ynthe
tic pr
oces
s
chole
sterol
bios
ynthe
tic pr
oces
s
steroi
d bios
ynthe
tic pr
oces
s
oxida
tion r
educ
tion
lipid
biosy
ntheti
c proc
ess
acyl-
CoA m
etabo
lic pr
oces
s
isopre
noid
biosy
ntheti
c proc
ess
cofac
tor m
etabo
lic pr
oces
s
fatty
acid
metabo
lic pr
oces
s
Fold
enr
ichm
ent
GO Term
A
D
> 3 2 1 0
-1 -2
> -3
Fold change?
?
?
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
ND HFD
Rel
ativ
e m
RN
ASrebf1
0.00.20.40.60.81.01.21.41.61.82.0
ND HFDR
elat
ive
mR
NA
Fasn
0.00.20.40.60.81.01.21.41.61.8
ND HFD
Rel
ativ
e m
RN
A
Ldlr
0.00.20.40.60.81.01.21.41.6
ND HFD
Rel
ativ
e m
RN
A
Hmgcr
P=0.06
WTKO
B
Glucose Insulin
Figure 8. A PTG-SREBP1/mTORC1 Feedback Mechanism Regulates Lipogenesis
Page 38 of 49
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SUPPLEMENTARY DATA
Supplementary Figure Legends
Supplementary Figure 1. Metabolic phenotypes of C57BL/6J mice fed normal chow
diet (ND) or high fat diet (HFD).
Male mice aged 8-10 weeks were put on HFD for 12 weeks (n=8 each group). (A)
Growth curve. (B) Fasting glucose level. (C) Fasting insulin level. (D) Glucose tolerance
test. (E) Insulin tolerance test. (F) Representative Western blots showing pAKT (S473)
levels from liver lysates of ND and HFD mice sacrificed after 16hrs fasting followed by
6hrs refeeding. (G) Representative Western blots showing pAKT (S473) levels from liver
lysates of ND and HFD mice fasted overnight and sacrificed 20 min after intraperitoneal
injections of insulin (2U/kg body weight). (H) Quantitative real-time PCR showing
hepatic gluconeogenic gene expression.
Values are expressed as mean ± SE.*indicates statistical significance by student t-
test (*P<0.05).
Supplementary Figure 2. Glycogen synthesis is similarly regulated in MEFs, 293
and liver cell lines.
Quantitative real-time PCR showing GL (A) and GM (B) gene expression in TSC: PTG
knockdown MEFs.
(C) Glycogen content in 293A or Hepa1c cells transiently overexpressing WT PTG or
PTG mutants incompetent to bind to GS or PP1.
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Supplementary Figure 3. PTG Ablation Results in Decreased Glycogen and Lipid
levels in vivo after Short-term HFD without Changing Glucose and Insulin
Sensitivity.
(A) Genotyping of transgenic mouse showing homozygous deletion of the PTG gene.
(B) PTG homozygous knockout mouse has improved survival rate at birth with more
backcross. Heterozygous PTG knockout mice maintained on a mixed background were
sequentially backcrossed with C57BL/6J mice and birth ratio of homozygous female,
male, and total PTG knockout mice were calculated from genotyping of over 200 pups
generated from heterozygous breeding pairs at each backcross generation. *indicates
statistical significance by CHITEST relative to expected Mendelian ratio of 25% each
group (*P<0.05).
(C) Growth curves and (D) food intake for wild type (WT) or PTG knockout (KO) mice.
Mice were fed a normal chow diet (ND) for 8 weeks before a high fat diet (HFD) feeding
of 24 weeks. Body weights were measured every other week. Food intake was measured
for consecutively three weeks (n=8-10 mice each group).
(E) Glycogen content and (F) TG levels in the liver of WT and PTG KO mice on HFD
for 12 weeks and fasted overnight (n=7-10 mice each group).
(G) Quantitative real time PCR showing relative mRNA expression level of PTG
(Ppp1r3c), SREBP1 (Srebf1) and GL (Ppp1r3c) in the same mice as in (E) and (F).
(H) Oral glucose tolerance test and (I) Insulin tolerance test of WT and PTG KO mice on
HFD for 12 weeks.
Supplementary Figure 4. GS and GP activity is not changed in PTG KO mice.
Page 40 of 49
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(A) Hepatic GS activity (-/+ G6P) and (B) GP activity (-/+AMP) from ND and HFD
PTG mice sacrificed after 16hrs fasting (n=8-10 mice each group).
Page 41 of 49
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0
50
100
150
200
250
HFD
Fasting glucose (mg/dL)
Fasting insulin (ng/ml)
0
100
200
300
400
500
600
0 15 30 45 60 75 90 105 120
Glucose (mg/dL)
HFD
ND
ND HFDND
Glucose (% of basal)
0
20
40
60
80
100
120
0 15 30 45 60 75 90 105 120
HFD
ND
Fasted Refed Fasted Refed
ND HFD
- - + + + - - + + +
ND HFD
insulin
20
22
24
26
28
30
32
34
36
38
40
0 2 4 6 8 10 12
Body Weight (g)
Weeks on HFD
ND
HFD
A B C
0
0.5
1.0
1.5
2.0
2.5
3.0
D E
F G
AKT
pAKT
(S473)
AKT
pAKT
(S473)
* ***
* *
*
*
*
* **
*
*
*
**
Time after injection (min) Time after injection (min)
0.2
0.4
0.6
0.8
1.0
1.2
Fasted Refed
Relative mRNA
PEPCK ND HFD
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Fasted Refed
Relative mRNA
G6PC
0
0.5
1.0
1.5
2.0
2.5
Fasted Refed
Relative mRNA
PDK4
0
H
Suppementary Figure 1. Metabolic phenotypes of C57BL/6J mice fed normal chow diet (ND) or high fat diet (HFD).
Page 42 of 49
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A
B
PTG: WT KO Het
PTG KO mouse ratio (%)
0
25
50
BC1 BC2 BC3
FemaleMaleTotal
300 bp D
* * *
200 bp
C
0
1
2
3
4
ND HFD
Food intake
WTKO
(g/mouse/day)
Body weight (g)
0 2 4 6 8 10 12 1416 18 20 22 24
60
50
40
30
20
10
0
HFD-WT
HFD-KO
ND-WT
ND-KO
Weeks on HFD
0
5
10
15
20
25
30
35
ND HFD
E
**
***
glycogen content
(µmol glucose/g tissue)
0
50
100
150
200
250
ND HFD
TG (µmol glycerol/g tissue)
**** *
050100150200250300350400450
Glucose (mg/dL)
WTKO
HFD
0
50
100
150
200
250
300
0 15 30 45 60 75 90 105 120
Glucose (mg/dL)
Time after injection (min)
WTKO
ND
0 15 30 45 60 75 90 105 120Time after injection (min)
0
20
40
60
80
100
120
0 15 30 45 60 75 90 105 120
WTKO
ND
Glucose (% of basal)
Time after injection (min)
0
20
40
60
80
100
120
Glucose (% of basal)
WT
KO
HFD
0 15 30 45 60 75 90 105 120Time after injection (min)
F G
H
Supplementary Figure 3. PTG Ablation Results in Decreased Glycogen and Lipid levels in vivo after
Short-term HFD without Changing Glucose and Insulin Sensitivity .
0.0
0.5
1.0
1.5
2.0
2.5
3.0
ND HFD
Relative mRNA
Ppp1r3c
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
ND HFD
Relative mRNA
Srebf1* *
*
WT
KO
0.0
0.5
1.0
1.5
2.0
2.5
ND HFD
Relative mRNA
Ppp1r3b**
WTKO
WTKO
I
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Supplementary Table 1. List of Real Time PCR Primers
*Triplicates of each sample were prepared and normalized to RPLP0 (primer1) to
determine relative expression levels. In the case of TSC2 knockout MEFs, we observed
significant changes in the levels using this primer so we used RPLP0 (primer2) for
normalization.
Name Gene Symbol Primer Sequence
FAS Fasn F: 5'-GGAGGTGGTGATAGCCGGTAT-3'
R: 5'-TGGGTAATCCATAGAGCCCAG-3'
HMGCR Hmgcr F: 5’- AGCTTGCCCGAATTGTATGTG-3’
R: 5’-TCTGTTGTGAACCATGTGACTTC -3’
LDLR Ldlr F: 5’- AGTGGCCCCGAATCATTGAC-3’
R: 5’-CTAACTAAACACCAGACAGAGGC -3’
PTG Ppp1r3c F: 5’-TGATCCATGTGCTAGATCCACG-3’
R: 5’-ACTCTGCGATTTGGCTTCCTG-3’
GYS1 Gys1 F: 5’-CGCTGGAAGGGTGAGCTTT-3’
R: 5’-GAAGTGGGCAACCACATACG-3’
GYS2 Gys2 F: 5’- ACCAAGGCCAAAACGACAG-3’
R: 5’-GGGCTCACATTGTTCTACTTGA-3’
GM Ppp1r3a F: 5’-GCTTCCCGGAGAGTTTCCTTT-3’
R: 5’-CACGGCTTTCTGGACTTGGA-3’
GL Ppp1r3b F: 5’-GTGGACATCCAATACAGCTACAG-3’
R: 5’- CCGAGAACACTTTCACCATTGT-3’
PEPCK Pck1 F: 5'-CTGCATAACGGTCTGGACTTC-3'
R: 5’-CAGCAACTGCCCGTACTCC-3’
PDK4 Pdk4 F: 5'-GGAGTGTTCACTAAGCGGTCA-3'
R: 5'-AGGGAGGTCGAGCTGTTCTC-3'
PKLR Pklr 5'-TCAAGGCAGGGATGAACATTG-3'
5'-CACGGGTCTGTAGCTGAGTG-3'
GCK Gck F: 5'-ATGGCTGTGGATACTACAAGGA-3'
R: 5'-TTCAGGCCACGGTCCATCT-3'
TNFa Tnf F: 5'-ACGGCATGGATCTCAAAGAC-3'
R: 5'-AGATAGCAAATCGGCTGACG-3'
F4/80 Emr1 F: 5'-CTGGGATCCTACAGCTGCTC-3'
R: 5'-AGGAGCCTGGTACATTGGTG-3'
CD11C Itgax F: 5'-CTGGATAGCCTTTCTTCTGCTG-3'
R: 5'-GCACACTGTGTCCGAACTCA-3'
Rantes Ccl5 F: 5'-GCTGCTTTGCCTACCTCTCC-3'
R: 5'-TCGAGTGACAAACACGACTGC-3'
MCP-1 Ccl2 F: 5'-TTAAAAACCTGGATCGGAACCAA-3' R: 5'-GCATTAGCTTCAGATTTACGGGT-3'
Page 46 of 49
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Supplementary Table 2. List of Genes Shown on the Heatmap from Figure 8C.
Gene Symbols Cyp7a1
Hmox1
Aco2
Apoa2
Mttp
Apoa5
Elovl3
Pal2g6
Irs2
Tm7sf2
Nr0b2
Pck1
Fdps
Insig1
Aldoc
Idi1
Sqle
Acss2
Acat2
Ebp
Fads1
Idh1
Dbi
Pmvk
Pgd
Ggps1
Pcyt1a
Ucp2
Acly
Ldlr
Fasn
Nss
Nsdhl
Mid1ip1
Cyb5b
Hsd17b7
Dhcr24
Fdft1
Tmem97
Rdh11
Srebf2
Dhcr7
Mvk
Thrsp
Mmab
Hmgcr
Pfkfb1
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Supplementary Method
Enzyme activity assay. GS activity was determined by measuring incorporation of 14C-
glucose from UDP- 14C-glucose into glycogen in the absence or presence of G6P as
described previously (5). GP activity was assayed by measuring incorporation of 14C-
glucose from 14C-glucose-1-phosphate into glycogen in the absence or presence of AMP
(6). Activity ratios represent the activity measured in the absence of the allosteric effector
(G6P for GS or AMP for GP) divided by that in the presence of the allosteric effector.
Primary hepatocyte isolation. Primary hepatocyte isolation and cultures were carried
out as previously described (7). Cells were starved in serum-free media for 3 hr prior to
the incubation with 14C-glucose for 1hr. Glucose conversion into glycogen, lipid and
CO2 assay was performed as described in (8,9). Values are normalized to the protein
content of hepatocytes and averaged from three independent experiments using primary
hepatocytes isolated from 3 WT and 4 PTG KO mice on ND individually.
Euglycemic hyperinsulinemic clamp experiments. Euglycemic hyperinsulinemic
studies were performed by the Animal Phenotyping Core of the Nutrition Obesity
Research Center from University of Michigan, as previously described (10). Liver
glycogen and lipids were extracted and 3H counts were calculated for in vivo glucose
incorporation experiment.
Supplementary References
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Diabetes
1. Crosson SM, Khan A, Printen J, Pessin JE, Saltiel AR. PTG gene deletion causes
impaired glycogen synthesis and. Journal of Clinical Investigation.
2003;111(9):1423–32.
2. Cheng A, Zhang M, Crosson SM, Bao ZQ, Saltiel AR. Regulation of the mouse
protein targeting to glycogen (PTG) promoter by the FoxA2 forkhead protein and
by 3’,5'-cyclic adenosine 5'-monophosphate in H4IIE hepatoma cells.
Endocrinology. 2006 Jul 1;147(7):3606–12.
3. Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, Bajraszewski N, et al. Loss
of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through
downregulation of PDGFR. The Journal of clinical investigation. 2003
Oct;112(8):1223–33.
4. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and
tissue-specific expression of transgenes delivered by lentiviral vectors. Science
(New York, N.Y.). 2002 Feb 1;295(5556):868–72.
5. Thomas JA, Schlender KK, Larner J. A rapid filter paper assay for UDPglucose-
glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-
glucose. Analytical biochemistry. 1968 Oct 24;25(1):486–99.
6. Gilboe DP, Larson KL, Nuttall FQ. Radioactive method for the assay of glycogen
phosphorylases. Analytical biochemistry. 1972 May;47(1):20–7.
7. Liu C, Li S, Liu T, Borjigin J, Lin JD. Transcriptional coactivator PGC-1alpha
integrates the mammalian clock and energy metabolism. Nature. 2007 May
24;447(7143):477–81.
8. Greenberg CC, Meredith KN, Yan L, Brady MJ. Protein targeting to glycogen
overexpression results in the specific enhancement of glycogen storage in 3T3-L1
adipocytes. The Journal of biological chemistry. 2003 Aug 15;278(33):30835–42.
9. Meng ZX, Yin Y, Lv JH, Sha M, Lin Y, Gao L, et al. Aberrant activation of liver
X receptors impairs pancreatic beta cell function through upregulation of sterol
regulatory element-binding protein 1c in mouse islets and rodent cell lines.
Diabetologia. 2012 Jun;55(6):1733–44.
10. Bracy DP, Ayala JE, Malabanan C, James FD, Ansari T, Fueger PT, et al.
Hyperinsulinemic-euglycemic clamps in conscious, unrestrained mice. Journal of
visualized experiments. 2011 Jan;(57).
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