Research Article Fomitopsis pinicola Jeseng Formulation ...downloads.hindawi.com/journals/ecam/2016/7312472.pdfsenticosus ,Allium tuberosum, and Viscum album coloratum ) at a ratio

Research ArticleA Fomitopsis pinicola Jeseng Formulation Hasan Antiobesity Effect and Protects against Hepatic Steatosis inMice with High-Fat Diet-Induced Obesity

Hoe-Yune Jung123 Yosep Ji4 Na-Ri Kim1 Do-Young Kim1

Kyong-Tai Kim2 and Bo-Hwa Choi1

1Pohang Center for Evaluation of Biomaterials Pohang Technopark Pohang 37668 Republic of Korea2Department of Life Science Division of Integrative Biosciences and Biotechnology POSTECH Pohang 37673 Republic of Korea3RampD Center NovMetaPharma Co Ltd Pohang 37668 Republic of Korea4School of Life Science Handong Global University Pohang 37554 Republic of Korea

Correspondence should be addressed to Bo-Hwa Choi bhchoipohangtporg

Received 1 January 2016 Revised 23 March 2016 Accepted 31 March 2016

Academic Editor Menaka C Thounaojam

Copyright copy 2016 Hoe-Yune Jung et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study investigated the antiobesity effect of an extract of the Fomitopsis pinicola Jeseng-containing formulation (FAVA) whichis a combination of four natural components Fomitopsis pinicola Jeseng Acanthopanax senticosus Viscum album coloratum andAllium tuberosum High-fat diet- (HFD-) fedmale C57BL6J mice were treated with FAVA (200mgkgday) for 12 weeks tomonitorthe antiobesity effect and amelioration of nonalcoholic fatty liver diseases (NAFLD) Body and white adipose tissue (WAT) weightswere reduced in FAVA-treated mice and a histological examination showed an amelioration of fatty liver in FAVA-treated micewithout decreasing food consumption Additionally FAVA reduced serum lipid profiles leptin and insulin levels comparedwith theHFD control groupThe FAVA extract suppressed lipogenic mRNA expression levels fromWAT concomitantly with the cholesterolbiosynthesis level in the liver These results demonstrate the inhibitory effects of FAVA on obesity and NAFLD in the diet-inducedobese (DIO) mouse model Therefore FAVAmay be an effective therapeutic candidate for treating obesity and fatty liver caused bya high-fat diet

1 Introduction

There is increasing consensus that obesity may be the maincause of various metabolic disorders Obesity is caused bythe combined effects of excess energy intake and reducedenergy expenditure It is one of the fastest growing disordersworldwide and is associated with various clinical symptomsin developed countries [1] such as hyperlipidemia insulinresistance and nonalcoholic fatty liver diseases (NAFLD) [2]It is well known that excessive fat consumption is implicatedin the development of obesity in mice [3] and long-termfeeding with a high-fat diet (HFD) can induce obesitytogether with hyperlipidemia insulin resistance andNAFLD[4] Hyperlipidemia is associated with high levels of lipidsand lipoproteins in the blood and causes atherosclerosis andacute pancreatitis [5] AlthoughNAFLD is the second leading

cause of death in the general population [6 7] there is nopharmacological agent known to reverse NAFLD Recentlyeffective medical interventions have been focused on themodification of risk factors such as diet andweight reduction[8]

Adipose tissue an important repository for energy stor-age regulates energy homeostasis Adipogenesis a differ-entiation process of adipocytes involves changes in geneexpression and cellular morphology Adipocyte hypertrophyresults from an excessive accumulation of lipids from theintake of inordinate energy sources such as HFD Duringadipogenesis peroxisome proliferator-activated receptor-120574(PPAR-120574) and CCAATenhancer-binding protein-120572 (CEBP-120572) play key roles as major transcriptional factors [9] Expres-sion of PPAR-120574 a transcription factor of the nuclear-receptorsuperfamily and CEBP-120572 a member of the CEBP family of

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 7312472 10 pageshttpdxdoiorg10115520167312472

2 Evidence-Based Complementary and Alternative Medicine

basic leucine zipper class of transcription factors increasesduring 3T3-L1 cell differentiation [10] Lipin is also a centralregulator of adipose tissue development Mammalian lipinproteins have been shown to control gene expression andto enzymatically convert phosphatidate into diacylglycerolan essential precursor in triacylglycerol and phospholipidsynthesis [11] Previous studies have established that lipin-1is required at an early step in adipocyte differentiation forthe induction of the adipogenic gene transcription programincluding the key regulator PPAR-120574 [12]

Acetoacetyl-CoA synthetase (AACS) regulation is relatedto cholesterol and lipid homeostasis [13] Sterol responseelement binding protein-2 (SREBP-2) may play an essentialrole in the transcriptional regulation of AACS SREBP-2 is a leucine zipper transcription factor that controls arate-limiting enzyme in cholesterol synthesis HMG-CoAreductase (HMGCR) when the factor binds sterol responseelement [14 15]

The effects ofAcanthopanax senticosusAllium tuberosumandViscum album coloratum have been studied for inhibitionof fatty acid synthase and prevention of obesity as wellas reducing hepatic steatosis [16ndash18] Fomitopsis pinicolaJeseng has been reported for antihyperglycemic effect indiabetic rats [19] In addition it has been reported that 120573-glucan-rich extract amajor component of Fomitopsis pinicolaJeseng effectively reduces adiposity [19 20] However to ourknowledge no reports are available on the effect of Fomitopsispinicola Jeseng on obesity

In this study we investigated the effect of FAVA in a high-fat diet-induced mouse modelThe FAVA is a combination ofherbal extracts (ie Fomitopsis pinicola JesengAcanthopanaxsenticosus Allium tuberosum and Viscum album coloratum)at a ratio of 5 3 1 1 This study investigated the effectof a mixture containing dietary components on metabolicdisorders including obesity hyperlipidemia and NAFLDusing a high-fat diet-induced obesity mouse model and themolecular mechanism level of adipogenesis and cholesterolbiosynthesis Our results indicate the great potential ofFAVA as a potential metabolic regulator of adipogenesis andcholesterol biosynthesis and as a potential therapeutic agentfor preventing or treating obesity and NAFLD

2 Materials and Methods

21 Preparation of FAVA The oriental medicinal and herbalmixture used in this experiment (FAVA) was prepared asdescribed previously [21ndash23] Briefly Acanthopanax sentico-sus and Allium tuberosumwere extracted with 80methanolwhile water extracted Fomitopsis pinicola Jeseng and Viscumalbum coloratum were purchased from commercial vendor(Mistle Biotech Co Ltd Korea) Each extraction of FAVAwas resuspended in distilled water (DW) in a ratio of 5030 10 and 10 respectively and prepared in appropriatediluent for further in vivo study

22 Animals and Diets Lean male C57BL6J mice (7weeks old) were purchased from Charles River LaboratoriesJapan Inc (Yokohama Japan) All animal experiments wereapproved by the Ethics Review Committee of the Pohang

Center for the Evaluation of Biomaterials Republic of KoreaAll mice were housed for 1 week under a 1212-h lightdarkcycle in a temperature- (22 plusmn 1∘C) and humidity- (55 plusmn 5)controlled room and fed standard laboratory chow and waterad libitum while FAVA orlistat and saline supplementationwere performed using oral gavage once a day To induceobesity the mice were fed a HFD (Rodent Diet D12492Research Diet New Brunswick NJ USA) consisting of60 kcal fat Control mice were fed a low-fat chow diet(Rodent Diet D12450B Research Diet New Brunswick NJUSA) consisting of 10 kcal fat Experimental mice weregiven FAVA or orlistat as a positive control (Chongqing ZeinPharmaceutical Co Ltd Chongqing China)Themice wererandomly divided into four groups (119899 = 8 per group) thatwere fed a low-fat chow diet (CHOW) a high-fat diet (HFD)HFD plus FAVA (200mgkgday) or HFD plus orlistat(60mgkgday) Animals were fed via oral feeding needlesfor 12 weeks and the CHOW and HFD group receivedan equivalent volume of saline Body weight was measuredonce a week and food intake was measured three times perweek during the course of the study At the conclusion ofthe in vivo experiment the mice were sacrificed by cervicalvertebral dislocation and the epididymal mesenteric andsubcutaneous fat pads and liver were collected and weighedThe epididymal fat pad samples were stored at minus80∘C untilanalysis

23 Serum Analysis Serum was collected by cardiac punc-ture stored for 20 minutes at room temperature for coagu-lation and then separated by centrifugation at 2000timesg for20 minutes The serum was stored at minus70∘C until analysisThe levels of triglycerides total cholesterol high-densitylipoprotein (HDL) cholesterol low-density lipoprotein (LDL)cholesterol glucose alanine transaminase (ALT) aspartatetransaminase (AST) BUN and creatinine in serum weremeasured by using an automated biochemical analyzer (BS-390 Mindray Bio-Medical Electronics Co Ltd China)

24 Measurement of Leptin and Insulin The leptin andinsulin concentrations in serumwere determined by amouseenzyme-linked immunosorbent assay (ELISA) kit (MorinagaInstitute of Biological Science Yokohama Japan)The prepa-ration of serum samples is described above

25 Abdominal Computed Tomography Analysis Experi-ments of micro-computed tomography (micro-CT) wereperformed with an animal positron emission tomography(PET)CTsingle photon emission computed tomography(SPECT) system (Inveon Siemens USA) prior to the sac-rifice of animals under 15ndash2 isoflurane in O

2anesthesia

Computed tomography pictures were further analyzed usingSiemens Inveon software to calculate the three-dimensionalvolume of the fat mass between lumbar vertebrae one to five

26 Liver Histology Liver tissues were immediately isolatedafter sacrifice For hematoxylin and eosin (HampE) staining thetissues were fixed in 10 formalin processed and embeddedin paraffin prior to sectioning (10 120583m) and staining The liver

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primers used in the reverse transcriptase-polymerase chain reaction analysis

Gene name Accession number Sequence

Lipin-1 NM 172950 Forward 51015840-TCA GAC ACT TTC AGT AAC TTC AC-31015840

Reverse 51015840-TAT CAG CCT TCC CAG CAG-31015840

CEBP-120572 NM 007678 Forward 51015840-CGT CTA AGA TGA GGG AGT C-31015840

Reverse 51015840-GGC ACA AGG TTA CTT CCT-31015840

PPAR-120574 NM 001127330 Forward 51015840-GAA AGA CAA CGG ACA AAT CAC-31015840

Reverse 51015840-GAA ACT GGC ACC CTT GAA-31015840

AACS NM 030210 Forward 51015840-AAG CCC AGA GTT ACG AGT AT-31015840

Reverse 51015840-ACA CAG GAA TAG AGG AGT TCT-31015840

HMGCR NM 008255 Forward 51015840-AGA ATA ATG TGC TAA GTA GTG CTA A-31015840

Reverse 51015840-GCC TCT CTG AAC AAA GAC TC-31015840

SREBP-2 NM 033218 Forward 51015840-GCG ACC AGG AAG AAG AGA-31015840

Reverse 51015840-ACA AAT CCC ACA GAG TCC A-31015840

120573-actin NM 007393 Forward 51015840-GGG AAG GTG ACA GCA TTG-31015840

Reverse 51015840-ATG AAG TAT TAA GGC GGA AGA TT-31015840

CEBP-120572 CCAATenhancer-binding protein-120572 PPAR-120574 peroxisome proliferator-activated receptor-120574 AACS acetoacetyl-CoA synthetase HMGCR HMG-CoA reductase and SREBP-2 sterol regulatory element binding protein-2

samples of 3 mice from each group (CHOW HFD HFD+ FAVA and HFD + ORLISTAT) were measured Brieflythe following criteria were used for scoring hepatic steatosisgrade 0 (no fatty liver) and grade 1 (mild fatty liver) ifhepatocytes occupied lt33 of the hepatic parenchyma [24]

27 RNA Preparation and Real-Time PCR Total RNAwas extracted by ReliaPrep RNA Tissue Miniprep System(Promega) according to the manufacturerrsquos instructionsRNA integrity was assessed by an automated microfluidics-based system (Bioanalyzer 2100 Agilent Palo Alto CAUSA) First-strand cDNA was synthesized with the iScriptcDNA Synthesis Kit (Bio-Rad Hercules CA USA) and real-time PCR was performed using an iCycler iQ Real-TimeDetection System (Bio-Rad) PCR reactions were conductedwith iQ SYBR Green Supermix (Bio-Rad) Real-time PCRanalysis was performed using an iCycler iQ Real-TimeDetection System (Bio-Rad) Amplification of real-time PCRwas performed according to the protocols of Jung et al [25]The reaction was performed at 95∘C for 3min followed by 39cycles of amplification (95∘C for 10 s 58∘C for 10 s and 72∘Cfor 30 s) A melting curve was produced to confirm a singlegene-specific peak and detect primerdimer formation byheating the samples from 65 to 95∘C in 05∘C increments witha dwell time at each temperature of 10 s while continuouslymonitoring fluorescence The mRNA levels of specific geneswere normalized to those of 120573-actin The primers used arelisted in Table 1

28 Statistical Analyses The data (mean plusmn SE) were analyzedusing GraphPad Prism (version 504 GraphPad SoftwareUSA) Unpaired two-tailed Studentrsquos 119905-tests were used toevaluate differences betweenmeans as indicated and 119901 valueslt 005 were considered significant

3 Results

31 Effects of FAVA on Body Weight Dietary Intake andFat Mass in White Adipose Tissue in HFD-Fed Mice Theeffects of FAVA on body weights are shown in Figure 1(a)During the 12-week experiment body weight was measuredweekly and food intake was measured every other day After9 weeks the body weight of the mice in the HFD groupwas significantly higher than that of the mice in the CHOWgroup (119901 lt 00005) The FAVA-treated group showed asignificant decrease in body weight compared with the HFDgroup At the end of the experiment the body weight ofthe mice fed FAVA was 97 plusmn 20 lower (119901 lt 005) thanthat of the mice in the HFD group whereas HF diet plusorlistat-fed mice weighed almost the same as the mice thatwere fed FAVA (Figure 1(a)) These effects of FAVA on bodyweight were not due to decreased food intake because theamount of kcal consumed per mouse over a 24-h periodremained unchanged (Figure 1(b)) These data indicate thatFAVAmight have antiobesity effects in vivo without affectingfood intake To investigate whether body weight loss wascaused by decreased adiposity the animals were sacrificedand the epididymal fat pad the mesenteric fat pad and thesubcutaneous fat pad were dissected and weighed FAVAsupplementation significantly suppressed the increase of fatmass in all white adipose tissues including mesenteric sub-cutaneous and epididymal adipose tissue (Figures 1(c)ndash1(e))

32 Effect of FAVA on Adiposity in HFD-Fed Mice Weperformed micro-CT imaging to assess the effect of FAVAon adiposity CT imaging showed a significant reduction inbody fat profiles with FAVA treatment (Figure 2(a)) Therewas a significant reduction in fat volume (Figure 2(b)) andtotal body fat percentage in FAVA-fed groups compared withthe HFD group (Figures 2(b) and 2(c))


0 1 2 3 4 5 6 7 8 9 10 11 1220

22

24

26

28

30

32

34

36

CHOWHFD

HFD + FAVAHFD + ORLISTAT

Time of treatments (week)

Body

wei

ght (

g)

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowastlowastlowastlowast

(a)

CHOW HFD FAVA ORLISTAT0

5

10

15

Food

inta

ke (k

cald

aym

ouse

)

CHOWHFD

(b)


500

1000

1500

2000

2500

Epid

idym

al fa

t pad

(mg)

CHOWHFD

lowast

(c)

0

200

400

600

800

CHOWHFD

Mes

ente

ric fa

t pad

(mg)

CHOW HFD FAVA ORLISTAT

lowastlowast

(d)


500

1000

1500

2000

2500

Subc

utan

eous

fat p

ad (m

g)

CHOWHFD

lowast

lowast

(e)

Figure 1 Effect of FAVA on body weight food intake and white fat pad in mice fed a high-fat diet for 12 weeks (a) Changes in body weightgain at each treatment period are shown (circle) CHOW chow diet (black triangle) HFD high-fat diet (red triangle) HFD + ORLISTAThigh-fat diet plus orlistat 60mgkg and (green triangle) HFD + FAVA high-fat diet plus FAVA 200mgkg (b) Average food intake expressedas kcalmouseday ((c)ndash(e)) Epididymal fat pad (c) mesenteric fat pad (d) and subcutaneous fat pad (e) weights expressed The valuesrepresent the mean plusmn standard error of mean (SEM) (119901 lt 005 and 119901 lt 0005 versus the CHOW group lowast119901 lt 005 lowastlowast119901 lt 0005 andlowastlowastlowast119901 lt 00005 versus the HFD group 119899 = 8 per group)


CHOW HFD

HFD + ORLISTAT HFD + FAVA

(a)


2000

4000

6000

8000

Fat v

olum

e (m

m3)

lowast

CHOWHFD

(b)


20

40

60

Fat m

ass

tota

l bod

y vo

lum

e (

)

lowast

CHOWHFD

(c)

Figure 2 Effect of FAVA on high-fat diet-induced adiposity (a) Micro-computed tomography (micro-CT) pictures were analyzed usingSiemens Inveon software to calculate the three-dimensional volume of the fat mass between vertebrae number one to five of mice fed a chowdiet (CHOW) high-fat diet (HFD)HFDwith 60mgkgday orlistat (ORLISTAT) orHFDwith 200mgkgday FAVA (FAVA) (b) Fat volumes(mm3) in mice are shown (c) Fat pad mass expressed as percentage of total body weight The values represent the mean plusmn SEM (119901 lt 005versus the CHOW group lowast119901 lt 005 versus the HFD group 119899 = 3)

33 Effects of FAVA on Serum Insulin Leptin and LipidProfiles in the Serum of HFD-Fed Mice The changes in theblood plasma parameters are shown in Figures 3(a)ndash3(e) Asshown in Figures 3(a) and 3(b) HFD-induced obese miceshowed significantly higher levels of serum insulin and leptinwhereas the FAVA group showed significantly decreasedlevels of serum insulin and leptin by 609 plusmn 81 and404 plusmn 30 respectively Concomitant reductions of seruminsulin and leptin levels were monitored in the orlistat groupin a similar manner Additionally the FAVA group showedlower levels of serum total cholesterol and the ratio of LDLcholesteroltotal cholesterol than those of the HFD group

34 Effects of FAVA onmRNALevels of Transcriptional Factorsin Epididymal Fat Pad Because FAVA extract reduced fatmass in all white adipose tissues and serum insulin levels(Figures 1 and 3) we evaluated the effect of FAVA on the

expression of various adipogenic and lipogenic genes [26]PPAR120574 and CEBP120572 are known to have roles in insulinsensitivity lipogenesis and lipolysis [27] Lipin-1 is alsothought to regulate the transcription of genes involved inadipocyte differentiation and fat synthesis and storage [28]To investigate the antiadipogenic mechanism the effectsof FAVA on mRNA expression levels of PPAR120574 CEBP120572and lipin-1 were determined in the epididymal fat pad Theexpression of both adipogenic genes PPAR120574 and CEBP120572was significantly decreased by FAVA (Figures 4(b) and 4(c))Additionally FAVA significantly suppressed lipin-1 expres-sion by 956plusmn05 compared with that of theHFD group andwas greater than that of the orlistat group as a positive control

35 Effects of FAVA onHepatic Histology of HFD-Fed C57BL6Mice and mRNA Levels of Cholesterol Biosynthesis in LiverA common characteristic among people with obesity is



1

2

3

4In

sulin

(ng

mL)

lowastlowastlowastlowast

CHOWHFD

(a)


10

20

30

Lept

in (n

gm

L)

lowast

lowastlowastlowast

CHOWHFD

(b)


50

100

150

200

250

Tota

l cho

leste

rol (

mg

dL)

lowastlowastlowast

lowast

CHOWHFD

(c)


02

04

06

08

HD

L-C

tota

l-C (r

atio

)lowast

lowast

CHOWHFD

(d)


002

004

006

008

010

LDL-

Cto

tal-C

(rat

io)

lowast

p = 008

CHOWHFD

(e)


50

100

150

200

250

Glu

cose

(mg

dL)

lowastlowast

CHOWHFD

(f)

Figure 3 Effect of FAVA on serum insulin leptin and lipid profiles in mice fed a high-fat diet Changes in insulin (a) leptin (b) totalcholesterol (c) the ratio of HDL cholesteroltotal cholesterol (d) and the ratio of LDL cholesteroltotal cholesterol (e) of the mice weremeasuredThe values represent themean plusmn SEM (119901 lt 005 119901 lt 0005 and 119901 lt 00005 versus the CHOWgroup lowast119901 lt 005 lowastlowast119901 lt 0005and lowastlowastlowast119901 lt 00005 versus the HFD group 119899 = 5sim7 per group)



10

20

30

40Re

lativ

e mRN

A le

vels

(Lip

in-1

)

lowastlowast

lowast

CHOWHFD

(a)


20

40

60

80

Relat

ive m

RNA

leve

ls (C

EBP

-120572)

lowastlowast

lowastlowast

CHOWHFD

(b)


10

20

30

40

50

Relat

ive m

RNA

leve

ls (P

PAR-120574

)

lowastlowast

lowast

CHOWHFD

(c)

Figure 4 mRNA expressions of transcription factors in the epididymal fat pad of animals treated with HFD or HFD + ORLISTAT (high-fat diet plus orlistat 60mgkg) or FAVA (high-fat diet plus FAVA 200mgkg) or chow as quantified by real-time PCR The graphs representmRNA expression of transcription factors Lipin-1 (a) ACC and CEBP-120572 (b) and PPAR-120574 The data represent the mean plusmn SEM (119901 lt 005and 119901 lt 0005 versus the CHOW group lowast119901 lt 005 and lowastlowast119901 lt 0005 versus the HFD group 119899 = 5)

the development of fatty liver [29 30] Therefore we alsoanalyzed the effect of FAVA on fatty liver developmentHistological evaluation is regarded as the ldquogold standardrdquofor assessing the presence and severity of NAFLD [31] Wehistologically evaluated liver sections to determine the extentto which FAVA attenuated hepatic steatosis developmentAs shown in Figure 5(a) mild fatty liver was observed inmice that were fed a high-fat diet without FAVA Howevera marked reduction in the degree of steatosis was shown inlivers from high-fat diet mice treated with FAVA MoreoverFAVA treatment also decreased total serum cholesterol inmice to 137 plusmn 34 (Figure 3(c)) Therefore we investigatedwhether SREBP-2 AACS and HMGCR RNA in the mouseliver were induced by FAVA Total RNA was prepared frommouse livers and SREBP-2 AACS and HMGCR mRNAlevels were quantified using real-time PCR SREBP-2 AACSand HMGCR mRNA levels were dramatically suppressed inthe mice that were fed FAVA (Figures 5(b) and 5(c))

4 Discussion

Our study is the first to demonstrate that FAVA preventsweight gain in HFD-induced obesity in C57Bl6 mice Ourresults showed that body weight gain in groups fed adiet supplemented with FAVA was reduced compared withcontrol HFD mice (Figure 1(a)) Epididymal mesentericand subcutaneous fat pads in C57BL6 mice were signif-icantly reduced by FAVA supplementation (Figures 1(c)ndash1(e)) There was a significant reduction in subcutaneousand abdominal fat mass in FAVA-fed groups compared withthe HFD group (Figures 2(a)ndash2(c)) Subcutaneous fat andabdominal fat are the major types of white adipose tissueAbdominal obesity is associated with an increased risk ofcardiovascular diseases and insulin resistance [32]This studyalso provides evidence that dietary supplementation of FAVAprotects against hepatic steatosis development (Figure 5(a))We have considered the possibility that the effect of FAVA


CHOW


HFD

(a)


1

2

3

Relat

ive m

RNA

leve

ls (A

ACS)

lowast

CHOWHFD

(b)


2

4

6

Relat

ive m

RNA

leve

ls (H

MG

CR)

lowastlowast

CHOWHFD

(c)


1

2

3

4

5

Relat

ive m

RNA

leve

ls (S

REBP

-2)

lowastlowast

lowast

CHOWHFD

(d)

Figure 5 Effect of FAVA on hepatic steatosis and mRNA expressions of cholesterol biosynthesis in the liver of mice (a) Hematoxylin andeosin staining of liver from mice fed chow diet (CHOW) high-fat diet (HFD) or high-fat diet supplemented with orlistat at 60mgkgday(HFD + ORLISTAT) or high-fat diet supplemented with FAVA at 200mgkgday (HFD + FAVA) (40x magnification) ((b)ndash(d)) The graphsrepresent mRNA expression of cholesterol synthesis factors AACS (b) HMGCR (c) and SREBP2 (d) which was analyzed by real-time PCRThe data represent the mean plusmn SEM (119901 lt 005 and 119901 lt 0005 versus the CHOW group lowast119901 lt 005 and lowastlowast119901 lt 0005 versus the HFD group119899 = 5)


may bemediated through food intake because decreased foodintake would be expected to significantly affect body weightwhich influences hepatic steatosis In this study howeverthere was no difference in food intake-induced increaseof body weight between the FAVA-fed and non-FAVA-fedgroups (Figure 1(b)) This result suggests that FAVA directlyprotected against obesity and hepatic steatosis independentof food intake

Obesity is most likely to cause hyperlipidemia whichis considered the leading cardiovascular risk The hallmarkof dyslipidemia in obesity is hypertriglyceridemia in combi-nation with the preponderance of high LDL and low HDLcholesterol [33] This study shows that in high-fat diet-fedmice FAVA supplementation significantly reduced serumlevels of cholesterol and the ratio of LDL cholesteroltotalcholesterol (Figures 3(c) and 3(d)) Furthermore insulinlevels were increased in the HFD group and were decreasedsignificantly by FAVA supplementation (Figure 3(a)) In thecase of prediabetes increases of blood glucose stimulate thesecretion of insulin and subsequently induce hyperinsuline-mia to a normal blood glucose range Hyperinsulinemiawhich is a biomarker of insulin resistance is frequentlyaccompanied by obesity [34] Leptin is a fat-derived hormonethat plays an important role in appetite control and energyexpenditure [35] It has been reported that the concentrationof serum leptin is associated with general adiposity andreflects the body fat content [36] In this report it wasdemonstrated that FAVA treatment suppressed the plasmaleptin level inmice fedwithHFD (Figure 3(b))Moreover theweight of adipose tissues strongly correlated with the plasmaleptin level These results confirm that FAVA treatmentexerted an antiobesity effect in the diet-induced obesityC57BL6 mouse model

PPAR-120574 a transcription factor predominantly expressedin adipose tissue plays an essential role in adipocyte dif-ferentiation lipid storage and glucose homeostasis [37]Additionally adipogenesis is highly regulated by two primaryadipogenic transcription factors PPAR-120574 and CEBPs [38]Among those factors PPAR-120574 is well known as the keyregulator of adipogenic transcription [10] PPAR-120574 is alsoknown to bind to the CEBP-120572 promoter region that inducesthe expression of CEBP-120572 [39] CEBP-120572 is a promis-ing candidate transcription factor for directly controllingadipocyte differentiation [40] We found that FAVA signifi-cantly downregulated PPAR-120574 and CEBP-120572mRNA levels inthe epididymal fat pad This effect might be explained in twoways FAVA either inhibited PPAR-120574 and CEBP-120572 or sup-pressed the upstream molecules Lipin-1 is also required inadipocyte differentiation for the induction of the adipogenicgene transcription [12] We found that FAVA could inhibitadipocyte differentiation through the suppression of lipin-1

Acetoacetyl-CoA synthetase (AACS) can facilitate theincorporation of ketones into lipogenesis [13] Hasegawa etal [13] demonstrated that the AACS gene which encodesthe ketone body-utilizing enzyme is transcriptionally regu-lated by SREBP-2 and the knockdown of SREBP-2 induceddownregulation of AACS and HMGCR gene expressionAdditionally ketone body metabolism via AACS plays anessential role in cholesterol homeostasis In this study we

showed that the treatment of mice with FAVA resulted ina decrease of SREBP2 AACS and HMGCR mRNA levelsTherefore our results suggest that FAVA improves obesityhyperlipidemia and NAFLD and that FAVA treatment mightbe a promising adjuvant therapy in the management of thesemetabolic disorders

5 Conclusions

FAVA had a marked inhibitory effect on the developmentof obesity and NAFLD in a high-fat diet-induced obesitymousemodel Inhibiting transcription factors and adipocyte-specific lipogenic genes and decreasing cholesterol synthesisare two possible mechanisms for the antiobesity effect ofFAVA This study suggests that FAVA might be a potentialdietary supplement for preventing obesity and NAFLD

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

Thework was supported by Current Subsidies of Pohang city

References

[1] B M Spiegelman and J S Flier ldquoObesity and the regulation ofenergy balancerdquo Cell vol 104 no 4 pp 531ndash543 2001

[2] C Couillard PMauriege P Imbeault et al ldquoHyperleptinemia ismore closely associated with adipose cell hypertrophy thanwithadipose tissue hyperplasiardquo International Journal of Obesity vol24 no 6 pp 782ndash788 2000

[3] M Rebuffe-Scrive R Surwit M Feinglos C Kuhn and JRodin ldquoRegional fat distribution and metabolism in a newmouse model (C57BL6J) of non-insulin-dependent diabetesmellitusrdquoMetabolism vol 42 no 11 pp 1405ndash1409 1993

[4] R S Surwit C M Kuhn C Cochrane J A McCubbin andMN Feinglos ldquoDiet-induced type II diabetes in C57BL6J micerdquoDiabetes vol 37 no 9 pp 1163ndash1167 1988

[5] N Ewald P D Hardt and H-U Kloer ldquoSevere hypertriglyc-eridemia and pancreatitis presentation andmanagementrdquoCur-rent Opinion in Lipidology vol 20 no 6 pp 497ndash504 2009

[6] Z M Younossi A M Diehl and J P Ong ldquoNonalcoholic fattyliver disease an agenda for clinical researchrdquo Hepatology vol35 no 4 pp 746ndash752 2002

[7] A Franzese P Vajro A Argenziano et al ldquoLiver involvementin obese children ultrasonography and liver enzyme levelsat diagnosis and during follow-up in an Italian populationrdquoDigestiveDiseases and Sciences vol 42 no 7 pp 1428ndash1432 1997

[8] J Medina L I Fernandez-Salazar L Garcıa-Buey and RMoreno-Otero ldquoApproach to the pathogenesis and treatment ofnonalcoholic steatohepatitisrdquo Diabetes Care vol 27 no 8 pp2057ndash2066 2004

[9] R F Morrison and S R Farmer ldquoInsights into the transcrip-tional control of adipocyte differentiationrdquo Journal of CellularBiochemistry vol 76 supplement 33 pp 59ndash67 1999

[10] E D Rosen C J Walkey P Puigserver and B M SpiegelmanldquoTranscriptional regulation of adipogenesisrdquo Genes and Devel-opment vol 14 no 11 pp 1293ndash1307 2000


[11] R Ugrankar Y Liu J Provaznik S Schmitt and M LehmannldquoLipin is a central regulator of adipose tissue development andfunction in Drosophila melanogasterrdquo Molecular and CellularBiology vol 31 no 8 pp 1646ndash1656 2011

[12] P Zhang K Takeuchi L S Csaki and K Reue ldquoLipin-1 phosphatidic phosphatase activity modulates phosphatidatelevels to promote peroxisome proliferator-activated receptor120574 (PPAR120574) gene expression during adipogenesisrdquo Journal ofBiological Chemistry vol 287 no 5 pp 3485ndash3494 2012

[13] S Hasegawa K Noda A Maeda M Matsuoka M Yamasakiand T Fukui ldquoAcetoacetyl-CoA synthetase a ketone body-utilizing enzyme is controlled by SREBP-2 and affects serumcholesterol levelsrdquoMolecular Genetics and Metabolism vol 107no 3 pp 553ndash560 2012

[14] S M Vallett H B Sanchez J M Rosenfeld and T F OsborneldquoA direct role for sterol regulatory element binding protein inactivation of 3-hydroxy-3-methylglutaryl coenzyme A reduc-tase generdquo The Journal of Biological Chemistry vol 271 no 21pp 12247ndash12253 1996

[15] X Hua C Yokoyama J Wu et al ldquoSREBP-2 a second basicndashhelixndashloopndashhelixndashleucine zipper protein that stimulates tran-scription by binding to a sterol regulatory elementrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 90 no 24 pp 11603ndash11607 1993

[16] Y-S Cha S-J Rhee and Y-R Heo ldquoAcanthopanax senticosusextract prepared from cultured cells decreases adiposity andobesity indices in C57BL6J mice fed a high fat dietrdquo Journalof Medicinal Food vol 7 no 4 pp 422ndash429 2004

[17] W X Tian X F Ma S Y Zhang Y H Sun and B H LildquoFatty acid synthase inhibitors from plants and their potentialapplication in the prevention of metabolic syndromerdquo ClinicalOncology and Cancer Research vol 8 no 1 pp 1ndash9 2011

[18] H-Y Jung Y-H Kim I-B Kim et al ldquoThe Korean mistletoe(Viscum album coloratum) extract has an antiobesity effectand protects against hepatic steatosis in mice with high-fat diet-induced obesityrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 168207 9 pages 2013

[19] S-I Lee J-S Kim S-H Oh K-Y Park H-G Lee and S-DKim ldquoAntihyperglycemic effect of Fomitopsis pinicola extractsin streptozotocin-induced diabetic ratsrdquo Journal of MedicinalFood vol 11 no 3 pp 518ndash524 2008

[20] Y Zhang L Xia W Pang et al ldquoA novel soluble 120573-13-d-glucanSalecan reduces adiposity and improves glucose tolerance inhigh-fat diet-fed micerdquo British Journal of Nutrition vol 109 no2 pp 254ndash262 2013

[21] M Sung H Y Jung J Choi S Lee B Choi and S S ParkldquoPreparation of functional healthy drinks by Acanthopanaxsenticosus extractsrdquo Journal of Life Science vol 24 no 9 pp 959ndash966 2014

[22] J-M Gu and S-S Park ldquoOptimization of endoglucanaseproduction from Fomitopsis pinicolamyceliardquoKorean Journal ofMicrobiology and Biotechnology vol 41 no 2 pp 145ndash152 2013

[23] M Lee B Ryu M Kim Y Lee and G Moon ldquoProtective effectof dietary buchu (Chinese chives) against oxidative damagefrom aging and ultraviolet irradiation in ICR mice skinrdquoNutraceuticals and Food vol 7 no 3 pp 238ndash244 2002

[24] R S Bruno C E Dugan J A Smyth D A DiNatale and S IKoo ldquoGreen tea extract protects leptin-deficient spontaneouslyobese mice from hepatic steatosis and injuryrdquo The Journal ofNutrition vol 138 no 2 pp 323ndash331 2008

[25] H-Y Jung J-C Shin S-M Park N-R Kim W Kwak andB-H Choi ldquoPinus densiflora extract protects human skin

fibroblasts against UVB-induced photoaging by inhibiting theexpression of MMPs and increasing type I procollagen expres-sionrdquo Toxicology Reports vol 1 pp 658ndash666 2014

[26] L Fajas ldquoAdipogenesis a cross-talk between cell proliferationand cell differentiationrdquo Annals of Medicine vol 35 no 2 pp79ndash85 2003

[27] C E Lowe S OrsquoRahilly and J J Rochford ldquoAdipogenesis at aglancerdquo Journal of Cell Science vol 124 no 16 pp 2681ndash26862011

[28] K A Fawcett N Grimsey R J F Loos et al ldquoEvaluating therole of LPIN1 variation in insulin resistance body weight andhuman lipodystrophy in UK populationsrdquoDiabetes vol 57 no9 pp 2527ndash2533 2008

[29] Y-X Wang C-H Lee S Tiep et al ldquoPeroxisome-proliferator-activated receptor delta activates fat metabolism to preventobesityrdquo Cell vol 113 no 2 pp 159ndash170 2003

[30] J E Schaffer ldquoLipotoxicity when tissues overeatrdquo CurrentOpinion in Lipidology vol 14 no 3 pp 281ndash287 2003

[31] E M Brunt ldquoPathology of nonalcoholic steatohepatitisrdquoHepa-tology Research vol 33 no 2 pp 68ndash71 2005

[32] A Wronska and Z Kmiec ldquoStructural and biochemical charac-teristics of various white adipose tissue depotsrdquo Acta Physiolog-ica vol 205 no 2 pp 194ndash208 2012

[33] B Klop J W F Elte and M C Cabezas ldquoDyslipidemia inobesity mechanisms and potential targetsrdquoNutrients vol 5 no4 pp 1218ndash1240 2013

[34] A G Tabak C Herder W Rathmann E J Brunner and MKivimaki ldquoPrediabetes a high-risk state for diabetes develop-mentrdquoThe Lancet vol 379 no 9833 pp 2279ndash2290 2012

[35] A M Brennan and C S Mantzoros ldquoDrug insight the roleof leptin in human physiology and pathophysiologymdashemergingclinical applicationsrdquo Nature Clinical Practice Endocrinology ampMetabolism vol 2 no 6 pp 318ndash327 2006

[36] H Staiger and H-U Haring ldquoAdipocytokines fat-derivedhumoral mediators of metabolic homeostasisrdquo Experimentaland Clinical Endocrinology and Diabetes vol 113 no 2 pp 67ndash79 2005

[37] Y-Y Sung T Yoon W-K Yang S J Kim D-S Kim and H KKim ldquoThe antiobesity effect of Polygonum aviculare L Ethanolextract in high-fat diet-induced obese micerdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID626397 11 pages 2013

[38] A Soukas N D Socci B D Saatkamp S Novelli and J MFriedman ldquoDistinct transcriptional profiles of adipogenesis invivo and in vitrordquoThe Journal of Biological Chemistry vol 276no 36 pp 34167ndash34174 2001

[39] E D Rosen C-H Hsu X Wang et al ldquoCEBP120572 inducesadipogenesis through PPAR120574 a unified pathwayrdquo Genes andDevelopment vol 16 no 1 pp 22ndash26 2002

[40] Z Wu Y Xie N L R Bucher and S R Farmer ldquoConditionalectopic expression of CEBP120573 in NIH-3T3 cells induces PPAR120574and stimulates adipogenesisrdquoGenes andDevelopment vol 9 no19 pp 2350ndash2363 1995

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


basic leucine zipper class of transcription factors increasesduring 3T3-L1 cell differentiation [10] Lipin is also a centralregulator of adipose tissue development Mammalian lipinproteins have been shown to control gene expression andto enzymatically convert phosphatidate into diacylglycerolan essential precursor in triacylglycerol and phospholipidsynthesis [11] Previous studies have established that lipin-1is required at an early step in adipocyte differentiation forthe induction of the adipogenic gene transcription programincluding the key regulator PPAR-120574 [12]

Acetoacetyl-CoA synthetase (AACS) regulation is relatedto cholesterol and lipid homeostasis [13] Sterol responseelement binding protein-2 (SREBP-2) may play an essentialrole in the transcriptional regulation of AACS SREBP-2 is a leucine zipper transcription factor that controls arate-limiting enzyme in cholesterol synthesis HMG-CoAreductase (HMGCR) when the factor binds sterol responseelement [14 15]

The effects ofAcanthopanax senticosusAllium tuberosumandViscum album coloratum have been studied for inhibitionof fatty acid synthase and prevention of obesity as wellas reducing hepatic steatosis [16ndash18] Fomitopsis pinicolaJeseng has been reported for antihyperglycemic effect indiabetic rats [19] In addition it has been reported that 120573-glucan-rich extract amajor component of Fomitopsis pinicolaJeseng effectively reduces adiposity [19 20] However to ourknowledge no reports are available on the effect of Fomitopsispinicola Jeseng on obesity

In this study we investigated the effect of FAVA in a high-fat diet-induced mouse modelThe FAVA is a combination ofherbal extracts (ie Fomitopsis pinicola JesengAcanthopanaxsenticosus Allium tuberosum and Viscum album coloratum)at a ratio of 5 3 1 1 This study investigated the effectof a mixture containing dietary components on metabolicdisorders including obesity hyperlipidemia and NAFLDusing a high-fat diet-induced obesity mouse model and themolecular mechanism level of adipogenesis and cholesterolbiosynthesis Our results indicate the great potential ofFAVA as a potential metabolic regulator of adipogenesis andcholesterol biosynthesis and as a potential therapeutic agentfor preventing or treating obesity and NAFLD

2 Materials and Methods

21 Preparation of FAVA The oriental medicinal and herbalmixture used in this experiment (FAVA) was prepared asdescribed previously [21ndash23] Briefly Acanthopanax sentico-sus and Allium tuberosumwere extracted with 80methanolwhile water extracted Fomitopsis pinicola Jeseng and Viscumalbum coloratum were purchased from commercial vendor(Mistle Biotech Co Ltd Korea) Each extraction of FAVAwas resuspended in distilled water (DW) in a ratio of 5030 10 and 10 respectively and prepared in appropriatediluent for further in vivo study

22 Animals and Diets Lean male C57BL6J mice (7weeks old) were purchased from Charles River LaboratoriesJapan Inc (Yokohama Japan) All animal experiments wereapproved by the Ethics Review Committee of the Pohang

Center for the Evaluation of Biomaterials Republic of KoreaAll mice were housed for 1 week under a 1212-h lightdarkcycle in a temperature- (22 plusmn 1∘C) and humidity- (55 plusmn 5)controlled room and fed standard laboratory chow and waterad libitum while FAVA orlistat and saline supplementationwere performed using oral gavage once a day To induceobesity the mice were fed a HFD (Rodent Diet D12492Research Diet New Brunswick NJ USA) consisting of60 kcal fat Control mice were fed a low-fat chow diet(Rodent Diet D12450B Research Diet New Brunswick NJUSA) consisting of 10 kcal fat Experimental mice weregiven FAVA or orlistat as a positive control (Chongqing ZeinPharmaceutical Co Ltd Chongqing China)Themice wererandomly divided into four groups (119899 = 8 per group) thatwere fed a low-fat chow diet (CHOW) a high-fat diet (HFD)HFD plus FAVA (200mgkgday) or HFD plus orlistat(60mgkgday) Animals were fed via oral feeding needlesfor 12 weeks and the CHOW and HFD group receivedan equivalent volume of saline Body weight was measuredonce a week and food intake was measured three times perweek during the course of the study At the conclusion ofthe in vivo experiment the mice were sacrificed by cervicalvertebral dislocation and the epididymal mesenteric andsubcutaneous fat pads and liver were collected and weighedThe epididymal fat pad samples were stored at minus80∘C untilanalysis

23 Serum Analysis Serum was collected by cardiac punc-ture stored for 20 minutes at room temperature for coagu-lation and then separated by centrifugation at 2000timesg for20 minutes The serum was stored at minus70∘C until analysisThe levels of triglycerides total cholesterol high-densitylipoprotein (HDL) cholesterol low-density lipoprotein (LDL)cholesterol glucose alanine transaminase (ALT) aspartatetransaminase (AST) BUN and creatinine in serum weremeasured by using an automated biochemical analyzer (BS-390 Mindray Bio-Medical Electronics Co Ltd China)

24 Measurement of Leptin and Insulin The leptin andinsulin concentrations in serumwere determined by amouseenzyme-linked immunosorbent assay (ELISA) kit (MorinagaInstitute of Biological Science Yokohama Japan)The prepa-ration of serum samples is described above

25 Abdominal Computed Tomography Analysis Experi-ments of micro-computed tomography (micro-CT) wereperformed with an animal positron emission tomography(PET)CTsingle photon emission computed tomography(SPECT) system (Inveon Siemens USA) prior to the sac-rifice of animals under 15ndash2 isoflurane in O

2anesthesia

Computed tomography pictures were further analyzed usingSiemens Inveon software to calculate the three-dimensionalvolume of the fat mass between lumbar vertebrae one to five

26 Liver Histology Liver tissues were immediately isolatedafter sacrifice For hematoxylin and eosin (HampE) staining thetissues were fixed in 10 formalin processed and embeddedin paraffin prior to sectioning (10 120583m) and staining The liver






















3 Results




0 1 2 3 4 5 6 7 8 9 10 11 1220

22

24

26

28

30

32

34

36

CHOWHFD



Body

wei

ght (

g)

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

lowast


(a)


5

10

15

Food

inta

ke (k

cald

aym

ouse

)

CHOWHFD

(b)


500

1000

1500

2000

2500

Epid

idym

al fa

t pad

(mg)

CHOWHFD

lowast

(c)

0

200

400

600

800

CHOWHFD

Mes

ente

ric fa

t pad

(mg)


lowastlowast

(d)


500

1000

1500

2000

2500

Subc

utan

eous

fat p

ad (m

g)

CHOWHFD

lowast

lowast

(e)



CHOW HFD


(a)


2000

4000

6000

8000

Fat v

olum

e (m

m3)

lowast

CHOWHFD

(b)


20

40

60

Fat m

ass

tota

l bod

y vo

lum

e (

)

lowast

CHOWHFD

(c)








1

2

3

4In

sulin

(ng

mL)


CHOWHFD

(a)


10

20

30

Lept

in (n

gm

L)

lowast

lowastlowastlowast

CHOWHFD

(b)


50

100

150

200

250

Tota

l cho

leste

rol (

mg

dL)

lowastlowastlowast

lowast

CHOWHFD

(c)


02

04

06

08

HD

L-C

tota

l-C (r

atio

)lowast

lowast

CHOWHFD

(d)


002

004

006

008

010

LDL-

Cto

tal-C

(rat

io)

lowast

p = 008

CHOWHFD

(e)


50

100

150

200

250

Glu

cose

(mg

dL)

lowastlowast

CHOWHFD

(f)




10

20

30

40Re

lativ

e mRN

A le

vels

(Lip

in-1

)

lowastlowast

lowast

CHOWHFD

(a)


20

40

60

80

Relat

ive m

RNA

leve

ls (C

EBP

-120572)

lowastlowast

lowastlowast

CHOWHFD

(b)


10

20

30

40

50

Relat

ive m

RNA

leve

ls (P

PAR-120574

)

lowastlowast

lowast

CHOWHFD

(c)



4 Discussion



CHOW


HFD

(a)


1

2

3

Relat

ive m

RNA

leve

ls (A

ACS)

lowast

CHOWHFD

(b)


2

4

6

Relat

ive m

RNA

leve

ls (H

MG

CR)

lowastlowast

CHOWHFD

(c)


1

2

3

4

5

Relat

ive m

RNA

leve

ls (S

REBP

-2)

lowastlowast

lowast

CHOWHFD

(d)








5 Conclusions


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































3 Results




0 1 2 3 4 5 6 7 8 9 10 11 1220

22

24

26

28

30

32

34

36

CHOWHFD



Body

wei

ght (

g)

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

lowast


(a)


5

10

15

Food

inta

ke (k

cald

aym

ouse

)

CHOWHFD

(b)


500

1000

1500

2000

2500

Epid

idym

al fa

t pad

(mg)

CHOWHFD

lowast

(c)

0

200

400

600

800

CHOWHFD

Mes

ente

ric fa

t pad

(mg)


lowastlowast

(d)


500

1000

1500

2000

2500

Subc

utan

eous

fat p

ad (m

g)

CHOWHFD

lowast

lowast

(e)



CHOW HFD


(a)


2000

4000

6000

8000

Fat v

olum

e (m

m3)

lowast

CHOWHFD

(b)


20

40

60

Fat m

ass

tota

l bod

y vo

lum

e (

)

lowast

CHOWHFD

(c)








1

2

3

4In

sulin

(ng

mL)


CHOWHFD

(a)


10

20

30

Lept

in (n

gm

L)

lowast

lowastlowastlowast

CHOWHFD

(b)


50

100

150

200

250

Tota

l cho

leste

rol (

mg

dL)

lowastlowastlowast

lowast

CHOWHFD

(c)


02

04

06

08

HD

L-C

tota

l-C (r

atio

)lowast

lowast

CHOWHFD

(d)


002

004

006

008

010

LDL-

Cto

tal-C

(rat

io)

lowast

p = 008

CHOWHFD

(e)


50

100

150

200

250

Glu

cose

(mg

dL)

lowastlowast

CHOWHFD

(f)




10

20

30

40Re

lativ

e mRN

A le

vels

(Lip

in-1

)

lowastlowast

lowast

CHOWHFD

(a)


20

40

60

80

Relat

ive m

RNA

leve

ls (C

EBP

-120572)

lowastlowast

lowastlowast

CHOWHFD

(b)


10

20

30

40

50

Relat

ive m

RNA

leve

ls (P

PAR-120574

)

lowastlowast

lowast

CHOWHFD

(c)



4 Discussion



CHOW


HFD

(a)


1

2

3

Relat

ive m

RNA

leve

ls (A

ACS)

lowast

CHOWHFD

(b)


2

4

6

Relat

ive m

RNA

leve

ls (H

MG

CR)

lowastlowast

CHOWHFD

(c)


1

2

3

4

5

Relat

ive m

RNA

leve

ls (S

REBP

-2)

lowastlowast

lowast

CHOWHFD

(d)








5 Conclusions


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1 2 3 4 5 6 7 8 9 10 11 1220

22

24

26

28

30

32

34

36

CHOWHFD



Body

wei

ght (

g)

lowast

lowast lowastlowast

lowast

lowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowast

lowast


(a)


5

10

15

Food

inta

ke (k

cald

aym

ouse

)

CHOWHFD

(b)


500

1000

1500

2000

2500

Epid

idym

al fa

t pad

(mg)

CHOWHFD

lowast

(c)

0

200

400

600

800

CHOWHFD

Mes

ente

ric fa

t pad

(mg)


lowastlowast

(d)


500

1000

1500

2000

2500

Subc

utan

eous

fat p

ad (m

g)

CHOWHFD

lowast

lowast

(e)



CHOW HFD


(a)


2000

4000

6000

8000

Fat v

olum

e (m

m3)

lowast

CHOWHFD

(b)


20

40

60

Fat m

ass

tota

l bod

y vo

lum

e (

)

lowast

CHOWHFD

(c)








1

2

3

4In

sulin

(ng

mL)


CHOWHFD

(a)


10

20

30

Lept

in (n

gm

L)

lowast

lowastlowastlowast

CHOWHFD

(b)


50

100

150

200

250

Tota

l cho

leste

rol (

mg

dL)

lowastlowastlowast

lowast

CHOWHFD

(c)


02

04

06

08

HD

L-C

tota

l-C (r

atio

)lowast

lowast

CHOWHFD

(d)


002

004

006

008

010

LDL-

Cto

tal-C

(rat

io)

lowast

p = 008

CHOWHFD

(e)


50

100

150

200

250

Glu

cose

(mg

dL)

lowastlowast

CHOWHFD

(f)




10

20

30

40Re

lativ

e mRN

A le

vels

(Lip

in-1

)

lowastlowast

lowast

CHOWHFD

(a)


20

40

60

80

Relat

ive m

RNA

leve

ls (C

EBP

-120572)

lowastlowast

lowastlowast

CHOWHFD

(b)


10

20

30

40

50

Relat

ive m

RNA

leve

ls (P

PAR-120574

)

lowastlowast

lowast

CHOWHFD

(c)



4 Discussion



CHOW


HFD

(a)


1

2

3

Relat

ive m

RNA

leve

ls (A

ACS)

lowast

CHOWHFD

(b)


2

4

6

Relat

ive m

RNA

leve

ls (H

MG

CR)

lowastlowast

CHOWHFD

(c)


1

2

3

4

5

Relat

ive m

RNA

leve

ls (S

REBP

-2)

lowastlowast

lowast

CHOWHFD

(d)








5 Conclusions


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CHOW HFD


(a)


2000

4000

6000

8000

Fat v

olum

e (m

m3)

lowast

CHOWHFD

(b)


20

40

60

Fat m

ass

tota

l bod

y vo

lum

e (

)

lowast

CHOWHFD

(c)








1

2

3

4In

sulin

(ng

mL)


CHOWHFD

(a)


10

20

30

Lept

in (n

gm

L)

lowast

lowastlowastlowast

CHOWHFD

(b)


50

100

150

200

250

Tota

l cho

leste

rol (

mg

dL)

lowastlowastlowast

lowast

CHOWHFD

(c)


02

04

06

08

HD

L-C

tota

l-C (r

atio

)lowast

lowast

CHOWHFD

(d)


002

004

006

008

010

LDL-

Cto

tal-C

(rat

io)

lowast

p = 008

CHOWHFD

(e)


50

100

150

200

250

Glu

cose

(mg

dL)

lowastlowast

CHOWHFD

(f)




10

20

30

40Re

lativ

e mRN

A le

vels

(Lip

in-1

)

lowastlowast

lowast

CHOWHFD

(a)


20

40

60

80

Relat

ive m

RNA

leve

ls (C

EBP

-120572)

lowastlowast

lowastlowast

CHOWHFD

(b)


10

20

30

40

50

Relat

ive m

RNA

leve

ls (P

PAR-120574

)

lowastlowast

lowast

CHOWHFD

(c)



4 Discussion



CHOW


HFD

(a)


1

2

3

Relat

ive m

RNA

leve

ls (A

ACS)

lowast

CHOWHFD

(b)


2

4

6

Relat

ive m

RNA

leve

ls (H

MG

CR)

lowastlowast

CHOWHFD

(c)


1

2

3

4

5

Relat

ive m

RNA

leve

ls (S

REBP

-2)

lowastlowast

lowast

CHOWHFD

(d)








5 Conclusions


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















1

2

3

4In

sulin

(ng

mL)


CHOWHFD

(a)


10

20

30

Lept

in (n

gm

L)

lowast

lowastlowastlowast

CHOWHFD

(b)


50

100

150

200

250

Tota

l cho

leste

rol (

mg

dL)

lowastlowastlowast

lowast

CHOWHFD

(c)


02

04

06

08

HD

L-C

tota

l-C (r

atio

)lowast

lowast

CHOWHFD

(d)


002

004

006

008

010

LDL-

Cto

tal-C

(rat

io)

lowast

p = 008

CHOWHFD

(e)


50

100

150

200

250

Glu

cose

(mg

dL)

lowastlowast

CHOWHFD

(f)




10

20

30

40Re

lativ

e mRN

A le

vels

(Lip

in-1

)

lowastlowast

lowast

CHOWHFD

(a)


20

40

60

80

Relat

ive m

RNA

leve

ls (C

EBP

-120572)

lowastlowast

lowastlowast

CHOWHFD

(b)


10

20

30

40

50

Relat

ive m

RNA

leve

ls (P

PAR-120574

)

lowastlowast

lowast

CHOWHFD

(c)



4 Discussion



CHOW


HFD

(a)


1

2

3

Relat

ive m

RNA

leve

ls (A

ACS)

lowast

CHOWHFD

(b)


2

4

6

Relat

ive m

RNA

leve

ls (H

MG

CR)

lowastlowast

CHOWHFD

(c)


1

2

3

4

5

Relat

ive m

RNA

leve

ls (S

REBP

-2)

lowastlowast

lowast

CHOWHFD

(d)








5 Conclusions


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















10

20

30

40Re

lativ

e mRN

A le

vels

(Lip

in-1

)

lowastlowast

lowast

CHOWHFD

(a)


20

40

60

80

Relat

ive m

RNA

leve

ls (C

EBP

-120572)

lowastlowast

lowastlowast

CHOWHFD

(b)


10

20

30

40

50

Relat

ive m

RNA

leve

ls (P

PAR-120574

)

lowastlowast

lowast

CHOWHFD

(c)



4 Discussion



CHOW


HFD

(a)


1

2

3

Relat

ive m

RNA

leve

ls (A

ACS)

lowast

CHOWHFD

(b)


2

4

6

Relat

ive m

RNA

leve

ls (H

MG

CR)

lowastlowast

CHOWHFD

(c)


1

2

3

4

5

Relat

ive m

RNA

leve

ls (S

REBP

-2)

lowastlowast

lowast

CHOWHFD

(d)








5 Conclusions


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CHOW


HFD

(a)


1

2

3

Relat

ive m

RNA

leve

ls (A

ACS)

lowast

CHOWHFD

(b)


2

4

6

Relat

ive m

RNA

leve

ls (H

MG

CR)

lowastlowast

CHOWHFD

(c)


1

2

3

4

5

Relat

ive m

RNA

leve

ls (S

REBP

-2)

lowastlowast

lowast

CHOWHFD

(d)








5 Conclusions


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusions


Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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