research article decaffeinated green coffee bean extract...

Research ArticleDecaffeinated Green Coffee Bean Extract AttenuatesDiet-Induced Obesity and Insulin Resistance in Mice

Su Jin Song Sena Choi and Taesun Park

Department of Food and Nutrition Brain Korea 21 PLUS Project Yonsei University 50 Yonsei-ro Seodaemun-guSeoul 120-749 Republic of Korea

Correspondence should be addressed to Taesun Park tsparkyonseiackr

Received 16 December 2013 Accepted 2 March 2014 Published 9 April 2014

Academic Editor Ravirajsinh Jadeja

Copyright copy 2014 Su Jin Song et al This 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 whether decaffeinated green coffee bean extract prevents obesity and improves insulin resistance andelucidated its mechanism of action Male C57BL6N mice (119873 = 48) were divided into six dietary groups chow diet HFDHFD-supplemented with 01 03 and 09 decaffeinated green coffee bean extract and 015 5-caffeoylquinic acid Basedon the reduction in HFD-induced body weight gain and increments in plasma lipids glucose and insulin levels the minimumeffective dose of green coffee bean extract appears to be 03 Green coffee bean extract resulted in downregulation of genesinvolved in WNT10b- and galanin-mediated adipogenesis and TLR4-mediated proinflammatory pathway and stimulation ofGLUT4 translocation to the plasma membrane in white adipose tissue Taken together decaffeinated green coffee bean extractappeared to reverse HFD-induced fat accumulation and insulin resistance by downregulating the genes involved in adipogenesisand inflammation in visceral adipose tissue

1 Introduction

Coffee is one of the most widely consumed beverages inthe world and therefore the potential health consequencesof coffee consumption are of great public interest Heavycoffee drinking may result in sleep disorders hypokalemiaand cardiac arrhythmias [1ndash4] At the same time severalepidemiologic studies have reported that the risk of Parkin-sonrsquos disease Alzheimerrsquos disease and certain types of canceris reduced in regular coffee consumers [5] In additioncoffee has recently received scientific attention as currentepidemiologic and in vivo studies have revealed its healthbenefits against obesity and metabolic disorders especiallytype 2 diabetes [6ndash10] These health advantages are mostlyderived from chlorogenic acids contained in coffee beans [11ndash14]

Adipogenesis is a process of mesenchymal precur-sor cells differentiating into adipocytes where peroxisomeproliferator-activated receptor 1205742 (PPAR1205742) and CCAATenhancer-binding protein 120572 (CEBP120572) are the master tran-scriptional regulators [1 15] Downstream targets for PPAR1205742

include adipocyte lipid binding protein (aP2) cluster ofdifferentiation 36 (CD36) lipoprotein lipase (LPL) and fattyacid synthase (FAS) which together induce lipid accumula-tion and metabolism Thus inactivation of these adipogenicregulators may be a novel way to suppress adipogenesis andultimately prevent obesity

Another critical aspect of adipose tissue is that it serves asan endocrine organ releasing biologically active adipokinesToll-like receptor (TLR) 2 and TLR4 induce the expressionof a large number of proinflammatory target genes It wasrecently discovered that TLR4 can sense free fatty acids(FFAs) engaging proinflammatory pathways that lead tosecretion of cytokines [16] Furthermore several studies havedemonstrated a causative relationship between inflammationand insulin resistance [17] JNK especially serves as a mainmediator that leads to insulin resistance by impairing GLUT4translocation Thus reducing the FFA level in blood andperipheral tissues such as the adipose tissue and musclesmight result in therapeutic effects against obesity by attenuat-ing not only adipogenesis but also inflammation and insulinresistance

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 718379 14 pageshttpdxdoiorg1011552014718379

2 Evidence-Based Complementary and Alternative Medicine

Raw coffee beans are rich in chlorogenic acids andcaffeine and their contents in coffee beans are significantlydecreased during the roasting and decaffeination processes[18] Green coffee bean extract used in the present studyis prepared from decaffeinated and unroasted coffee beansmaking it a novel source of chlorogenic acids and eliminatingthe possible side effects of caffeine [1ndash3]There is no report ontoxicological studies on green coffee bean extract In a clinicaltrial decaffeinated green coffee bean extract induced weightloss in overweight volunteers provided with 400mgdayfor 60 days [19] Yet further investigation on toxic doseof green coffee bean extract is required in animal modelsCho et al revealed that 5-caffeoylquinic acid (CQA) arepresentative chlorogenic acid in green coffee beans exhibitsantiobesity properties in mice fed a HFD [12] Anotherstudy reported that decaffeinated green coffee bean extractdelivered through drinking water for 20 weeks significantlyimproved HFD-induced insulin resistance in mice howeverthe dose delivered to mice was not clearly indicated and itsmolecular mechanism on improving insulin sensitivity hasnot been examined [10] Also the antiobesity effect of decaf-feinated green coffee bean extract has not yet been reportedinHFD-induced obesemiceTherefore the aims of this studywere to investigate whether decaffeinated green coffee beanextract exerts protective effects against visceral obesity andinsulin resistance in mice fed a HFD and to evaluate whetherthese effects are derived from 5-CQA Furthermore weexplored the potential molecular mechanisms of the healthbenefits of decaffeinated green coffee bean extract focusingon the gene expression involved in adipogenesis and insulinresistance in white adipose tissue (WAT)

2 Materials and Methods

21 Extraction and HPLC Analysis of Decaffeinated GreenCoffee Bean Extract The decaffeinated green coffee beanextract utilized for this study was provided by Naturex Inc(Avignon France) under the trade name Svetol Svetol wasobtained by extracting decaffeinated raw green coffee (Coffeacanephora robusta) beans with 30 ethanol at 70∘C for2 h To determine 5-CQA (IUPAC numbering) content indecaffeinated green coffee bean extract HPLC analysis wasperformed via a Supelco C18 column (250 times 46mm 5 120583minner diameter) at 40∘Cwith a flow rate of 14mLmin using agradientmobile phase composed ofwater (A) and acetonitrile(B)Themobile phasewas 95 5mixture of componentsA andB as the initial condition of the chromatography the sampleinjection volume was 2 120583L The absorption spectrum of 5-CQA was monitored at 330 nm using the photodiode arraydetector

22 Animal Care and Experimental Protocol Forty-eightmale C57BL6N mice (Orient Gyeonggi-do Republic ofKorea) were housed in standard cages and placed in a roomwhere the temperature was maintained at 23 plusmn 1∘C relativehumidity at 50 plusmn 1 and the light at a 12 h lightdark cycleDuring a 1-week acclimatization period all mice consumeda commercial diet and tap water ad libitum Afterwards

they were divided into six weight-matched groups (119899 = 8)the chow diet (CD) high-fat diet (HFD) 01 03 and09 green coffee bean extract-supplemented diet (GCD)and 015 5-CQA-supplemented diet (CQD) groups (SigmaMO USA) The HFD was composed of 200 g of fatkg (170 gof lard plus 30 g of corn oil) and 1 (ww) cholesterol TheGCD was identical to the HFD except that it included 0103 or 09 green coffee bean extract The CQD was alsoidentical to the HFD except that it contained 015 5-CQAThe diets were given in the form of pellets for eleven weeks

Food intake of themicewas recorded daily and their bodyweights were measured weekly during the feeding periodAt the end of the experimental period the animals wereanesthetized with ether following a 12 h fasting period Bloodsampleswere drawn from the abdominal aorta into an EDTA-coated tube and plasma samples were obtained by centrifu-gation at 1000timesg for 15min at 4∘C Visceral fat pads fromfour different regions (epididymal perirenal mesenteric andretroperitoneal regions) were excised rinsedwith phosphate-buffered saline (PBS) and stored at minus80∘C until analysisAll animal experiments adhered to the Korean Food andDrug Administration (KFDA) guidelinesThe protocols werereviewed and approved by the Institutional Animal Care andUse Committee (IACUC) of the Yonsei Laboratory AnimalResearch Center (YLARC) (Permit no 2013-0104) All micewere maintained in the specific pathogen-free facility of theYLARC

23 Histological Analysis The epididymal fat pads were fixedin neutral buffered formalin and embedded in paraffinsectioned at thicknesses of 5 120583m The tissue sections werestained with hematoxylin and eosin (HampE)

24 Biochemical Analysis The plasma concentrations oftriglycerides (TG) FFA total cholesterol (TC) and glucosewere measured enzymatically using commercial kits (Bio-Clinical System Gyeonggi-do Republic of Korea) Plasmaleptin adiponectin interleukin-6 (IL-6) monocyte chemoat-tractant protein-1 (MCP-1) and insulin levels were analyzedusing an ELISA kit (Millipore MA USA) The homeostasismodel assessment of basal insulin resistance (HOMA-IR)was used to calculate an index from the product of thefasting concentrations of plasma glucose (mmolL) andinsulin (pmolL) divided by 225 Lower HOMA-IR valuesindicate greater insulin sensitivity and higher HOMA-IRvalues indicate insulin resistance

25 Oral Glucose Tolerance Test An oral glucose tolerancetest (OGTT gavage with 2 g glucose10mL per kg bodyweight) was performed 2 weeks before the end of thetreatment on 18 h fastedmice by administering glucose orallyBlood was collected from the tail vein at 0 15 30 60 90 and120min following glucose administration to determine bloodglucose

26 Semiquantitative Reverse Transcriptase Polymerase ChainReaction (RT-PCR) Total RNA was isolated from the epi-didymal adipose tissue of eachmousewith Trizol (Invitrogen

Evidence-Based Complementary and Alternative Medicine 3

CA USA) Of the total RNA 4 120583L was reverse-transcribedto cDNA using the Superscript II kit (Invitrogen) accordingto the manufacturerrsquos instructions Table 1 shows the forward(F) and reverse (R) primer sequences The PCR procedurewas designed as follows 10min at 94∘C 30ndash35 cycles at 94∘Cfor 30 s 55∘C for 30 s 72∘C for 1min and 10min of incubationat 72∘C Next 4 120583L of each PCR reaction mixture was mixedwith 1 120583L of 6-fold-concentrated loading buffer and thenloaded onto a 2 agarose gel containing ethidium bromideThe mRNA levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels whichwere used as an internal control

27 Western Blot Analysis The epididymal adipose tissuesof each mouse were homogenized in an extraction buffercontaining 100mM Tris-HCl pH 74 5mM EDTA 50mMsodium pyrophosphate 50mMNaF 100mM orthovanadate1 Triton X-100 1mM phenylmethanesulfonyl fluoride2 120583gmL aprotinin 1120583gmL pepstatin A and 1 120583gmL leu-peptin The tissue homogenates were centrifuged at 1300timesgfor 20min at 4∘C The protein concentrations of the tissueextracts were measured via Bradford assay (Bio-Rad CAUSA) The protein samples were separated by SDS-PAGEand electrophoretically transferred to nitrocellulose mem-branes (Amersham Buckinghamshire UK) The sampleswere incubated overnight and hybridized with primary anti-bodies (diluted 1 1000) at 4∘C Antibodies to the followingproteins were purchased from the indicated sources 120573-catenin 120573-actin (Santa Cruz Biotechnology CA USA) c-JunN-terminal kinase (JNK) p-JNK (Tyr183) insulin receptorsubstrate (IRS) p-IRS (Ser307) protein kinase B (AKT) p-AKT (Thr308) GLUT4 and GAPDH (Cell Signaling Tech-nology MA USA) The membranes were incubated with thecorresponding secondary antibody Next immunoreactivesignals were detected via a chemiluminescent detectionsystem (Amersham Buckinghamshire UK) and quantifiedusing Quantity One analysis software (Bio-Rad)

28 Statistical Analysis The data on body weight gainplasma biochemistries and adipocyte diameter is expressedas the mean plusmn SEM of 8 mice The RT-PCR and Westerndata are means from 119899 = 8plusmn SEM of three independentexperiments (119899 = 2 3 per experiment) for each group Datawere analyzed by one-way analysis of variance (ANOVA)followed by Duncanrsquos multiple range tests 119875 values lt 005were considered statistically significant

3 Results

31 HPLCAnalysis ofDecaffeinatedGreenCoffee BeanExtractThe extraction yield of decaffeinated green coffee beans was15TheHPLC analysis (Figure 1) revealed that decaffeinatedgreen coffee bean extract (Svetol) contained 164 5-CQA

32 Body and Visceral Fat-Pad Weights After 11 weeks ofexperimental feeding the final body weight gain was dose-dependently decreased in the 01GCD and 03GCD groups(Figure 2(a)) Food intake did not differ among experimental

(min)0 5 10 15 20 25 30 35

Nor

m

0

200

400

600

800

140

73-

chlo

roge

nic

acid

DAD1 A sig = 3304 ref = off(chlorogenic acid20130722 2013-07-22 17-15-59201307220000019D)

Figure 1 The HPLC chromatogram of decaffeinated green coffeebean extract The peak was assigned based on the isolation of 5-CQA

groups during the 11-week feeding period (Figure 2(b)) andthe food efficiency ratio (FER) was significantly decreased inmice fed the 03GCDwhen compared with mice fed the HFD(Figure 2(c))The total visceral fat-pad weight of mice fed theHFD was reduced when the mice were supplemented with03 green coffee bean extract (Figures 2(d) and 2(e)) Nofurther reduction in body weight gain and visceral fat-padweightwas noted in the 09GCDgroupMoreover 03 greencoffee bean extract decreased body weight gain and visceraladiposity as much as 015 5-CQA did Based on the resultsabove 03 appears to be the minimum effective dose atwhich green coffee bean extract reduces bodyweight gain andvisceral fat-pad weight Therefore the histological analysisof epididymal adipose tissue sections by HampE staining wasdone with the 03GCD group among the green coffee beanextract supplemented groups The staining data showed thatthe average adipocyte diameter was significantly smaller inthe 03GCD and CQD groups when compared with the HFDgroup (Figures 2(f) and 2(g))

33 Plasma Biochemistries Mice fed a 03GCD exhibited sig-nificantly lower levels of plasma lipids leptin and cytokinesand significantly higher levels of adiponectin than those feda HFD (Figure 3) Yet mice fed a 09GCD showed no furtherdecreases in these plasma measures The changes in plasmabiomarkers related to obesity and inflammation that occurredin the 03GCD group were similar to those observed with theCQD group

34 Glucose Utilization and Insulin Sensitivity We investi-gated whether green coffee bean extract influenced insulinsensitivity OGTT was performed after 9 weeks of dietsupplementation to determine the effect of green coffee beanextract and 5-CQA on glucose tolerance in HFD-fed mice(Figure 4(a)) Integrated plasma glucose concentration ascalculated by the area under the curve (AUC) was signifi-cantly reduced in 03GCD-fed mice than in HFD-fed mice(Figure 4(b)) Moreover 03GCD-fed mice demonstrated asimilar reduction level in the integrated glucose concen-tration as CQD-fed mice did Furthermore when fastingplasma glucose and insulin levels were measured at the end


Table 1 Primer sequences and PCR conditions

Gene description Primers Sequences (51015840-31015840) 119879119898

(∘C) Size (bp)

SFRP 5 F CTTGGTGTCCTTGCGCTTTA 61 155R CTGATGGCCTCATGGAACAG

DKK2 F TCATTCCCTGTTCTTCAGCG 55 144R GCATTTCCTTCAGATTGGCA

WNT10b F TTTTGGCCACTCCTCTTCCT 61 183R TCCTTTTCCAACCGAAAACC

Galanin F GAGCCTTGATCCTGCACTGA 60 121R AGTGGCTGACAGGGTCACAA

GalR1 F CCAAGGGGGTATCCCAGTAA 60 147R GGCCAAACACTACCAGCGTA

GalR2 F ATAGTGGTGCTCATGCTGGAA 60 134R AGGCTGGATCGAGGGTTCTA

PKC120575 F CTGAGCGCTGCAAGAAGAAC 60 146R TGGAAACTTTGATCCTGCACTGA

Cyc-D F TGGGAAGTTTTGTTGGGTCA 55 144R TCCTTGTCCAGGTAATGCCA

PPAR1205742 F TTCGGAATCAGCTCTGTGGA 55 148R CCATTGGGTCAGCTCTTGTG

CEBP120572 F AAGGCCAAGAAGTCGGTGGA 55 189R CCATAGTGGAAGCCTGATGC

FAS F TTGCCCGAGTCAGAGAACC 55 171R CGTCCACAATAGCTTCATAGC

Leptin F CTCCAAGGTTGTCCAGGGTT 55 143R AAAACTCCCCACACAATGGG

CD36 F ATGACGTGGCAAAGAACAGC 55 160R GAAGGCTCAAAGATGCCTCC

aP2 F ACATGAAAGTGGGAGTG 55 128R AAGTACTCTCTGACCGGATG

TLR2 F TCTAAAGTCGATCCGCGACAT 55 344R TACCCAGCTCGCTCACTACGT

TLR4 F ACCTCTGCCTTCACTACAGA 486 223R AGGGACTTCTCAACCTTCTC

TNF120572 F TGTCTCAGCCTCTTCTCATT 55 156R AGATGATCTGAGTGTGAGGG

MCP1 F CCAGCAAGATGATCCCAATG 55 450R CTTCTTGGGGTCAGCACAGA

IFN120572 F ATGGCTAGGCTCTGTGCTTTCCT 58 638R GGGCTCTCCAGATTTCTGCTCTG

IFN120573 F CCACAGCCCTCTCCATCAACTATAAGC 56 372R AGCTCTTCAACTGGAGAGCAGTTGAGG

IL-6 F TTGCCTTCTTGGGACTGATG 55 162R CCACGATTTCCCAGAGAACA

GAPDH F AGAACATCATCCCTGCATCC 60 321R TCCACCACCCTGTTGCTGTA

SFRP 5 secreted frizzled-related protein 5 DKK2 Dickkopf 2 WNT10b wingless-type MMTV integration site family member 10B GalR1 galanin receptor1 PKC120575 protein kinase C delta Cyc-D cyclin D PPAR1205742 peroxisome proliferator-activated receptor gamma CEBP120572 CCAATenhancer-binding proteinalpha FAS fatty acid synthase CD36 fatty acid translocase aP2 adipocyte protein2 TLR Toll-like receptor TNF120572 tumor necrosis factor alpha IFNinterferon MCP1 monocyte chemoattractant protein 1 IL-6 interleukin-6 GAPDH gyceraldehyde-3-phosphate dehydrogenase


Body weight gain (g11wks)

d

ab

c cc

4

11

18

25

CD HFD 01 03 09 CQDGCD

(a)

ab b b b b

00

11

22

33

44

CD HFD 01 03 09 CQD

Food intake (gday)

GCD

(b)

c

a ab b b

000

003

006

009

012

CD HFD 01 03 09 CQD

FER

GCD

(c)

CD HFD 03GCD CQD

Epididymal

Perirenal

Retroperitoneal

Mesenteric

(d)

cd d c

d

a

aa

a

a

a

abab

b

ab

b

cc

b

c

b

bcbc

b

c

b

abcbc

b

bc

0

1

2

3

4

5

Epid

idym

al

Perir

enal

Retro

perit

onea

l

Mes

ente

ric

Tota

l

Visceral fat weight (g)

CD

HFD01GCD03GCD09GCD

CQD

(e)

CD HFD 03GCD CQD

(f)

Adipocyte diameter (120583m)

c

a

bb

40

60

80

100

120

CD HFD 03GCD CQD

(g)

Figure 2 Effects of green coffee bean extract and 5-CQA supplementation on body weight gain food efficiency ratio (FER) and visceralfat-pad weights of mice fed a HFDMice were fed the experimental diets for 11 weeks (a) Body weight gain (b) food intake (c) FER ((d) (e))visceral fat-padweights (f) representative pictures ofHampE-stained fat cells frommice epididymal adipose tissue (times100) and (g) densitometricanalysis of adipocyte diameter in epididymal tissue Data represent mean plusmn SEM 119899 = 8 Mean values indicated with different letters indicatestatistical significance (119875 lt 005) FER = body weight gain for experimental period (g)food intake for experimental period (g)

of the feeding period 03GCD group exhibited significantreductions in plasma glucose and insulin levels in comparisonto the HFD group (Figures 4(c) and 2(d)) Along with theplasma glucose and insulin levels the HOMA-IR valuesindicated that insulin sensitivity was improved significantlyin 03GCD group (Figure 4(e)) No further decreases in thesemarkers related to glucose utilization and insulin sensitivity

were shown in 09GCD-fed mice The effect of 03GCD todecrease plasma glucose and insulin levels and to improveinsulin sensitivity was quantitatively similar to that ofCQD

35 Expression of Genes Related to Adipogenesis We exploredthe potential mechanisms by which green coffee bean extract


bc

ab

bc c bc

02

05

08

11

14

CD HFD 01 03 09 CQD

Triglyceride (mM)

GCD

(a)

b

a a

b b b

4000

5000

6000

7000

8000

CD HFD 01 03 09 CQD

Free fatty acid (mEqL)

GCD

(b)

c

a a

bb b

203040506070

CD HFD 01 03 09 CQD

Total cholesterol (mM)

GCD

(c)

e

ab

cd c

00

60

120

180

240

300

CD HFD 01 03 09 CQD

Leptin (ngmL)

GCD

(d)

a

ed

c bc

70

100

130

160

190

220

CD HFD 01 03 09 CQD

Adiponectin (ngmL)

GCD

(e)

b

a

b b b b

00

200

400

600

800

CD HFD 01 03 09 CQD

IL-6 (pgmL)

GCD

(f)

ba

a

b bb

500

560

620

680

740

800

CD HFD 01 03 09 CQD

MCP-1 (pgmL)

GCD

(g)

Figure 3 Effects of green coffee bean extract and 5-CQA supplementation onplasma levels of lipids leptin adiponectin and proinflammatorycytokines in mice fed a HFD (a) TG (b) FFA (c) TC (d) leptin (e) adiponectin (f) IL-6 and (g) MCP-1 Bars represent mean plusmn SEM 119899 = 8Mean values indicated with different letters indicate statistical significance (119875 lt 005)

may attenuate HFD-induced adipogenesis in the epididymaladipose tissue of 03GCD-fed mice We found that green cof-fee bean extract decreased the expression of secreted frizzled-receptor protein 5 (SFRP5) and dickkopf 2 (DKK2) andincreased expression ofwingless-typeMMTV integration sitefamily 10b (WNT10b) (Figure 5(a)) Western blot analysisindicated that 120573-catenin levels were significantly elevated

by green coffee bean extract supplementation (Figure 5(b))Furthermore green coffee bean extract significantly reducedmRNA levels of galanin galanin receptor (GalR) 1 GalR2protein kinase C (PKC) 120575 and cyclin-D (Figure 5(c)) AsFigure 5(d) demonstrates the mRNA levels of adipogenictranscription factors PPAR1205742 and CEBP120572 were signifi-cantly downregulated in mice fed the green coffee bean


OGTT

CD HFD01GCD 03GCD09GCD CQD

5090

130170210250290330

0 30 60 90 120Time (min)

Glu

cose

(mm

olL

)

(a)

d

a

abc

bc bc

27

33

39

45

51

57


AUC (mmolLmiddoth)

(b)

b

a a

b b b

100

150

200

250

300

CD HFD 01 03 09 CQD

Glucose (mmolL)

GCD

(c)

c

a ab b b

00

03

06

09

12

15

CD HFD 01 03 09 CQD

Insulin (ngmL)

GCD

(d)

c

a a

b bb

0

03

06

09

12

15

CD HFD 01 03 09 CQD

HOMA-IR (mmolL)

GCD

(e)

Figure 4 Effect of green coffee bean extract and 5-CQA supplementation on glucose utilization and insulin sensitivity in mice fed a HFD(a) OGTT (b) AUC (c) glucose (d) insulin (e) HOMA-IR Bars represent mean plusmn SEM 119899 = 8 Mean values indicated with different lettersindicate statistical significance (119875 lt 005)

extract comparedwith those inmice fed theHFDThemRNAlevels of key adipogenic genes such as FAS leptin CD36and aP2 were significantly decreased in green coffee beanextract supplemented groups (Figure 5(d)) Moreover thesesignificant changes in the expression of adipogenic genes thatoccurred in the 03GCDgroupwere similar to those observedin the CQD group (Figure 5(d))

36 Expression of Inflammation-Related Signaling MoleculesWe examined the anti-inflammatory effects of green coffee

bean extract in the epididymal adipose tissue of mice main-tained on the HFD Gene expression of inflammatory media-tors such as TLR2 and TLR4 was downregulated in 03GCD-fed mice when compared with HFD-fed mice (Figure 6(a))Furthermore immunoblot results indicated that JNK phos-phorylation in GCD-fedmice occurred significantly less thanin HFD-fed mice (Figure 6(b)) The expression of proinflam-matory cytokines including tumor necrosis factor (TNF)120572 MCP-1 interferon (IFN) 120572 IFN120573 and IL-6 in GCD-fed mice was significantly downregulated in comparison


SFRP5

DKK2

WNT10b

GAPDH

CD HFD 03GCD CQD

b b a

a

a

b

b

bab

b

a

0

05

1

15

2

25

3

SFRP5 DKK2 WNT10b

CDHFD

03GCDCQD

Relat

ive g

ene e

xpre

ssio

n

(a)

GAPDH

CDHFD

03GCDCQD

a

c

a

b

02

04

06

08

10

12

CD HFD 03GCD CQD

CD HFD 03GCD CQD120573-Catenin

120573-C

aten

inG

APD

H

(b)

CDHFD

03GCDCQD

GalaninGalR1GalR2

Cyc-DGAPDH

b c b b b

aa

a

a ab b bb bb c

b b b

005

115

225

335

4

Galanin GalR1 GalR2 Cyc-DRelat

ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

PKC120575

PKC120575

(c)

FASLeptinCD36

aP2GAPDH

b b c b b b

a

a

a a

a a

bb

bb

bbb b b b b

005

115

225

335

445

FAS Leptin CD36 aP2

Relat

ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

CDHFD

03GCDCQD

bc

PPAR1205742CEBP120572

PPAR1205742 CEBP120572

(d)

Figure 5 Effects of green coffee bean extract and 5-CQA supplementation on genes regulating adipogenesis in mice fed the HFD (a) Theexpression of WNT10b-related genes in the epididymal adipose tissue was determined by RT-PCR and normalized to that of GAPDH (b)Protein levels of 120573-catenin and GAPDH in the epididymal adipose tissue were determined byWestern blotting (c) and (d)The expression ofgalanin-related and adipogenic target genes in the epididymal adipose tissue was determined by RT-PCR and normalized to that of GAPDHData represent the results of three independent experiments (119899 = 2 3 mice per experiment) 119875 lt 005 indicates statistical significance Valuesare the mean plusmn SEM 119899 = 8 for each group


TLR2

TLR4

GAPDH

CD HFD 03GCD CQD

b b

a

a

bb

b b

0

05

1

15

2

25

3

35

TLR2 TLR4

Relat

ive g

ene e

xpre

ssio

n

CDHFD

03GCDCQD

(a)

p-JNK

JNK

CD HFD 03GCD CQD

b

a

b

b

06

09

12

15

18

24

21

CD HFD 03GCD CQD

p-JN

KJN

K (fo

ld ch

ange

ver

sus C

D)

CDHFD

03GCDCQD

(b)

MCP1

IL-6GAPDH

b b b b b

a

a

a a

a

bb b b bb

b b b b

012345

MCP1 IL-6Relat

ive g

ene e

xpre

ssio

n

CD HFD

03G

CD

CQD

TNF120572

TNF120572

IFN120572

IFN120572

IFN120573

IFN120573

CDHFD

03GCDCQD

(c)

Figure 6 Effects of green coffee bean extract and 5-CQA supplementation on the expression of gene involved in inflammation in mice fed aHFD (a)The expression of TLR2 and TLR4 in epididymal adipose tissue was determined by RT-PCR and normalized to that of GAPDH (b)Protein levels of p-JNK and JNK in the epididymal adipose tissuewere determined byWestern blotting (c)The expression of proinflammatorycytokine genes in the epididymal adipose tissue was determined by RT-PCR and normalized to that of GAPDH Data represent the results ofthree independent experiments (119899 = 2 3 mice per experiment) 119875 lt 005 indicates statistical significance Values are the mean plusmn SEM 119899 = 8for each group

with those in HFD-fed mice (Figure 6(c)) These significantchanges of gene expression related to inflammation exhibitedin 03GCD-fed mice were also shown in CQD-fed mice(Figure 6)

37 Expression of Genes Related to Insulin Resistance Weevaluated the protein levels of genes involved in insulinresistance in the epididymal fat tissue of 03GCD-fed miceWestern blot analysis revealed that phosphorylation of IRS-1(Ser307) and AKT (Thr308) was significantly decreased andincreased respectively in GCD-fed mice when comparedto HFD-fed mice (Figures 7(a) and 7(b)) The amount ofGLUT4 found in the membrane fraction of the epididymaladipocytes was significantly increased whereas the amountof total cellular GLUT4 in cytosol remained the same in green

coffee bean extract-fed mice when compared with HFD-fedmice (Figure 7(c)) Moreover 03GCD was as effective inimproving insulin sensitivity and enhancing GLUT4 translo-cation to the membrane as CQD was (Figure 7)

4 Discussion

In the present study decaffeinated green coffee bean extracthas demonstrated a significant weight-lowering effect inHFD-fed mice Among the green coffee bean extract dosages(01 03 and 09) 03 green coffee bean extract wasproved to be the minimum effective dose for preventingbody weight gain fat accumulation and insulin resistance inmice fed the HFD for 11 weeks No further dose-dependentdecreases in body weight gain visceral fat-pad weights and


CD HFD 03GCD CQD

050

080

110

140

170

200

CD HFD 03GCD CQD

p-IR

S1to

tal I

RS1

b

a

bb

p-IRS1(Ser307)

IRS1

CDHFD

03GCDCQD

(a)

04

06

08

10

12

CD HFD 03GCD CQD

p-A

KTto

tal A

KT

c

a bb

p-AKT (Thr308)

AKT

CD HFD 03GCD CQD

CDHFD

03GCDCQD

(b)

Glut4(M)

Glut4(T)

GAPDH

CD HFD 03GCD CQD

c

a

b

a

b

a

00

03

06

09

12

15

Glut4(M) Glut4(T)

Relat

ive p

rote

in ex

pres

sion

aa120573-Actin

CDHFD

03GCDCQD

(c)

Figure 7 Effects of green coffee bean extract and 5-CQA supplementation on the proteins involved in GLUT4 translocation in mice fed aHFD Protein levels of p-IRS1 p-AKT plasma membrane GLUT4 and corresponding total proteins in the epididymal adipose tissue weredetermined by Western blotting Data represent the results of three independent experiments (119899 = 2 3 mice per experiment) 119875 lt 005indicates statistical significance Values are the mean plusmn SEM 119899 = 8 for each group

plasma lipids and glucose profiles were noted at 09 greencoffee bean extract dosageThedose of 03 green coffee beanextract (300mg green coffee bean extractkg diet) in micecorresponds to approximately 1460mg60 kg body weightin human when calculated on the basis of normalization tobody surface area as recommended by Reagan-Shaw et al[20] In order to obtain 1460mg of decaffeinated green coffeebean extract 97 grams of decaffeinated green coffee beansis required as calculated from its extraction yield of 15According to Moon the content of total chlorogenic acidsis reduced by approximately 90 in dark roasted beans [18]This implies that 10 timesmore dark roasted beans (97 grams)are required to produce similar weight-reducing effects as thedecaffeinated green coffee beans

Green coffee beans are a rich source of polyphenolsespecially chlorogenic acids Of a variety of chlorogenic acids5-CQA has been known to protect tissues from oxidativestressmodulate glucosemetabolism andmediate antiobesityeffect [12 21 22] In the present study 5-CQA was the mostabundant and active component contained in green coffeebean extract and exerted a significant weight-lowering effectinHFD-fedmice (Figure 1) Based onour assumption that theantiobesity effect of decaffeinated green coffee bean extractwas expected to be dose dependent and be derived from5-CQA the dose of 5-CQA in CQD was chosen to matchthe amount of 5-CQA contained in the 09GCD (the highestdose of decaffeinated green coffee bean extract)Neverthelessweight-suppressing and insulin-sensitizing effects of green


P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1

Target genesTarget genes

FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

Figure 8 The possible molecular mechanisms of decaffeinated green coffee bean extract in attenuating adipogenesis inflammation andinsulin resistance induced by HFD Decaffeinated green coffee bean extract reverses HFD-induced changes in expression of genes involved inWNT10b- and galanin-mediated adipogenesis cascades in the epididymal adipose tissue The downstream adipogenic transcription factors(PPAR1205742 and CEBP120572) and their target genes were also suppressed by decaffeinated green coffee bean extract in the epididymal adiposetissue Decaffeinated green coffee bean extract reverses the HFD-induced changes in the expression of genes related to TLR24-mediatedproinflammatory signaling cascades and proteins involved in GLUT4 translocation in the epididymal adipose tissue

coffee bean extract plateaued at 03 supplementation The03GCD (containing 005 5-CQA) significantly reducedbody weight gain and improved insulin sensitivity as much asthe CQD (containing 015 5-CQA) did This effect could bepossibly due to other polyphenols contained in green coffeebean extract which exert beneficial effects against obesity andinsulin resistance The extent to which 03GCD decreasedbody weight gain and increased insulin sensitivity in micefed the HFD was also shown in 09GCD Exhibiting nofurther preventive effect against obesity and insulin resistancein 09GCD green coffee bean extract seems to reach itsmaximum effect at 31 and 24 reductions in body weightgain and fasting plasma glucose respectively in HFD-fedmice

As food intake did not differ among the experimentalgroups we can hypothesize that the weight-reducing effectof green coffee bean extract is mediated by the inhibitionof adipogenesis Indeed we have confirmed from mRNAexpression levels that adipogenic target genes of PPAR1205742 andCEBP120572 were downregulated in mice fed the green coffeebean extract Several upstream molecules such as WNT10bgalanin fibroblast growth factor 1 and bone morphogenetic

proteins induce PPAR1205742 and CEBP120572 [23ndash25] Of the variousupstream molecules the adipogenic mechanism of WNT10bhas been thoroughly researched both in vivo and in vitro [23]WNT signaling initiates as WNT10b a glycoprotein bindsto fizzled receptors (FZDR) and lipoprotein-receptor-relatedprotein (LRP) [23] Upon WNT10b signaling activation120573-catenin phosphorylation and its subsequent degradationare prevented [26] Then hypophosphorylated 120573-cateninaccumulates in the cytoplasm and translocates into thenucleus where it binds to the T-cell-specific transcriptionfactorlymphoid-enhancer-binding factor (TCFLEF) andsuppresses PPAR1205742 and CEBP120572 The WNT10b signalingpathway is suppressed by extracellular antagonists such asSFRP5 and DKK2 Secreted from adipocytes SFRP5 andDKK2 inhibit WNT10b signaling in adipose tissue via anautocrineparacrine mechanism (Figure 8) SFRP5 binds andsequesters WNT10b from FZD receptors whereas DKK2binds and disrupts LRP from binding to FZDR [27 28]Mice fed the green coffee bean extract exhibited lower geneexpression of SFRP5 and DKK2 compared with those fed theHFD only Transcriptional regulation on SFRP5 and DKK2genes has not yet been explored in WAT Yet Qi et al


have reported that epigenetic silencing of SFRP5 mediatedby hypermethylation causes constitutive activation of WNTsignaling and colorectal tumorigenesis in colorectal tumorcells [29] Epigenetic regulation of the SFRP5 gene in adiposetissue has not been reported however similar epigeneticsilencing of the SFRP5 gene could possibly induce WNTsignaling in adipose tissue Perhaps green coffee bean extractplays a role in epigenetic silencing of SFRP5 resultingin lower SFRP5 expression in adipose tissue but whetherit directly or indirectly modulates its expression requiresfurther investigation

Galanin a 2930-residue neuropeptide regulates foodconsumption memory neurogenesis and neuroendocrinefunction by binding to receptors in the hypothalamic regions[30] It is well known that galanin is expressed and widelydistributed in the central nervous system However recentstudies have revealed that galanin expression has also beenobserved in peripheral tissues such as stomach WAT andtaste buds [24 31] In our previous study we discoveredthat diet-induced obese mice exhibited the upregulation ofgalanin and its receptors in WAT [24] As a result increasedlevels of galanin activate galanin receptor subtypes whichare members of the G protein-coupled receptor familySignaling properties of GalR1 and GalR2 are somewhatdifferent but both eventually lead to the activation of ERKUpon prolonged HFD consumption the activation of GalR1increases mitogen-activated protein kinase (MAPK) activityin a PKC-independent manner by coupling to Gi-type G-proteins via G120573120574 subunits and induces extracellular signal-regulated kinases 12 (ERK) activation [32] The activation ofGalR2 coupled to Gq11-type G-proteins increases phospho-lipase C (PLC) activation PLC cleaves phosphatidylinositol45-bisphsphate into inositol 145-triphosphate and diacylglycerol a PKC120575 activator Subsequently PKC120575 induces theactivation of ERK which increases the expression of PPAR1205742and CEBP120572 and promotes adipogenesis The mRNA levelsof genes involved in galanin-mediated adipogenesis werereduced in mice fed green coffee bean extract Green coffeebean extract appears to suppress adipogenesis by reversingHFD-induced increased expression of galanin its receptorsand adipogenic transcription factors

Since leptin secretion from adipose tissues is positivelycorrelatedwithTGaccumulation in adipocytes plasma leptinlevel is a biomarker in assessing obesity in both experimentalanimals and humans [33 34] Increments of serum leptinlevels usually occur together with adipocyte hypertrophyIn the present study HFD-fed mice demonstrated higherplasma leptin levels and larger adipocyte size than CD-fedmice Yet green coffee bean extract seems to decrease plasmaleptin and its mRNA expression along with the averageadipocyte diameter in HFD-fed mice

Lower levels of plasma proinflammatory cytokines suchas IL-6 and MCP-1 were observed in mice fed green cof-fee bean extract when compared with mice fed the HFDGenerally the levels of plasma cytokines correlate with theexpression of proinflammatory adipokines in WAT [35 36]Adipocytes under normal or healthy conditions referredto as ldquoleanrdquo fat cells engage metabolic pathways leavingimmune-response pathways inactive [36] However when

ldquoleanrdquo fat cells are constantly exposed to overnutrition andstore excess fat beyond a ldquocritical sizerdquo they become ldquofatrdquofat cells which are a good source of adipokines [37] Inthis modified milieu from a metabolic to proinflammatoryenvironment inflammatory mediators such as TLR2 andTLR4 are chronically expressed in adipocytes [36] TLRs playa role in the innate immune response which discriminatesbetween ldquoself rdquo and ldquononself rdquo and activates proinflammatoryprocesses upon stimulation via pathogen-associated molec-ular patterns such as lipopolysaccharides [38] Activation ofTLRs induces phosphorylation of JNK and recruitment ofactivator protein-1 (AP-1) which acts as a transcriptionalactivator of proinflammatory cytokines such as TNF120572MCP1IFN120572 IFN120573 and IL-6 [16 39ndash41] Our data revealed thatthe expression of TLR2 and TLR4 was reduced in mice fedthe green coffee bean extract when compared with those fedthe HFD This implies that green coffee bean extract mayattenuateWAT from becoming a HFD-induced inflamed andmodified environment Recently it was reported that fattyacids particularly saturated fatty acids (C140 C160 andC180) serve as ligands to TLR4 [16 40] In the present studymice fed the green coffee bean extract exhibited lower plasmaFFA levels when compared with those fed a HFD From thiswe suggest that green coffee bean extractmay have attenuatedactivation of TLR4-mediated signaling pathway and thereforeresulted in the decreased expression of proinflammatorycytokines confirmed by their mRNA expression levels Col-lectively we speculate that green coffee bean extract maysuppress proinflammatory responses by not only decreasingthe expression of TLR2 and TLR4 but also decreasing ligand-induced TLR4-mediated inflammatory signaling

Fasting plasma glucose and insulin levels and OGTTresults demonstrated that insulin sensitivity was enhanced ingreen coffee bean extract fed mice Improvement in insulinsensitivity was further confirmed by Western blot analysis ofinsulin signaling-related molecules and membrane GLUT4Mice fed the green coffee bean extract exhibited increasedphosphorylation of AKT and translocation of GLUT4 fromthe cytosol to the membrane when compared with thosefed a HFD Whether green coffee bean extract has a directinfluence on GLUT4 translocation requires further inves-tigation However we speculate that the beneficial effectsagainst insulin resistance could be associated with decreasedinflammatory responses Recent studies have suggested thatJNK an important stress sensor plays a crucial role in the reg-ulation of HFD-induced insulin resistance and inflammation[36] When WAT becomes inflamed JNK is phosphorylatedand p-JNK inactivates IRS-1 by phosphorylating its serineresidue [39] Serine phosphorylation on IRS-1 blocks GLUT4translocation and therefore impairs insulin sensitivity [42]Thus green coffee bean extract may have reversed HFD-induced insulin resistance by decreasing JNK activation andincreasing GLUT4 translocation

In conclusion the present study shows that green coffeebean extract significantly reduces visceral fat-pad accumu-lation and improves insulin resistance in mice fed the HFDat a minimum effective dose of 03 supplementation Wesuggest that 5-CQA and other polyphenols in green coffeebean extract may bring an additive effect in decreasing


body weight gain and increasing insulin sensitivity Thesebeneficial effects are possibly due to the downregulationof genes associated with adipogenesis and inflammation inthe WATofmice Taken as awhole decaffeinated green coffeebean extract may be used as a therapeutic agent that preventsobesity and metabolic syndrome

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research was supported by the SRC program (Center forFood amp Nutritional Genomics Grant no 2008-0062618) ofthe National Research Foundation (NRF) of Korea funded bythe Ministry of Education Science and Technology

References

[1] A Smith ldquoEffects of caffeine on human behaviorrdquo Food andChemical Toxicology vol 40 no 9 pp 1243ndash1255 2002

[2] C C Appel and T D Myles ldquoCaffeine-induced hypokalemicparalysis in pregnancyrdquo Obstetrics amp Gynecology vol 97 no 5part 2 pp 805ndash807 2001

[3] P W Curatolo and D Robertson ldquoThe health consequences ofcaffeinerdquo Annals of Internal Medicine vol 98 no 5 part 1 pp641ndash653 1983

[4] M C Cornelis ldquoCoffee intakerdquo Progress in Molecular Biologyand Translational Science vol 108 pp 293ndash322 2012

[5] A Rosso J Mossey and C F Lippa ldquoCaffeine neuroprotectivefunctions in cognition and Alzheimerrsquos diseaserdquo AmericanJournal of Alzheimerrsquos Disease amp Other Dementias vol 23 no5 pp 417ndash422 2008

[6] R M van Dam and F B Hu ldquoCoffee consumption and risk oftype 2 diabetes a systematic reviewrdquoThe Journal of theAmericanMedical Association vol 294 no 1 pp 97ndash104 2005

[7] J A Greenberg C N Boozer and A Geliebter ldquoCoffeediabetes and weight controlrdquo The American Journal of ClinicalNutrition vol 84 no 4 pp 682ndash693 2006

[8] K Tanaka S Nishizono S Tamaru et al ldquoAnti-obesity andhypotriglyceridemic properties of coffee bean extract in SDratsrdquo Food Science and Technology Research vol 15 no 2 pp147ndash152 2009

[9] T Murase K Misawa Y Minegishi et al ldquoCoffee polyphenolssuppress diet-induced body fat accumulation by downregu-lating SREBP-1c and related molecules in C57BL6J micerdquoAmerican Journal of Physiology Endocrinology andMetabolismvol 300 no 1 pp E122ndashE133 2011

[10] L Ho M Varghese J Wang et al ldquoDietary supplementationwith decaffeinated green coffee improves diet-induced insulinresistance and brain energy metabolism in micerdquo NutritionalNeuroscience vol 15 no 1 pp 37ndash45 2012

[11] K W Ong A Hsu and B K H Tan ldquoChlorogenic acidstimulates glucose transport in skeletal muscle via AMPKactivation a contributor to the beneficial effects of coffee ondiabetesrdquo PLoS ONE vol 7 no 3 Article ID e32718 2012

[12] A-S Cho S-M Jeon M-J Kim et al ldquoChlorogenic acidexhibits anti-obesity property and improves lipid metabolism

in high-fat diet-induced-obese micerdquo Food and Chemical Toxi-cology vol 48 no 3 pp 937ndash943 2010

[13] H Hemmerle H-J Burger P Below et al ldquoChlorogenic acidand synthetic chlorogenic acid derivatives novel inhibitors ofhepatic glucose-6-phosphate translocaserdquo Journal of MedicinalChemistry vol 40 no 2 pp 137ndash145 1997

[14] D V R de Sotillo and M Hadley ldquoChlorogenic acid modifiesplasma and liver concentrations of cholesterol triacylglyceroland minerals in (fafa) Zucker ratsrdquo The Journal of NutritionalBiochemistry vol 13 no 12 pp 717ndash726 2002

[15] S M Rangwala and M A Lazar ldquoTranscriptional control ofadipogenesisrdquo Annual Review of Nutrition vol 20 pp 535ndash5592000

[16] H Shi M V Kokoeva K Inouye I Tzameli H Yin and JS Flier ldquoTLR4 links innate immunity and fatty acid-inducedinsulin resistancerdquoThe Journal of Clinical Investigation vol 116no 11 pp 3015ndash3025 2006

[17] H Xu G T Barnes Q Yang et al ldquoChronic inflammation in fatplays a crucial role in the development of obesity-related insulinresistancerdquoThe Journal of Clinical Investigation vol 112 no 12pp 1821ndash1830 2003

[18] J-K Moon H S Yoo and T Shibamoto ldquoRole of roastingconditions in the level of chlorogenic acid content in coffeebeans correlation with coffee acidityrdquo Journal of Agriculturaland Food Chemistry vol 57 no 12 pp 5365ndash5369 2009

[19] O Dellalibera B Lemaire and S Lafay ldquoSvetol green coffeeextract induces weight loss and increases the lean to fat massratio in volunteers with overweight problemrdquo Phytotherapievol 4 no 4 pp 194ndash197 2006

[20] S Reagan-Shaw M Nihal and N Ahmad ldquoDose translationfrom animal to human studies revisitedrdquo The FASEB Journalvol 22 no 3 pp 659ndash661 2008

[21] Z Zhao H S Shin H Satsu M Totsuka and M Shimizu ldquo5-caffeoylquinic acid and caffeic acid down-regulate the oxidativestress- and TNF-120572-induced secretion of interleukin-8 fromCaco-2 cellsrdquo Journal of Agricultural and Food Chemistry vol56 no 10 pp 3863ndash3868 2008

[22] B K Bassoli P Cassolla G R Borba-Murad et al ldquoChlorogenicacid reduces the plasma glucose peak in the oral glucosetolerance test effects on hepatic glucose release and glycaemiardquoCell Biochemistry and Function vol 26 no 3 pp 320ndash328 2008

[23] C Christodoulides C Lagathu J K Sethi and A Vidal-PuigldquoAdipogenesis andWNT signallingrdquo Trends in Endocrinology ampMetabolism vol 20 no 1 pp 16ndash24 2009

[24] A Kim and T Park ldquoDiet-induced obesity regulates thegalanin-mediated signaling cascade in the adipose tissue ofmicerdquo Molecular Nutrition amp Food Research vol 54 no 9 pp1361ndash1370 2010

[25] T J Schulz and Y-H Tseng ldquoEmerging role of bone mor-phogenetic proteins in adipogenesis and energy metabolismrdquoCytokine amp Growth Factor Reviews vol 20 no 5-6 pp 523ndash5312009

[26] X He ldquoA Wnt-Wnt situationrdquo Developmental Cell vol 4 no 6pp 791ndash797 2003

[27] A Rauch and S Mandrup ldquoLighting the fat furnace withoutSFRP5rdquo The Journal of Clinical Investigation vol 122 no 7 pp2349ndash2352 2012

[28] B Gustafson and U Smith ldquoThe WNT inhibitor dickkopf1 and bone morphogenetic protein 4 rescue adipogenesis inhypertrophic obesity in humansrdquo Diabetes vol 61 no 5 pp1217ndash1224 2012


[29] J Qi Y-Q Zhu J Luo and W-H Tao ldquoHypermethylation andexpression regulation of secreted frizzled-related protein genesin colorectal tumorrdquoWorld Journal of Gastroenterology vol 12no 44 pp 7113ndash7117 2006

[30] I Saar J Runesson I McNamara J Jarv J K Robinson andU Langel ldquoNovel galanin receptor subtype specific ligands infeeding regulationrdquoNeurochemistry International vol 58 no 6pp 714ndash720 2011

[31] Y Seta S Kataoka T Toyono and K Toyoshima ldquoExpressionof galanin and the galanin receptor in rat taste budsrdquo Archivesof Histology and Cytology vol 69 no 4 pp 273ndash280 2006

[32] R Lang A L Gundlach and B Kofler ldquoThe galanin peptidefamily receptor pharmacology pleiotropic biological actionsand implications in health and diseaserdquo Pharmacology amp Ther-apeutics vol 115 no 2 pp 177ndash207 2007

[33] M Maffei J Halaas E Ravussin et al ldquoLeptin levels in humanand rodentmeasurement of plasma leptin and obRNA in obeseand weight-reduced subjectsrdquoNatureMedicine vol 1 no 11 pp1155ndash1161 1995

[34] RN JadejaM CThounaojamUV Ramani R VDevkar andA V Ramachandran ldquoAnti-obesity potential of Clerodendronglandulosum Coleb leaf aqueous extractrdquo Journal of Ethnophar-macology vol 135 no 2 pp 338ndash343 2011

[35] S E Shoelson J Lee and A B Goldfine ldquoInflammation andinsulin resistancerdquoThe Journal of Clinical Investigation vol 116no 7 pp 1793ndash1801 2006

[36] M F Gregor and G S Hotamisligil ldquoInflammatory mecha-nisms in obesityrdquo Annual Review of Immunology vol 29 pp415ndash445 2011

[37] SMittra V S Bansal and P K Bhatnagar ldquoFrom a glucocentricto a lipocentric approach towards metabolic syndromerdquo DrugDiscovery Today vol 13 no 5-6 pp 211ndash218 2008

[38] C Erridge ldquoEndogenous ligands of TLR2 and TLR4 agonistsor assistantsrdquo Journal of Leukocyte Biology vol 87 no 6 pp989ndash999 2010

[39] M T A Nguyen H Satoh S Favelyukis et al ldquoJNK andtumor necrosis factor-120572mediate free fatty acid-induced insulinresistance in 3T3-L1 adipocytesrdquo The Journal of BiologicalChemistry vol 280 no 42 pp 35361ndash35371 2005

[40] J K Kim ldquoFat uses a TOLL-road to connect inflammation anddiabetesrdquo Cell Metabolism vol 4 no 6 pp 417ndash419 2006

[41] S-J Kim Y Choi Y-H Choi and T Park ldquoObesity activatestoll-like receptor-mediated proinflammatory signaling cascadesin the adipose tissue of micerdquo The Journal of NutritionalBiochemistry vol 23 no 2 pp 113ndash122 2012

[42] R Feinstein H Kanety M Z Papa B Lunenfeld and AKarasik ldquoTumor necrosis factor-120572 suppresses insulin-inducedtyrosine phosphorylation of insulin receptor and its substratesrdquoThe Journal of Biological Chemistry vol 268 no 35 pp 26055ndash26058 1993

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


Raw coffee beans are rich in chlorogenic acids andcaffeine and their contents in coffee beans are significantlydecreased during the roasting and decaffeination processes[18] Green coffee bean extract used in the present studyis prepared from decaffeinated and unroasted coffee beansmaking it a novel source of chlorogenic acids and eliminatingthe possible side effects of caffeine [1ndash3]There is no report ontoxicological studies on green coffee bean extract In a clinicaltrial decaffeinated green coffee bean extract induced weightloss in overweight volunteers provided with 400mgdayfor 60 days [19] Yet further investigation on toxic doseof green coffee bean extract is required in animal modelsCho et al revealed that 5-caffeoylquinic acid (CQA) arepresentative chlorogenic acid in green coffee beans exhibitsantiobesity properties in mice fed a HFD [12] Anotherstudy reported that decaffeinated green coffee bean extractdelivered through drinking water for 20 weeks significantlyimproved HFD-induced insulin resistance in mice howeverthe dose delivered to mice was not clearly indicated and itsmolecular mechanism on improving insulin sensitivity hasnot been examined [10] Also the antiobesity effect of decaf-feinated green coffee bean extract has not yet been reportedinHFD-induced obesemiceTherefore the aims of this studywere to investigate whether decaffeinated green coffee beanextract exerts protective effects against visceral obesity andinsulin resistance in mice fed a HFD and to evaluate whetherthese effects are derived from 5-CQA Furthermore weexplored the potential molecular mechanisms of the healthbenefits of decaffeinated green coffee bean extract focusingon the gene expression involved in adipogenesis and insulinresistance in white adipose tissue (WAT)

2 Materials and Methods

21 Extraction and HPLC Analysis of Decaffeinated GreenCoffee Bean Extract The decaffeinated green coffee beanextract utilized for this study was provided by Naturex Inc(Avignon France) under the trade name Svetol Svetol wasobtained by extracting decaffeinated raw green coffee (Coffeacanephora robusta) beans with 30 ethanol at 70∘C for2 h To determine 5-CQA (IUPAC numbering) content indecaffeinated green coffee bean extract HPLC analysis wasperformed via a Supelco C18 column (250 times 46mm 5 120583minner diameter) at 40∘Cwith a flow rate of 14mLmin using agradientmobile phase composed ofwater (A) and acetonitrile(B)Themobile phasewas 95 5mixture of componentsA andB as the initial condition of the chromatography the sampleinjection volume was 2 120583L The absorption spectrum of 5-CQA was monitored at 330 nm using the photodiode arraydetector

22 Animal Care and Experimental Protocol Forty-eightmale C57BL6N mice (Orient Gyeonggi-do Republic ofKorea) were housed in standard cages and placed in a roomwhere the temperature was maintained at 23 plusmn 1∘C relativehumidity at 50 plusmn 1 and the light at a 12 h lightdark cycleDuring a 1-week acclimatization period all mice consumeda commercial diet and tap water ad libitum Afterwards

they were divided into six weight-matched groups (119899 = 8)the chow diet (CD) high-fat diet (HFD) 01 03 and09 green coffee bean extract-supplemented diet (GCD)and 015 5-CQA-supplemented diet (CQD) groups (SigmaMO USA) The HFD was composed of 200 g of fatkg (170 gof lard plus 30 g of corn oil) and 1 (ww) cholesterol TheGCD was identical to the HFD except that it included 0103 or 09 green coffee bean extract The CQD was alsoidentical to the HFD except that it contained 015 5-CQAThe diets were given in the form of pellets for eleven weeks

Food intake of themicewas recorded daily and their bodyweights were measured weekly during the feeding periodAt the end of the experimental period the animals wereanesthetized with ether following a 12 h fasting period Bloodsampleswere drawn from the abdominal aorta into an EDTA-coated tube and plasma samples were obtained by centrifu-gation at 1000timesg for 15min at 4∘C Visceral fat pads fromfour different regions (epididymal perirenal mesenteric andretroperitoneal regions) were excised rinsedwith phosphate-buffered saline (PBS) and stored at minus80∘C until analysisAll animal experiments adhered to the Korean Food andDrug Administration (KFDA) guidelinesThe protocols werereviewed and approved by the Institutional Animal Care andUse Committee (IACUC) of the Yonsei Laboratory AnimalResearch Center (YLARC) (Permit no 2013-0104) All micewere maintained in the specific pathogen-free facility of theYLARC

23 Histological Analysis The epididymal fat pads were fixedin neutral buffered formalin and embedded in paraffinsectioned at thicknesses of 5 120583m The tissue sections werestained with hematoxylin and eosin (HampE)

24 Biochemical Analysis The plasma concentrations oftriglycerides (TG) FFA total cholesterol (TC) and glucosewere measured enzymatically using commercial kits (Bio-Clinical System Gyeonggi-do Republic of Korea) Plasmaleptin adiponectin interleukin-6 (IL-6) monocyte chemoat-tractant protein-1 (MCP-1) and insulin levels were analyzedusing an ELISA kit (Millipore MA USA) The homeostasismodel assessment of basal insulin resistance (HOMA-IR)was used to calculate an index from the product of thefasting concentrations of plasma glucose (mmolL) andinsulin (pmolL) divided by 225 Lower HOMA-IR valuesindicate greater insulin sensitivity and higher HOMA-IRvalues indicate insulin resistance

25 Oral Glucose Tolerance Test An oral glucose tolerancetest (OGTT gavage with 2 g glucose10mL per kg bodyweight) was performed 2 weeks before the end of thetreatment on 18 h fastedmice by administering glucose orallyBlood was collected from the tail vein at 0 15 30 60 90 and120min following glucose administration to determine bloodglucose

26 Semiquantitative Reverse Transcriptase Polymerase ChainReaction (RT-PCR) Total RNA was isolated from the epi-didymal adipose tissue of eachmousewith Trizol (Invitrogen





3 Results



(min)0 5 10 15 20 25 30 35

Nor

m

0

200

400

600

800

140

73-

chlo

roge

nic

acid









(∘C) Size (bp)


























d

ab

c cc

4

11

18

25


(a)

ab b b b b

00

11

22

33

44

CD HFD 01 03 09 CQD

Food intake (gday)

GCD

(b)

c

a ab b b

000

003

006

009

012

CD HFD 01 03 09 CQD

FER

GCD

(c)

CD HFD 03GCD CQD

Epididymal

Perirenal

Retroperitoneal

Mesenteric

(d)

cd d c

d

a

aa

a

a

a

abab

b

ab

b

cc

b

c

b

bcbc

b

c

b

abcbc

b

bc

0

1

2

3

4

5

Epid

idym

al

Perir

enal

Retro

perit

onea

l

Mes

ente

ric

Tota

l


CD

HFD01GCD03GCD09GCD

CQD

(e)

CD HFD 03GCD CQD

(f)


c

a

bb

40

60

80

100

120

CD HFD 03GCD CQD

(g)






bc

ab

bc c bc

02

05

08

11

14

CD HFD 01 03 09 CQD

Triglyceride (mM)

GCD

(a)

b

a a

b b b

4000

5000

6000

7000

8000

CD HFD 01 03 09 CQD


GCD

(b)

c

a a

bb b

203040506070

CD HFD 01 03 09 CQD


GCD

(c)

e

ab

cd c

00

60

120

180

240

300

CD HFD 01 03 09 CQD

Leptin (ngmL)

GCD

(d)

a

ed

c bc

70

100

130

160

190

220

CD HFD 01 03 09 CQD

Adiponectin (ngmL)

GCD

(e)

b

a

b b b b

00

200

400

600

800

CD HFD 01 03 09 CQD

IL-6 (pgmL)

GCD

(f)

ba

a

b bb

500

560

620

680

740

800

CD HFD 01 03 09 CQD

MCP-1 (pgmL)

GCD

(g)





OGTT


5090

130170210250290330

0 30 60 90 120Time (min)

Glu

cose

(mm

olL

)

(a)

d

a

abc

bc bc

27

33

39

45

51

57


AUC (mmolLmiddoth)

(b)

b

a a

b b b

100

150

200

250

300

CD HFD 01 03 09 CQD

Glucose (mmolL)

GCD

(c)

c

a ab b b

00

03

06

09

12

15

CD HFD 01 03 09 CQD

Insulin (ngmL)

GCD

(d)

c

a a

b bb

0

03

06

09

12

15

CD HFD 01 03 09 CQD

HOMA-IR (mmolL)

GCD

(e)






SFRP5

DKK2

WNT10b

GAPDH

CD HFD 03GCD CQD

b b a

a

a

b

b

bab

b

a

0

05

1

15

2

25

3

SFRP5 DKK2 WNT10b

CDHFD

03GCDCQD

Relat

ive g

ene e

xpre

ssio

n

(a)

GAPDH

CDHFD

03GCDCQD

a

c

a

b

02

04

06

08

10

12

CD HFD 03GCD CQD


120573-C

aten

inG

APD

H

(b)

CDHFD

03GCDCQD

GalaninGalR1GalR2

Cyc-DGAPDH

b c b b b

aa

a

a ab b bb bb c

b b b

005

115

225

335

4


ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

PKC120575

PKC120575

(c)

FASLeptinCD36

aP2GAPDH

b b c b b b

a

a

a a

a a

bb

bb

bbb b b b b

005

115

225

335

445

FAS Leptin CD36 aP2

Relat

ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

CDHFD

03GCDCQD

bc

PPAR1205742CEBP120572

PPAR1205742 CEBP120572

(d)



TLR2

TLR4

GAPDH

CD HFD 03GCD CQD

b b

a

a

bb

b b

0

05

1

15

2

25

3

35

TLR2 TLR4

Relat

ive g

ene e

xpre

ssio

n

CDHFD

03GCDCQD

(a)

p-JNK

JNK

CD HFD 03GCD CQD

b

a

b

b

06

09

12

15

18

24

21

CD HFD 03GCD CQD

p-JN

KJN

K (fo

ld ch

ange

ver

sus C

D)

CDHFD

03GCDCQD

(b)

MCP1

IL-6GAPDH

b b b b b

a

a

a a

a

bb b b bb

b b b b

012345

MCP1 IL-6Relat

ive g

ene e

xpre

ssio

n

CD HFD

03G

CD

CQD

TNF120572

TNF120572

IFN120572

IFN120572

IFN120573

IFN120573

CDHFD

03GCDCQD

(c)





4 Discussion



CD HFD 03GCD CQD

050

080

110

140

170

200

CD HFD 03GCD CQD

p-IR

S1to

tal I

RS1

b

a

bb

p-IRS1(Ser307)

IRS1

CDHFD

03GCDCQD

(a)

04

06

08

10

12

CD HFD 03GCD CQD

p-A

KTto

tal A

KT

c

a bb

p-AKT (Thr308)

AKT

CD HFD 03GCD CQD

CDHFD

03GCDCQD

(b)

Glut4(M)

Glut4(T)

GAPDH

CD HFD 03GCD CQD

c

a

b

a

b

a

00

03

06

09

12

15

Glut4(M) Glut4(T)

Relat

ive p

rote

in ex

pres

sion

aa120573-Actin

CDHFD

03GCDCQD

(c)





P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1


FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results



(min)0 5 10 15 20 25 30 35

Nor

m

0

200

400

600

800

140

73-

chlo

roge

nic

acid









(∘C) Size (bp)


























d

ab

c cc

4

11

18

25


(a)

ab b b b b

00

11

22

33

44

CD HFD 01 03 09 CQD

Food intake (gday)

GCD

(b)

c

a ab b b

000

003

006

009

012

CD HFD 01 03 09 CQD

FER

GCD

(c)

CD HFD 03GCD CQD

Epididymal

Perirenal

Retroperitoneal

Mesenteric

(d)

cd d c

d

a

aa

a

a

a

abab

b

ab

b

cc

b

c

b

bcbc

b

c

b

abcbc

b

bc

0

1

2

3

4

5

Epid

idym

al

Perir

enal

Retro

perit

onea

l

Mes

ente

ric

Tota

l


CD

HFD01GCD03GCD09GCD

CQD

(e)

CD HFD 03GCD CQD

(f)


c

a

bb

40

60

80

100

120

CD HFD 03GCD CQD

(g)






bc

ab

bc c bc

02

05

08

11

14

CD HFD 01 03 09 CQD

Triglyceride (mM)

GCD

(a)

b

a a

b b b

4000

5000

6000

7000

8000

CD HFD 01 03 09 CQD


GCD

(b)

c

a a

bb b

203040506070

CD HFD 01 03 09 CQD


GCD

(c)

e

ab

cd c

00

60

120

180

240

300

CD HFD 01 03 09 CQD

Leptin (ngmL)

GCD

(d)

a

ed

c bc

70

100

130

160

190

220

CD HFD 01 03 09 CQD

Adiponectin (ngmL)

GCD

(e)

b

a

b b b b

00

200

400

600

800

CD HFD 01 03 09 CQD

IL-6 (pgmL)

GCD

(f)

ba

a

b bb

500

560

620

680

740

800

CD HFD 01 03 09 CQD

MCP-1 (pgmL)

GCD

(g)





OGTT


5090

130170210250290330

0 30 60 90 120Time (min)

Glu

cose

(mm

olL

)

(a)

d

a

abc

bc bc

27

33

39

45

51

57


AUC (mmolLmiddoth)

(b)

b

a a

b b b

100

150

200

250

300

CD HFD 01 03 09 CQD

Glucose (mmolL)

GCD

(c)

c

a ab b b

00

03

06

09

12

15

CD HFD 01 03 09 CQD

Insulin (ngmL)

GCD

(d)

c

a a

b bb

0

03

06

09

12

15

CD HFD 01 03 09 CQD

HOMA-IR (mmolL)

GCD

(e)






SFRP5

DKK2

WNT10b

GAPDH

CD HFD 03GCD CQD

b b a

a

a

b

b

bab

b

a

0

05

1

15

2

25

3

SFRP5 DKK2 WNT10b

CDHFD

03GCDCQD

Relat

ive g

ene e

xpre

ssio

n

(a)

GAPDH

CDHFD

03GCDCQD

a

c

a

b

02

04

06

08

10

12

CD HFD 03GCD CQD


120573-C

aten

inG

APD

H

(b)

CDHFD

03GCDCQD

GalaninGalR1GalR2

Cyc-DGAPDH

b c b b b

aa

a

a ab b bb bb c

b b b

005

115

225

335

4


ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

PKC120575

PKC120575

(c)

FASLeptinCD36

aP2GAPDH

b b c b b b

a

a

a a

a a

bb

bb

bbb b b b b

005

115

225

335

445

FAS Leptin CD36 aP2

Relat

ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

CDHFD

03GCDCQD

bc

PPAR1205742CEBP120572

PPAR1205742 CEBP120572

(d)



TLR2

TLR4

GAPDH

CD HFD 03GCD CQD

b b

a

a

bb

b b

0

05

1

15

2

25

3

35

TLR2 TLR4

Relat

ive g

ene e

xpre

ssio

n

CDHFD

03GCDCQD

(a)

p-JNK

JNK

CD HFD 03GCD CQD

b

a

b

b

06

09

12

15

18

24

21

CD HFD 03GCD CQD

p-JN

KJN

K (fo

ld ch

ange

ver

sus C

D)

CDHFD

03GCDCQD

(b)

MCP1

IL-6GAPDH

b b b b b

a

a

a a

a

bb b b bb

b b b b

012345

MCP1 IL-6Relat

ive g

ene e

xpre

ssio

n

CD HFD

03G

CD

CQD

TNF120572

TNF120572

IFN120572

IFN120572

IFN120573

IFN120573

CDHFD

03GCDCQD

(c)





4 Discussion



CD HFD 03GCD CQD

050

080

110

140

170

200

CD HFD 03GCD CQD

p-IR

S1to

tal I

RS1

b

a

bb

p-IRS1(Ser307)

IRS1

CDHFD

03GCDCQD

(a)

04

06

08

10

12

CD HFD 03GCD CQD

p-A

KTto

tal A

KT

c

a bb

p-AKT (Thr308)

AKT

CD HFD 03GCD CQD

CDHFD

03GCDCQD

(b)

Glut4(M)

Glut4(T)

GAPDH

CD HFD 03GCD CQD

c

a

b

a

b

a

00

03

06

09

12

15

Glut4(M) Glut4(T)

Relat

ive p

rote

in ex

pres

sion

aa120573-Actin

CDHFD

03GCDCQD

(c)





P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1


FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(∘C) Size (bp)


























d

ab

c cc

4

11

18

25


(a)

ab b b b b

00

11

22

33

44

CD HFD 01 03 09 CQD

Food intake (gday)

GCD

(b)

c

a ab b b

000

003

006

009

012

CD HFD 01 03 09 CQD

FER

GCD

(c)

CD HFD 03GCD CQD

Epididymal

Perirenal

Retroperitoneal

Mesenteric

(d)

cd d c

d

a

aa

a

a

a

abab

b

ab

b

cc

b

c

b

bcbc

b

c

b

abcbc

b

bc

0

1

2

3

4

5

Epid

idym

al

Perir

enal

Retro

perit

onea

l

Mes

ente

ric

Tota

l


CD

HFD01GCD03GCD09GCD

CQD

(e)

CD HFD 03GCD CQD

(f)


c

a

bb

40

60

80

100

120

CD HFD 03GCD CQD

(g)






bc

ab

bc c bc

02

05

08

11

14

CD HFD 01 03 09 CQD

Triglyceride (mM)

GCD

(a)

b

a a

b b b

4000

5000

6000

7000

8000

CD HFD 01 03 09 CQD


GCD

(b)

c

a a

bb b

203040506070

CD HFD 01 03 09 CQD


GCD

(c)

e

ab

cd c

00

60

120

180

240

300

CD HFD 01 03 09 CQD

Leptin (ngmL)

GCD

(d)

a

ed

c bc

70

100

130

160

190

220

CD HFD 01 03 09 CQD

Adiponectin (ngmL)

GCD

(e)

b

a

b b b b

00

200

400

600

800

CD HFD 01 03 09 CQD

IL-6 (pgmL)

GCD

(f)

ba

a

b bb

500

560

620

680

740

800

CD HFD 01 03 09 CQD

MCP-1 (pgmL)

GCD

(g)





OGTT


5090

130170210250290330

0 30 60 90 120Time (min)

Glu

cose

(mm

olL

)

(a)

d

a

abc

bc bc

27

33

39

45

51

57


AUC (mmolLmiddoth)

(b)

b

a a

b b b

100

150

200

250

300

CD HFD 01 03 09 CQD

Glucose (mmolL)

GCD

(c)

c

a ab b b

00

03

06

09

12

15

CD HFD 01 03 09 CQD

Insulin (ngmL)

GCD

(d)

c

a a

b bb

0

03

06

09

12

15

CD HFD 01 03 09 CQD

HOMA-IR (mmolL)

GCD

(e)






SFRP5

DKK2

WNT10b

GAPDH

CD HFD 03GCD CQD

b b a

a

a

b

b

bab

b

a

0

05

1

15

2

25

3

SFRP5 DKK2 WNT10b

CDHFD

03GCDCQD

Relat

ive g

ene e

xpre

ssio

n

(a)

GAPDH

CDHFD

03GCDCQD

a

c

a

b

02

04

06

08

10

12

CD HFD 03GCD CQD


120573-C

aten

inG

APD

H

(b)

CDHFD

03GCDCQD

GalaninGalR1GalR2

Cyc-DGAPDH

b c b b b

aa

a

a ab b bb bb c

b b b

005

115

225

335

4


ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

PKC120575

PKC120575

(c)

FASLeptinCD36

aP2GAPDH

b b c b b b

a

a

a a

a a

bb

bb

bbb b b b b

005

115

225

335

445

FAS Leptin CD36 aP2

Relat

ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

CDHFD

03GCDCQD

bc

PPAR1205742CEBP120572

PPAR1205742 CEBP120572

(d)



TLR2

TLR4

GAPDH

CD HFD 03GCD CQD

b b

a

a

bb

b b

0

05

1

15

2

25

3

35

TLR2 TLR4

Relat

ive g

ene e

xpre

ssio

n

CDHFD

03GCDCQD

(a)

p-JNK

JNK

CD HFD 03GCD CQD

b

a

b

b

06

09

12

15

18

24

21

CD HFD 03GCD CQD

p-JN

KJN

K (fo

ld ch

ange

ver

sus C

D)

CDHFD

03GCDCQD

(b)

MCP1

IL-6GAPDH

b b b b b

a

a

a a

a

bb b b bb

b b b b

012345

MCP1 IL-6Relat

ive g

ene e

xpre

ssio

n

CD HFD

03G

CD

CQD

TNF120572

TNF120572

IFN120572

IFN120572

IFN120573

IFN120573

CDHFD

03GCDCQD

(c)





4 Discussion



CD HFD 03GCD CQD

050

080

110

140

170

200

CD HFD 03GCD CQD

p-IR

S1to

tal I

RS1

b

a

bb

p-IRS1(Ser307)

IRS1

CDHFD

03GCDCQD

(a)

04

06

08

10

12

CD HFD 03GCD CQD

p-A

KTto

tal A

KT

c

a bb

p-AKT (Thr308)

AKT

CD HFD 03GCD CQD

CDHFD

03GCDCQD

(b)

Glut4(M)

Glut4(T)

GAPDH

CD HFD 03GCD CQD

c

a

b

a

b

a

00

03

06

09

12

15

Glut4(M) Glut4(T)

Relat

ive p

rote

in ex

pres

sion

aa120573-Actin

CDHFD

03GCDCQD

(c)





P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1


FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















d

ab

c cc

4

11

18

25


(a)

ab b b b b

00

11

22

33

44

CD HFD 01 03 09 CQD

Food intake (gday)

GCD

(b)

c

a ab b b

000

003

006

009

012

CD HFD 01 03 09 CQD

FER

GCD

(c)

CD HFD 03GCD CQD

Epididymal

Perirenal

Retroperitoneal

Mesenteric

(d)

cd d c

d

a

aa

a

a

a

abab

b

ab

b

cc

b

c

b

bcbc

b

c

b

abcbc

b

bc

0

1

2

3

4

5

Epid

idym

al

Perir

enal

Retro

perit

onea

l

Mes

ente

ric

Tota

l


CD

HFD01GCD03GCD09GCD

CQD

(e)

CD HFD 03GCD CQD

(f)


c

a

bb

40

60

80

100

120

CD HFD 03GCD CQD

(g)






bc

ab

bc c bc

02

05

08

11

14

CD HFD 01 03 09 CQD

Triglyceride (mM)

GCD

(a)

b

a a

b b b

4000

5000

6000

7000

8000

CD HFD 01 03 09 CQD


GCD

(b)

c

a a

bb b

203040506070

CD HFD 01 03 09 CQD


GCD

(c)

e

ab

cd c

00

60

120

180

240

300

CD HFD 01 03 09 CQD

Leptin (ngmL)

GCD

(d)

a

ed

c bc

70

100

130

160

190

220

CD HFD 01 03 09 CQD

Adiponectin (ngmL)

GCD

(e)

b

a

b b b b

00

200

400

600

800

CD HFD 01 03 09 CQD

IL-6 (pgmL)

GCD

(f)

ba

a

b bb

500

560

620

680

740

800

CD HFD 01 03 09 CQD

MCP-1 (pgmL)

GCD

(g)





OGTT


5090

130170210250290330

0 30 60 90 120Time (min)

Glu

cose

(mm

olL

)

(a)

d

a

abc

bc bc

27

33

39

45

51

57


AUC (mmolLmiddoth)

(b)

b

a a

b b b

100

150

200

250

300

CD HFD 01 03 09 CQD

Glucose (mmolL)

GCD

(c)

c

a ab b b

00

03

06

09

12

15

CD HFD 01 03 09 CQD

Insulin (ngmL)

GCD

(d)

c

a a

b bb

0

03

06

09

12

15

CD HFD 01 03 09 CQD

HOMA-IR (mmolL)

GCD

(e)






SFRP5

DKK2

WNT10b

GAPDH

CD HFD 03GCD CQD

b b a

a

a

b

b

bab

b

a

0

05

1

15

2

25

3

SFRP5 DKK2 WNT10b

CDHFD

03GCDCQD

Relat

ive g

ene e

xpre

ssio

n

(a)

GAPDH

CDHFD

03GCDCQD

a

c

a

b

02

04

06

08

10

12

CD HFD 03GCD CQD


120573-C

aten

inG

APD

H

(b)

CDHFD

03GCDCQD

GalaninGalR1GalR2

Cyc-DGAPDH

b c b b b

aa

a

a ab b bb bb c

b b b

005

115

225

335

4


ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

PKC120575

PKC120575

(c)

FASLeptinCD36

aP2GAPDH

b b c b b b

a

a

a a

a a

bb

bb

bbb b b b b

005

115

225

335

445

FAS Leptin CD36 aP2

Relat

ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

CDHFD

03GCDCQD

bc

PPAR1205742CEBP120572

PPAR1205742 CEBP120572

(d)



TLR2

TLR4

GAPDH

CD HFD 03GCD CQD

b b

a

a

bb

b b

0

05

1

15

2

25

3

35

TLR2 TLR4

Relat

ive g

ene e

xpre

ssio

n

CDHFD

03GCDCQD

(a)

p-JNK

JNK

CD HFD 03GCD CQD

b

a

b

b

06

09

12

15

18

24

21

CD HFD 03GCD CQD

p-JN

KJN

K (fo

ld ch

ange

ver

sus C

D)

CDHFD

03GCDCQD

(b)

MCP1

IL-6GAPDH

b b b b b

a

a

a a

a

bb b b bb

b b b b

012345

MCP1 IL-6Relat

ive g

ene e

xpre

ssio

n

CD HFD

03G

CD

CQD

TNF120572

TNF120572

IFN120572

IFN120572

IFN120573

IFN120573

CDHFD

03GCDCQD

(c)





4 Discussion



CD HFD 03GCD CQD

050

080

110

140

170

200

CD HFD 03GCD CQD

p-IR

S1to

tal I

RS1

b

a

bb

p-IRS1(Ser307)

IRS1

CDHFD

03GCDCQD

(a)

04

06

08

10

12

CD HFD 03GCD CQD

p-A

KTto

tal A

KT

c

a bb

p-AKT (Thr308)

AKT

CD HFD 03GCD CQD

CDHFD

03GCDCQD

(b)

Glut4(M)

Glut4(T)

GAPDH

CD HFD 03GCD CQD

c

a

b

a

b

a

00

03

06

09

12

15

Glut4(M) Glut4(T)

Relat

ive p

rote

in ex

pres

sion

aa120573-Actin

CDHFD

03GCDCQD

(c)





P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1


FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















bc

ab

bc c bc

02

05

08

11

14

CD HFD 01 03 09 CQD

Triglyceride (mM)

GCD

(a)

b

a a

b b b

4000

5000

6000

7000

8000

CD HFD 01 03 09 CQD


GCD

(b)

c

a a

bb b

203040506070

CD HFD 01 03 09 CQD


GCD

(c)

e

ab

cd c

00

60

120

180

240

300

CD HFD 01 03 09 CQD

Leptin (ngmL)

GCD

(d)

a

ed

c bc

70

100

130

160

190

220

CD HFD 01 03 09 CQD

Adiponectin (ngmL)

GCD

(e)

b

a

b b b b

00

200

400

600

800

CD HFD 01 03 09 CQD

IL-6 (pgmL)

GCD

(f)

ba

a

b bb

500

560

620

680

740

800

CD HFD 01 03 09 CQD

MCP-1 (pgmL)

GCD

(g)





OGTT


5090

130170210250290330

0 30 60 90 120Time (min)

Glu

cose

(mm

olL

)

(a)

d

a

abc

bc bc

27

33

39

45

51

57


AUC (mmolLmiddoth)

(b)

b

a a

b b b

100

150

200

250

300

CD HFD 01 03 09 CQD

Glucose (mmolL)

GCD

(c)

c

a ab b b

00

03

06

09

12

15

CD HFD 01 03 09 CQD

Insulin (ngmL)

GCD

(d)

c

a a

b bb

0

03

06

09

12

15

CD HFD 01 03 09 CQD

HOMA-IR (mmolL)

GCD

(e)






SFRP5

DKK2

WNT10b

GAPDH

CD HFD 03GCD CQD

b b a

a

a

b

b

bab

b

a

0

05

1

15

2

25

3

SFRP5 DKK2 WNT10b

CDHFD

03GCDCQD

Relat

ive g

ene e

xpre

ssio

n

(a)

GAPDH

CDHFD

03GCDCQD

a

c

a

b

02

04

06

08

10

12

CD HFD 03GCD CQD


120573-C

aten

inG

APD

H

(b)

CDHFD

03GCDCQD

GalaninGalR1GalR2

Cyc-DGAPDH

b c b b b

aa

a

a ab b bb bb c

b b b

005

115

225

335

4


ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

PKC120575

PKC120575

(c)

FASLeptinCD36

aP2GAPDH

b b c b b b

a

a

a a

a a

bb

bb

bbb b b b b

005

115

225

335

445

FAS Leptin CD36 aP2

Relat

ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

CDHFD

03GCDCQD

bc

PPAR1205742CEBP120572

PPAR1205742 CEBP120572

(d)



TLR2

TLR4

GAPDH

CD HFD 03GCD CQD

b b

a

a

bb

b b

0

05

1

15

2

25

3

35

TLR2 TLR4

Relat

ive g

ene e

xpre

ssio

n

CDHFD

03GCDCQD

(a)

p-JNK

JNK

CD HFD 03GCD CQD

b

a

b

b

06

09

12

15

18

24

21

CD HFD 03GCD CQD

p-JN

KJN

K (fo

ld ch

ange

ver

sus C

D)

CDHFD

03GCDCQD

(b)

MCP1

IL-6GAPDH

b b b b b

a

a

a a

a

bb b b bb

b b b b

012345

MCP1 IL-6Relat

ive g

ene e

xpre

ssio

n

CD HFD

03G

CD

CQD

TNF120572

TNF120572

IFN120572

IFN120572

IFN120573

IFN120573

CDHFD

03GCDCQD

(c)





4 Discussion



CD HFD 03GCD CQD

050

080

110

140

170

200

CD HFD 03GCD CQD

p-IR

S1to

tal I

RS1

b

a

bb

p-IRS1(Ser307)

IRS1

CDHFD

03GCDCQD

(a)

04

06

08

10

12

CD HFD 03GCD CQD

p-A

KTto

tal A

KT

c

a bb

p-AKT (Thr308)

AKT

CD HFD 03GCD CQD

CDHFD

03GCDCQD

(b)

Glut4(M)

Glut4(T)

GAPDH

CD HFD 03GCD CQD

c

a

b

a

b

a

00

03

06

09

12

15

Glut4(M) Glut4(T)

Relat

ive p

rote

in ex

pres

sion

aa120573-Actin

CDHFD

03GCDCQD

(c)





P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1


FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OGTT


5090

130170210250290330

0 30 60 90 120Time (min)

Glu

cose

(mm

olL

)

(a)

d

a

abc

bc bc

27

33

39

45

51

57


AUC (mmolLmiddoth)

(b)

b

a a

b b b

100

150

200

250

300

CD HFD 01 03 09 CQD

Glucose (mmolL)

GCD

(c)

c

a ab b b

00

03

06

09

12

15

CD HFD 01 03 09 CQD

Insulin (ngmL)

GCD

(d)

c

a a

b bb

0

03

06

09

12

15

CD HFD 01 03 09 CQD

HOMA-IR (mmolL)

GCD

(e)






SFRP5

DKK2

WNT10b

GAPDH

CD HFD 03GCD CQD

b b a

a

a

b

b

bab

b

a

0

05

1

15

2

25

3

SFRP5 DKK2 WNT10b

CDHFD

03GCDCQD

Relat

ive g

ene e

xpre

ssio

n

(a)

GAPDH

CDHFD

03GCDCQD

a

c

a

b

02

04

06

08

10

12

CD HFD 03GCD CQD


120573-C

aten

inG

APD

H

(b)

CDHFD

03GCDCQD

GalaninGalR1GalR2

Cyc-DGAPDH

b c b b b

aa

a

a ab b bb bb c

b b b

005

115

225

335

4


ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

PKC120575

PKC120575

(c)

FASLeptinCD36

aP2GAPDH

b b c b b b

a

a

a a

a a

bb

bb

bbb b b b b

005

115

225

335

445

FAS Leptin CD36 aP2

Relat

ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

CDHFD

03GCDCQD

bc

PPAR1205742CEBP120572

PPAR1205742 CEBP120572

(d)



TLR2

TLR4

GAPDH

CD HFD 03GCD CQD

b b

a

a

bb

b b

0

05

1

15

2

25

3

35

TLR2 TLR4

Relat

ive g

ene e

xpre

ssio

n

CDHFD

03GCDCQD

(a)

p-JNK

JNK

CD HFD 03GCD CQD

b

a

b

b

06

09

12

15

18

24

21

CD HFD 03GCD CQD

p-JN

KJN

K (fo

ld ch

ange

ver

sus C

D)

CDHFD

03GCDCQD

(b)

MCP1

IL-6GAPDH

b b b b b

a

a

a a

a

bb b b bb

b b b b

012345

MCP1 IL-6Relat

ive g

ene e

xpre

ssio

n

CD HFD

03G

CD

CQD

TNF120572

TNF120572

IFN120572

IFN120572

IFN120573

IFN120573

CDHFD

03GCDCQD

(c)





4 Discussion



CD HFD 03GCD CQD

050

080

110

140

170

200

CD HFD 03GCD CQD

p-IR

S1to

tal I

RS1

b

a

bb

p-IRS1(Ser307)

IRS1

CDHFD

03GCDCQD

(a)

04

06

08

10

12

CD HFD 03GCD CQD

p-A

KTto

tal A

KT

c

a bb

p-AKT (Thr308)

AKT

CD HFD 03GCD CQD

CDHFD

03GCDCQD

(b)

Glut4(M)

Glut4(T)

GAPDH

CD HFD 03GCD CQD

c

a

b

a

b

a

00

03

06

09

12

15

Glut4(M) Glut4(T)

Relat

ive p

rote

in ex

pres

sion

aa120573-Actin

CDHFD

03GCDCQD

(c)





P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1


FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SFRP5

DKK2

WNT10b

GAPDH

CD HFD 03GCD CQD

b b a

a

a

b

b

bab

b

a

0

05

1

15

2

25

3

SFRP5 DKK2 WNT10b

CDHFD

03GCDCQD

Relat

ive g

ene e

xpre

ssio

n

(a)

GAPDH

CDHFD

03GCDCQD

a

c

a

b

02

04

06

08

10

12

CD HFD 03GCD CQD


120573-C

aten

inG

APD

H

(b)

CDHFD

03GCDCQD

GalaninGalR1GalR2

Cyc-DGAPDH

b c b b b

aa

a

a ab b bb bb c

b b b

005

115

225

335

4


ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

PKC120575

PKC120575

(c)

FASLeptinCD36

aP2GAPDH

b b c b b b

a

a

a a

a a

bb

bb

bbb b b b b

005

115

225

335

445

FAS Leptin CD36 aP2

Relat

ive g

ene e

xpre

ssio

nCD HFD

03G

CD

CQD

CDHFD

03GCDCQD

bc

PPAR1205742CEBP120572

PPAR1205742 CEBP120572

(d)



TLR2

TLR4

GAPDH

CD HFD 03GCD CQD

b b

a

a

bb

b b

0

05

1

15

2

25

3

35

TLR2 TLR4

Relat

ive g

ene e

xpre

ssio

n

CDHFD

03GCDCQD

(a)

p-JNK

JNK

CD HFD 03GCD CQD

b

a

b

b

06

09

12

15

18

24

21

CD HFD 03GCD CQD

p-JN

KJN

K (fo

ld ch

ange

ver

sus C

D)

CDHFD

03GCDCQD

(b)

MCP1

IL-6GAPDH

b b b b b

a

a

a a

a

bb b b bb

b b b b

012345

MCP1 IL-6Relat

ive g

ene e

xpre

ssio

n

CD HFD

03G

CD

CQD

TNF120572

TNF120572

IFN120572

IFN120572

IFN120573

IFN120573

CDHFD

03GCDCQD

(c)





4 Discussion



CD HFD 03GCD CQD

050

080

110

140

170

200

CD HFD 03GCD CQD

p-IR

S1to

tal I

RS1

b

a

bb

p-IRS1(Ser307)

IRS1

CDHFD

03GCDCQD

(a)

04

06

08

10

12

CD HFD 03GCD CQD

p-A

KTto

tal A

KT

c

a bb

p-AKT (Thr308)

AKT

CD HFD 03GCD CQD

CDHFD

03GCDCQD

(b)

Glut4(M)

Glut4(T)

GAPDH

CD HFD 03GCD CQD

c

a

b

a

b

a

00

03

06

09

12

15

Glut4(M) Glut4(T)

Relat

ive p

rote

in ex

pres

sion

aa120573-Actin

CDHFD

03GCDCQD

(c)





P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1


FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TLR2

TLR4

GAPDH

CD HFD 03GCD CQD

b b

a

a

bb

b b

0

05

1

15

2

25

3

35

TLR2 TLR4

Relat

ive g

ene e

xpre

ssio

n

CDHFD

03GCDCQD

(a)

p-JNK

JNK

CD HFD 03GCD CQD

b

a

b

b

06

09

12

15

18

24

21

CD HFD 03GCD CQD

p-JN

KJN

K (fo

ld ch

ange

ver

sus C

D)

CDHFD

03GCDCQD

(b)

MCP1

IL-6GAPDH

b b b b b

a

a

a a

a

bb b b bb

b b b b

012345

MCP1 IL-6Relat

ive g

ene e

xpre

ssio

n

CD HFD

03G

CD

CQD

TNF120572

TNF120572

IFN120572

IFN120572

IFN120573

IFN120573

CDHFD

03GCDCQD

(c)





4 Discussion



CD HFD 03GCD CQD

050

080

110

140

170

200

CD HFD 03GCD CQD

p-IR

S1to

tal I

RS1

b

a

bb

p-IRS1(Ser307)

IRS1

CDHFD

03GCDCQD

(a)

04

06

08

10

12

CD HFD 03GCD CQD

p-A

KTto

tal A

KT

c

a bb

p-AKT (Thr308)

AKT

CD HFD 03GCD CQD

CDHFD

03GCDCQD

(b)

Glut4(M)

Glut4(T)

GAPDH

CD HFD 03GCD CQD

c

a

b

a

b

a

00

03

06

09

12

15

Glut4(M) Glut4(T)

Relat

ive p

rote

in ex

pres

sion

aa120573-Actin

CDHFD

03GCDCQD

(c)





P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1


FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CD HFD 03GCD CQD

050

080

110

140

170

200

CD HFD 03GCD CQD

p-IR

S1to

tal I

RS1

b

a

bb

p-IRS1(Ser307)

IRS1

CDHFD

03GCDCQD

(a)

04

06

08

10

12

CD HFD 03GCD CQD

p-A

KTto

tal A

KT

c

a bb

p-AKT (Thr308)

AKT

CD HFD 03GCD CQD

CDHFD

03GCDCQD

(b)

Glut4(M)

Glut4(T)

GAPDH

CD HFD 03GCD CQD

c

a

b

a

b

a

00

03

06

09

12

15

Glut4(M) Glut4(T)

Relat

ive p

rote

in ex

pres

sion

aa120573-Actin

CDHFD

03GCDCQD

(c)





P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1


FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















P

LRP

P

Adipogenesis

AP-1

PJNK

Inflammation

Nucleus

HFDGCD

FAS

aP2

LeptinCD36

MCP1


FZDR

SFRP5

DKK2WNT10b

LEFTCF

ERK

GalR1 GalR2

FFA

TLR2 TLR4

IL-6

Cyc-D

Galanin

AP-1

FFA

P

AKT

GLUT4vesicle

GLUT4

GLUT4 translocation

P IRS1(Ser307)

RI

120573-Catenin

120573-Catenin

120573-Catenin

PPAR1205742

PKC120575

CEBP120572

TNF120572

IFN120572IFN120573

















Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























Acknowledgment


References


















































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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