research article olive leaf extract attenuates obesity in...

Research ArticleOlive Leaf Extract Attenuates Obesity in High-Fat Diet-FedMice by Modulating the Expression of Molecules Involved inAdipogenesis and Thermogenesis

Ying Shen Su Jin Song Narae Keum 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 2 October 2013 Revised 12 December 2013 Accepted 17 December 2013 Published 28 January 2014

Academic Editor Yoshiyuki Kimura

Copyright copy 2014 Ying Shen 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

The present study aimed to investigate whether olive leaf extract (OLE) prevents high-fat diet (HFD)-induced obesity in mice andto explore the underlying mechanisms Mice were randomly divided into groups that received a chow diet (CD) HFD or 015OLE-supplemented diet (OLD) for 8 weeks OLD-fedmice showed significantly reduced body weight gain visceral fat-pad weightsand plasma lipid levels as compared with HFD-fed mice OLE significantly reversed the HFD-induced upregulation of WNT10b-and galanin-mediated signaling molecules and key adipogenic genes (PPAR120574 CEBP120572 CD36 FAS and leptin) in the epididymaladipose tissue of HFD-fed mice Furthermore the HFD-induced downregulation of thermogenic genes involved in uncoupledrespiration (SIRT1 PGC1120572 and UCP1) and mitochondrial biogenesis (TFAM NRF-1 and COX2) was also significantly reversedby OLE These results suggest that OLE exerts beneficial effects against obesity by regulating the expression of genes involved inadipogenesis and thermogenesis in the visceral adipose tissue of HFD-fed mice

1 Introduction

Obesity is an energy balance disorder in which energyintake exceeds energy expenditure resulting in excessiveaccumulation of white adipose tissue (WAT) [1] Adipoge-nesis is the process by which the mesenchymal precursorcells differentiate into adipocytes [2] Research over the pastdecade has established the Wnt120573-catenin signaling pathwayas an important regulator of adipogenesis [3] WNT10bexpression inhibits adipogenesis by suppressing the expres-sion of transcription factors proliferator-activated receptorgamma (PPAR120574) and CCAATenhancer-binding proteinalpha (CEBP120572) [4] thereby inhibiting WAT developmentin vivo Galanin has also been reported to play an essentialrole in regulating adipogenesis in animal models [5] HFD-induced adipogenesis in the visceral adipose tissue of micecan be activated through the expression of galanin andits receptors together with the expression of the down-stream molecules such as Ras c-Raf protein kinase C delta(PKC120575) and extracellular signal-regulated kinases (ERKs)

that stimulate the activation of PPAR120574 and CEBP120572 [5]Thustherapeutic agents that increase WNT10b expression anddecrease galanin expression may suppress the expression ofadipogenic molecules thereby inhibiting adipogenesis [6]

Unlike WAT brown adipose tissue (BAT) is an essentialcomponent of energy expenditure as it dissipates energy inthe form of heat [7] a process known as thermogenesisAdipose mitochondria play a pivotal role in adaptive ther-mogenesis the key adipose-specific metabolic pathway thatis regulated by peroxisome proliferator-activated receptor 120574coactivator-1alpha (PGC-1120572) [8]This pathway oxidizes lipidsand dissipates heat energy as a result of the uncouplingof the mitochondrial electron transport chain due to ATPproduction by uncoupling protein 1 (UCP1) Thermogenesisis elevated in the mitochondria-rich BAT and it is alsoobserved in WAT which contains brown-like cells [9 10]Thermogenesis has been demonstrated to be modified bymacronutrient content in the diet dietary carbohydrateand fat type [11 12] Phytochemicals like resveratrol [13]and epigallocatechin gallate [14] have been reported to

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 971890 12 pageshttpdxdoiorg1011552014971890

2 Evidence-Based Complementary and Alternative Medicine

increase energy expenditure and thermogenesis in animalsand humans Therefore it is possible that novel therapeuticagents activating or elevating the expression of PGC-1120572 andUCP1 would have crucial impacts on increasing the energyexpenditure to ultimately prevent obesity

Olive (Olea europaea) leaf has been widely used intraditional remedies as well as in human diet as an extractin herbal tea and in the powder form in the European andMediterranean countries [15] Olive leaf extract (OLE) ismarketed as a natural nutraceutical with wide-ranging healthbenefits In recent years research has mainly focused on theeffects of extracts from olive leaves related to the preventionof hypertension [16] atherosclerosis [17] cancer [18] diabetes[19] and cardiovascular diseases [20 21] Olive leaves containseveral different compounds collectively termed as olivebiophenols which impart the therapeutic properties Themost abundant biophenol is oleuropein followed by otherbiophenols such as verbascoside luteolin rutin catechinand hydroxytyrosol in lower quantities [22] Our previousstudy demonstrated that the addition of oleuropein to HFDdecreased body weight gain and improved the lipid pro-files in the plasma of mice [23] Although several studieshave investigated the biochemical roles of olive leaves andolive leaves preparations their protective effect against diet-induced obesity has never been reported Therefore theobjective of this study was to investigate the weight-reducingeffect of a well-defined OLE by supplementation in HFD-induced mice and to investigate the underlying mechanismsof this effect with a particular focus on the expression ofmolecules involved in adipogenesis and thermogenesis

2 Materials and Method

21 Materials Dry OLE was obtained from FrutaromSwitzerland Ltd (Wadenswil Switzerland) under the tradename of Benolea The extract was manufactured from thedried leaves of Olea europaea L applying an ethanol (80mm) extraction procedure After a patented filtration pro-cess (EFLA Hyperpure) the crude extract was dried Finally15 (mm) Acaciae Gummi Ph Eur was added as a carriertogether with lt05 (mm) of silica colloidalis anhydrica PhEur The extract is characterized by a DER of 3ndash7 = 1 (5 =1) Characteristic component is 16ndash24 (mm) oleuropeinSibutramine was purchased from Sigma-Aldrich (MA USA)

22 High-Performance Liquid Chromatography (HPLC) Anal-ysis of OLE HPLC analysis was conducted using the Nova-Pak C18 column (150mm times 39mm 60AWaters AssociatesHarrow UK) at 25∘C at a flow rate of 1mLmin by using agradient mobile phase (water methanol in 600 400 ratio)as the initial condition of the chromatography The sampleinjection volume used was 10 120583L The absorption signal ofoleuropein was examined at 233 nm by using the UV detector(Agilent Technologies DE USA)

23 Animal and Diets Five-week-old male C57BL6N mice(Orient Bio Gyeonggi-do Korea)were housed in a pathogen-free facility at 21plusmn 20∘C with 50plusmn 5 relative humidity and a

Table 1 Composition of the experimental diets

Ingredients HFD(gkg diet)

OLD(gkg diet)

SD(gkg diet)

Casein 200 200 200DL-methionine 3 3 3Corn starch 111 1095 1109Sucrose 370 370 370Cellulose 50 50 50Corn oil 30 30 30Lard 170 170 170Mineral mixture1 42 42 42Vitamin mixture2 12 12 12Choline bitartrate 2 2 2Cholesterol 10 10 10tert-Butylhydroquinone3 004 004 004

Olive leaf extract mdash 15 mdashSibutramine mdash mdash 01Total (g) 1000 1000 1000Fat ( calories) 40 40 40Total energy(kcalkg diet) 4616 4616 46161AIN-76A mineral mix2AIN-76A vitamin mix3Antioxidant agent 001 g50 g lipids

12 h lightdark cycle the mice were provided access to rodentchow and tap water ad libitum for 1 weekThereafter themicewere divided into the following 4 groups matched for bodyweight (119899 = 8 in each group) the chow diet (CD) high-fatdiet (HFD) 015 (ww)OLE-supplemented diet (OLD) and001 (ww) sibutramine-supplemented diet (SD) groupsThe HFD consisted of 200 g fatkg body weight (170 g of lardand 30 g of corn oil) and 1 (ww) cholesterol The OLD wasidentical to the HFD and contained 015 OLE The SD wasalso identical to the HFD except that it was supplementedwith 001 sibutramineThe composition of the experimentaldiets including total energy (kcalkg diet) is shown in Table 1All the diets were provided in the form of pellets for 8 weeks

The food intake was monitored daily and body weightswere recorded on a weekly basis At the end of the experi-mental period the mice were anesthetized with diethyl etherafter they were fasted for 16 h Blood samples were drawnfrom the inferior vena cava into an ethylene-diamine-tetra-acetic acid (EDTA)-coated tube and the plasma sampleswere obtained by centrifuging the blood at 4000timesg for15min at 4∘C The epididymal retroperitoneal mesentericand perirenal fat-pads were dissected removed weighedand immediately snap-frozen in liquid nitrogen and storedat minus80∘C until further use All animal experiments wereperformed in accordance with the Korea Food and DrugAdministration (KFDA) guidelines All the experimentalprotocols were reviewed and approved by the InstitutionalAnimal Care and Use Committee (IACUC) of the YonseiLaboratory Animal Research Center (YLARC)

Evidence-Based Complementary and Alternative Medicine 3

80

60

40

20

0

0

2 4 6 8 10 12 14 16 18

(min)

(mAu

)

Ole

urop

ein

DAD1 A Sig = 233 4 Ref = off (OLEUROPEIN20130605KDH 2013-06-07 16-32-30201306070000002D)

Figure 1 High-performance liquid chromatography (HPLC) chro-matogram of the olive leaf extract (OLE) The peak was assignedbased on the isolation of oleuropein

24 Histological Analysis The epididymal fat-pads werefixed in 10 buffered formalin and embedded in paraffinblocks The paraffin blocks were sectioned and stained withhematoxylin-eosin (HampE)The sectional areas of the epididy-mal fat-pads were analyzed to quantify the size of the fatdroplets

25 Biochemical Analysis Plasma concentrations of totalcholesterol triglyceride HDL cholesterol free fatty acidand glucose were enzymatically determined using commer-cial kits (Bio-Clinical System Gyeonggi-do South Korea)Plasma levels of LDL + VLDL cholesterol were calculatedby subtracting the HDL cholesterol level from the totalcholesterol level Plasma level of leptin was measured usinga commercially available mouse enzyme-linked immunosor-bent assay (ELISA) kit (Millipore MA USA)

26 RNA Extraction and Semiquantitative Reverse Transcrip-tase-Polymerase Chain Reaction (RT-PCR) Total RNA wasisolated from the epididymal adipose tissue of each mouseby using TRIzol (Invitrogen CA USA) and was reverse-transcribed using the superscript II kit (Invitrogen) accordingto the manufacturerrsquos instructions The GenBank accessionnumbers of the relevant templates and the forward (F) andreverse (R) primer sequences are shown in Table 2 The PCRwas programmed as follows 10min at 94∘C 30ndash35 cyclesof 94∘C for 30 s 55∘C for 30 s 72∘C for 1min and 10minincubation at 72∘C Then 5120583L of each PCR product wasmixed with 1 120583L of 6-fold concentrated loading buffer thissolution was run on a 2 agarose gel containing ethidiumbromide The band intensities were quantified using theQuantity One Analysis Software (Bio-Rad CA USA) ThemRNA levels were normalized to that of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript

27 Western Blotting The epididymal adipose tissue samplesobtained from each mouse were homogenized in an extrac-tion buffer containing 100mM Tris-HCl (pH 74) 5mMEDTA 50mMNaCl 50mM sodium pyrophosphate 50mM

NaF 100mM orthovanadate 1 Triton X-100 1mM phenyl-methanesulfonyl fluoride 2120583gmL aprotinin 1120583gmL pep-statin A and 1 120583gmL leupeptin The homogenates were cen-trifuged at 1300timesg for 20min at 4∘CThe protein concentra-tions of the tissue extracts were measured by Bradford assay(Bio-Rad)The protein samples were separated by 8 sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(PAGE) and then electrophoretically transferred to nitro-cellulose membranes (Amersham Buckinghamshire UK)Thenitrocellulosemembranes were incubated overnight withprimary antibodies (diluted 1 1000) at 4∘C Antibodies tothe following proteins were commercially obtained fromthe indicated sources 120573-catenin and 120573-actin from SantaCruz Biotechnology (Santa Cruz CA USA) and extracellularsignal-related kinase (ERK) phospho-ERK (Thr202Tyr204)cyclicAMP-responsive element-binding protein (CREB) andphospho-CREB (Ser133) from Cell Signaling Technology(MA USA) The membranes were incubated with the cor-responding secondary antibodies and the immunoreactivesignals were detected using a chemiluminescent detectionsystem (Amersham) followed by quantification using theQuantity One Analysis Software (Bio-Rad)

28 Statistical Analysis The results on body weight gainplasma biochemistries and adipocyte diameter are presentedas themeanplusmn standard error ofmean (SEM) of 8mice in eachgroup The RT-PCR and western blotting data are shown asthe means plusmn SEM of three independent experiments (119899 = 2or 3 per experiment) for each group cumulatively including 8miceThe statistical significance of differencewas determinedusing one-way analysis of variance followed by Duncanrsquosmultiple-range tests All statistical analyses were performedwith SPSS 210 software 119875 le 005 was considered statisticallysignificant

3 Results

31 HPLC Analysis of OLE HPLC analysis revealed thatthe OLE used in the present study Benolea contained 16oleuropein (Figure 1)

32 Effects of OLE on Body and Visceral Fat-Pad WeightsHFD-fed mice showed significant increases in final bodyweight (38 119875 lt 005) and cumulative body weight gain(162 119875 lt 005) as compared with CD-fed mice OLD-fedmice showed a 37 reduction in final body weight and an80 decrease in body weight gain as compared with HFD-fed mice (Figures 2(a) and 2(b)) The food intake was alsosignificantly lower in OLD-fed mice (minus26 119875 lt 005) thanin HFD-fedmice during the 8-week feeding periodThe foodefficiency ratio (FER) (minus74 119875 lt 005) of OLD-fed mice wassignificantly lower than that of HFD-fed mice (Figures 2(c)and 2(d)) The cumulative caloric intake over 8 weeks was26 less in OLD-fed mice than in HFD-fed mice (660 versus887 kcal) The OLD group also showed significantly lowervalues for total visceral (epididymal mesenteric perirenaland retroperitoneal) fat-pad weights (minus72 119875 lt 005) thanthose observed in the HFD group Histological sections from


Table 2 Primer sequences and RT-PCR conditions

Gene description Primers Sequences (51015840 rarr 31015840) 119879119898

(∘C) Size (bp)

Peroxisome proliferator-activated receptor 120574 (PPAR120574) F TTCGGAATCAGCTCTGTGGA 55 148R CCATTGGGTCAGCTCTTGTG

CCAATenhancer binding-protein 120572 (CEBP120572) F AAGGCCAAGAAGTCGGTGGA 55 189R CCATAGTGGAAGCCTGATGC

Leptin F CTCCAAGGTTGTCCAGGGTT 55 143R AAAACTCCCCACAGAATGGG

Cluster of differentiation 36 (CD36) F ATGACGTGGCAAAGAACAGC 55 160R GAAGGCTCAAAGATGCCTCC

Fatty acid synthase (FAS) F TTGCCCGAGTCAGAGAACC 55 171R CGTCCACAATAGCTTCATAGC

Wingless-type MMTV integration site family member 10b (WNT10b) F TTTTGGCCACTCCTCTTCCT 61 183R TCCTTTTCCAACCGAAAACC

Secreted frizzled-related protein 5 (SFRP5) F CTGATGGCCTCATGGAACAG 55 155R CTTGGTGTCCTTGCGCTTTA

Galanin F GAGCCTTGATCCTGCACTGA 60 121R AGTGGCTGACAGGGTCACAA

Galanin receptor 1 (GalR1) F CCAAGGGGGTATCCCAGTAA 55 147R GGCCAAACACTACCAGCGTA

Galanin receptor 2 (GalR2) F ATAGTGGTGCTCATGCTGGAA 60 134R AGGCTGGATCGAGGGTTCTA

Protein kinase C 120575 (PKC120575) F CTGAGCGCTGCAAGAAGAAC 60 146R TGGAAACTTTGATCCTGCACTGA

Peroxisome proliferative activated receptor 120574 coactivator 1120572 (PGC-1120572) F TAAATCTGCGGGATGATGGA 55 117R GTTTCGTTCGACCTGCGTAA

Uncoupling protein 1 (UCP1) F GGTTTTGCACCACACTCCTG 55 111R ACATGGACATCGCACAGCTT

Cyclin D (cyc-D) F TGGGAAGTTTTGTTGGGTCA 55 144R TCCTTGTCCAGGTAATGCCA

E2F1 F CCTGGAGCATGTTAAAGAAG 55 102R CCTCGAGACCAAAGTGATAG

Sirtuin 1 (SIRT1) F AGCTCCTTGGAGACTGCGAT 55 182R ATGAAGAGGTGTTGGTGGCA

Cytochrome C oxidase subunit 2 (COX2) F CCGAGTCGTTCTGCCAATAG 55 159R AACCCTGGTCGGTTTGATGT

Mitochondrial transcription factor A (TFAM) F AGTGTGGCAGTCCATAGGCA 55 123R CAGTGCTTTTAGCACGCTCC

Nuclear respiratory factor-1 (NRF-1) F TTTCATGGACCCAGGCATTA 55 119R TGGTGGCCTGAGTTTGTGTT

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) F AGAACATCATCCCTGCATCC 55 321R TCCACCACCCTGTTGCTGTA

the epididymal adipose tissue of HFD-fed mice presented agreater adipocyte diameter as compared with CD-fed micethis change in the adipocyte size was significantly reversedby OLE supplementation (minus47 119875 lt 005) (Figures 2(e) and2(f)) OLE had greater effects on lowering body weight foodintake total visceral fat-pad weights and adipocyte size thandid sibutramine (Figures 2(a)ndash2(f))

33 Effects of OLE on Plasma Biochemistries OLE supple-mentation resulted in significantly lower concentrations of

triglyceride (minus55 119875 lt 005) total cholesterol (minus28119875 lt 005) LDL + VLDL cholesterol (minus62 119875 lt 005)FFA (minus42 119875 lt 005) glucose (minus17 119875 lt 005) andleptin (minus69 119875 lt 005) in the plasma of HFD-fed mice(Figures 3(a)ndash3(f))The effect of OLE on reducing the plasmalevels of triglyceride total cholesterol and LDL + VLDLcholesterol was significantly greater than that of sibutramine(Figures 3(a)ndash3(c)) OLE significantly reduced the plasmaFFA and leptin levels similar to sibutramine (Figures 3(d)and 3(f)) however OLE was less effective in reducing theplasma glucose level than was sibutramine (Figure 3(e))


b b c c cc c c c

a aa

aa

aa a

a

b b bb

bc d d d

bb b

bb

b b bb

20

25

30

35

40

0 4 7 14 21 28 35 42 49 56

CDHFD

OLDSD

Body weight change (g)

Days

(a)

c

a

d

b

0

5

10

15

20

CD HFD OLD SD

Body weight gain (g8wks)

(b)

CD HFD OLD SD

ab

cb

0

1

2

3

4

5 Food intake (gday)

(c)

c

a

c

b

000002004006008010

CD HFDOLD SD

FER

(d)

cc c c

c

a

a a a

a

cc c c

cb

b b b

b

0

1

2

3

4

Epididymal Perirenal Retroperitoneal Mesenteric Total

CDHFD

OLDSD

Visceral fat-pad weights (g)

(e)

CD

SDOLD

HFD

100120583m 100120583m

100120583m100120583m

b

a

c

b

0

20

40

60

80

100

CD HFD OLD SD

Adipocyte diameter (120583m)

(f)

Figure 2 Effects of OLE on body weight gain food efficiency ratio (FER) and visceral fat-pad weights of HFD-fed mice Mice were fedCD HFD OLD or SD for 8 weeks Changes in the (a) body weight (b) body weight gain (c) food intake (d) FER and (e) visceral fat-padweights (f) Histological analysis of the epididymal adipose tissue from each group was conducted to quantify the size of adipocyte (Left)Representative pictures of hematoxylin-eosin (HampE)-stained fat cells from the mice epididymal adipose tissue (times100) (Right) Densitometricanalysis of the average adipocyte diameter in the epididymal adipose tissue of mice Values are presented as means plusmn SEM (119899 = 8) Meanvalues indicated with different letters indicate statistical significance (119875 lt 005) FER = Body weight gain for experimental period (g)foodintake for experimental period (g)

34 Effects of OLE on the Expression ofMoleculesThat RegulateAdipogenesis We investigated whether OLE affected HFD-induced activation of adipogenesis in the epididymal adiposetissue of mice In the present study we found that OLE

significantly upregulated the expression of wingless-typeMMTV integration site family member 10b (WNT10b) (79119875 lt 005) and low-density lipoprotein receptor-relatedprotein 5 (LRP5) (26 119875 lt 005) and downregulated


b

a

c

b

00

03

06

09

12

15

CD HFD OLD SD

Triglyceride (mmolL)

(a)

d

a

cb

0

2

4

6

CD HFD OLD SD

Total cholesterol (mmolL)

(b)

c

a

c

b

0

1

2

3

CD HFD OLD SD

LDL + VLDL cholesterol (mmolL)

(c)

CD HFD OLD SD

ab

a

b b

0

200

400

600

800Free fatty acid (120583EqL)

(d)

CD HFD OLD SD

c

a

b

c

0

5

10

15Glucose (mmolL)

(e)

b

a

bb

0

6

12

18

24

30

CD HFD OLD SD

Leptin (ngmL)

(f)

Figure 3 Effects of OLE on plasma levels of lipids glucose and leptin in HFD-fed mice (a) Triglyceride (b) total cholesterol (c) LDL +VLDL cholesterol (d) free fatty acid (e) glucose and (f) leptin Bars represent the mean plusmn SEM (119899 = 8) Mean values indicated with differentletters indicate statistical significance (119875 lt 005)

the expression of secreted frizzled-related protein 5 (SFRP5)(minus59 119875 lt 005) in the epididymal adipose tissue of HFD-fed mice (Figure 4(a)) Western blotting of the epididymaladipose tissue of mice revealed a significantly higher proteinlevel of 120573-catenin (300 119875 lt 005) in OLD-fed mice relativeto HFD-fedmice (Figure 4(b)) Furthermore compared withHFD-fed mice OLD-fed mice showed significantly lowerexpression of galanin (minus65 119875 lt 005) galanin receptor1 (GalR1) (minus62 119875 lt 005) galanin receptor 2 (GalR2)(minus62 119875 lt 005) PKC120575 (minus74 119875 lt 005) cyclinD (Cyc-D) (minus68 119875 lt 005) and E2F1 (minus25 119875 lt005) in the epididymal adipose tissue of mice (Figure 4(c))OLE supplementation resulted in decreased protein levels ofphosphorylated ERK (Thr202Tyr204) (minus38 119875 lt 005) inHFD-fed mice (Figure 4(d)) The mRNA levels of CEBP120572PPAR120574 and its target genes (CD36 FAS and leptin) weresignificantly downregulated in OLD-fed mice as comparedwith those in HFD-fed mice (Figure 4(e))

35 Effects of OLE on the Expression of Molecules Involved inThermogenesis We next assessed whether OLE can inducethermogenesis in the epididymal adipose tissue of miceThe mRNA levels of mitochondrial protein cytochrome Coxidase subunit 2 (COX2) (93 119875 lt 005) mitochondrial

transcription factor A (TFAM) (71 119875 lt 005) nuclearrespiratory factor-1 (NRF-1) (53119875 lt 005) sirtuin 1 (SIRT1)(87 119875 lt 005) PGC-1120572 (89 119875 lt 005) and UCP1 (77119875 lt 005) were significantly elevated inOLD-fedmice than inHFD-fedmice (Figure 5(a)) OLE supplementation increasedthe protein level of phosphorylatedCREB (450119875 lt 005) inthe epididymal adipose tissue of HFD-fedmice (Figure 5(b))

4 Discussion

OLE is best known for its blood pressure-lowering effect inthe global market of functional food industry In 2008 OLEunder the trade name Benolea was approved by KFDA asa health functional food that exerts blood pressure-loweringeffect at a daily recommended dose of 500ndash1000mg In addi-tion a clinical trial conducted by Perrinjaquet-Moccetti et alconfirmed that no adverse event was observed at a daily givendose of 1000mg [21] The concentration of OLE (015) usedin the present study was in the same range as it was equivalentto an intake of approximately 732mg60 kg human bodyweight when calculated on the basis of normalization to thebody surface area as described by the recommendations byReagan-Shaw et al [24] and the US Food and Drug Adminis-tration (httpwwwfdagovcdercanceranimalframehtm)


b a a

a

CD

SFRP5

WNT10b

LRP5

GAPDH

HFD OLD CD HFD OLD

b cca

b

00

05

10

15

20

25

SFRP5 WNT10b LRP5

CDHFDOLD

Relat

ive g

ene e

xpre

ssio

n

(a)

CD HFD OLD

a

b

a

00

05

10

15

20

CD HFD OLD

Relat

ive p

rote

in ex

pres

sion

120573-Catenin

120573-Actin

(b)

b b b b b b

a a a aa

a

b b bc

b

b

00051015202530

Galanin

Galanin

GalR1

GalR1

GalR2

GalR2

Cyc-D

Cyc-D

E2F1

E2F1

CD

CD

HFD

GAPDH

HFD

OLD

OLD CD HFD OLD

Rela

tive g

ene

expr

essio

n

PKC120575

PKC120575

(c)

CD HFD OLD

b

a

b

00

05

10

15

20

CD HFD OLD

Total ERK

(Thr202Tyr204)

Rela

tive p

rote

inex

pres

sion

p-ERK

(d)

b b b b ba a a

a a

b b b cb

00

05

10

15

20

25

CD36

CD36

FAS

FAS

Leptin

Leptin

GAPDH

CDHFDOLD

CD HFD OLD

PPAR120574

PPAR120574

CEBP120572

CEBP120572

Relat

ive g

ene e

xpre

ssio

n

(e)

Figure 4 Effects of OLE on genes regulating adipogenesis in the epididymal adipose tissue of HFD-fed mice (a) Gene expression of SFRP5WNT10b and LRP5 (b) protein levels of 120573-catenin (c) mRNA expression of genes involved in the galanin-mediated signaling pathway (d)protein levels of p-ERK and total ERK (e) expression of PPAR1205742 CEBP120572 and their target genes Reverse transcriptase-polymerase chainreaction (RT-PCR) data represent the relative density normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Proteinlevels were normalized to the 120573-actin level Data represent the results of three independent experiments (119899 = 2 or 3 per experiment) 119875 lt 005indicates statistical significance Values are the means plusmn SEM 119899 = 8 for each group


a a ab b a a

c cb c

c bb b

aa

ba

00

05

10

15

COX2

COX2

TFAM

TFAM

NRF-1

NRF-1

SIRT1

SIRT1

UCP1

GAPDH

UCP1Re

lativ

e gen

eex

pres

sion

PGC-1120572

PGC-1120572

CDHFDOLD

CD HFD OLD CD HFD OLD

(a)

a

b

a

00

05

10

15

20

CD HFD OLD

CD HFD OLD

Rela

tive p

rote

inex

pres

sion

CREB

p-CREB

(b)

Figure 5 Effects of OLE on the expression of genes involved in thermogenesis in the epididymal adipose tissue of HFD-fed mice (a) Theexpression of genes involved in uncoupled respiration and mitochondrial biogenesis was determined by RT-PCR and normalized to that ofGAPDH (b) protein levels of p-CREB and total CREB were determined by western blotting and normalized to 120573-actin protein levels Datarepresent the results of three independent experiments (119899 = 2 or 3 per experiment) 119875 lt 005 indicates statistical significance Values are themeans plusmn SEM 119899 = 8 for each group

Sibutramine is a monoamine-reuptake inhibitor [25] whichwas approved as an antiobesity drug by the FDA in 1997 andwas marketed until 2010 In the present study sibutraminewas used as a positive control to evaluate the antiobesityeffects of OLE

OLE significantly decreased visceral fat-pad weight andplasma levels of triglyceride and FFA in HFD-fed miceThe decreased visceral fat accumulation might result in thereduced plasma level of FFAThe lower content of FFA whichflows into the liver through the portal vein may cause lesstriglyceride synthesis in the liver resulting in the decreasedplasma level of triglyceride in OLD-fed mice Histologicalanalysis revealed a significantly decreased adipocyte size andan increased number of adipocyte in the OLD group than inthe HFD group

Several olive leaf constituents have been reported to exertbeneficial effects against obesity both in vitro and in vivoOleuropein [23 26] hydroxytyrosol [26 27] luteolin [28]apigenin [29 30] rutin [31] and caffeic acid [32] were foundto decrease the accumulation of intracellular lipid as detectedby Oil Red O staining and the expression of adipogenictranscription factors such as PPAR120574 and CEBP120572 in 3T3-L1 cells Hydroxytyrosol also exerts beneficial effects on thepromotion of mitochondrial biogenesis via stimulation ofthe PGC-1120572 and its downstream target genes in 3T3-L1 cells[27] Oleuropein and rutin decreased body weight gain andimproved plasma lipid profiles and hepatic steatosis in HFD-fedmice [23 31] Caffeic acid also exhibited antiobesity effectsby reducing body and visceral fat-pad weights plasma levelsof lipids and obesity-related hormones such as leptin andinsulin in HFD-fed mice [32] On the basis of the results ofthese studies we speculate that the weight-lowering effects of

OLE can be derived from the combination of various weight-suppressing components

Oleuropein the bitter principle in olive leaf accountedfor 16 of the OLE used in this study In our previousresearch 003 oleuropein supplementation significantlyreduced body weight gain (minus55) and plasma lipid levelsin HFD-fed mice for 10 weeks [23] In the present studyOLD (015 OLE-supplemented diet) contained 0024oleuropein and significantly decreased body weight gain(minus80) and obesity-related plasma biomarkers in mice after8 weeks of feeding Although OLD in the present studycontained less oleuropein and was fed for a shorter periodit exerted better weight-lowering effects than those exhibitedby oleuropein-supplemented diet in our previous study [23]In addition OLE significantly reduced food intake in thepresent study whereas oleuropein alone did not affect foodintake in mice [23] Therefore we suggest that the bodyweight-lowering effect of OLE can be derived not only fromoleuropein but also from other active components present inolive leaves that may act concomitantly to reduce food intakeand body weight gain

OLE significantly reduced food intake by 26 inHFD-fedmice for 8 weeks Several animal studies on OLE investigatedits effects on glucose metabolism ultraviolet B irradiation-induced skin changes and wound healing [33ndash35] Unfor-tunately none of the studies presented data for daily foodintake It is well-known that GLP-1 is a satiety-signaling hor-mone that inhibits food intake by upregulating the expressionof anorexigenic neuropeptides such as proopiomelanocortin(POMC) and cocaine- and amphetamine-regulated tran-script (CART) [36] In the present study the plasma GLP-1 level was not measured because freshly collected plasma


samples were not treated with dipeptidyl peptidase IV (DPPIV) inhibitor without DPP IV inhibitor GLP-1 is rapidlydegraded in plasma Green et al demonstrated that oralgavage of dichloromethane extract of olive leaves improvedglucose metabolism by significantly increasing plasma GLP-1 concentrations in mice [33] Among the constituents ofOLE apigenin is known to inhibit food intake at the dosageof 005 by increasing the expression of POMC and CARTin HFD-fed mice [29] OLD used in the present study maycontain much less apigenin than 005 as in an earlierreport leaf extract of Olea europaea L contained 137apigenin [37] Further investigation is needed to confirmwhether the food intake-reducing effect of OLE is exerted byits apigenin component

Food efficiency ratio (FER) which is the ratio of bodyweight gain (g) to food intake (g) assesses utilization offood consumed The unchanged FER demonstrates thatthe weight-lowering effect of a certain antiobesity agent iscompletely attributable to reduction in food intake In thepresent study OLE significantly decreased FER in HFD-fed mice indicating that weight-lowering effect of OLE ismediated not only by the reduction of food intake but alsoby its direct effects on adipocytes The effect of OLE inreducing the expression of adipogenic genes could be derivedfrom both the direct inhibition of adipogenesis and thereduction of total caloric intake PPAR120574 and CEBP120572 [38]are the chief regulators that act synergistically to inducethe expression of downstream target genes of adipogene-sis including FAS CD36 and leptin Animal studies havereported that WNT10b [4] and galanin [5] played importantroles in regulating the expression of PPAR1205742 and CEBP120572WNTs secreted proteins that act through autocrine andparacrine mechanisms serve as a functional ldquobrakerdquo duringpreadipocyte recruitment into the differentiation program[3] Of the 19 possible WNTs WNT10b has been most clearlyimplicated as the endogenous WNT involved in regulatingadipogenesis [39] HFD decreased the binding of WNT10bto FZD receptors and to LRP56 coreceptors leading to theactivation of the cytoplasmic 120573-catenin degradation [6] Thedecreased cytosolic accumulation and nuclear translocationof 120573-catenin led to decreased transcriptional coactivationof the T-cell factor (TCF)lymphoid-enhancer factor (LEF)transcription factors and eventually upregulated the expres-sion of PPAR120574 and CEBP120572 [40] OLE significantly reversedthe HFD-induced decrease in the expression of WNT10band LRP5 which may be associated with increased cytosolicaccumulation of 120573-catenin

In addition it is noteworthy that the WNT signalingpathway is regulated by endogenous WNT inhibitors [3]SFRP5 a WNT inhibitor directly binds to and sequestersWNT10b fromFZD receptors Epigenetic activation of SFRP5in WAT increased susceptibility to obesity in HFD-fed mice[41] In the present study SFRP5 expression was significantlyreduced in the epididymal adipose tissue of OLD-fed micethan in HFD-fed mice Therefore we presume that OLE-mediated downregulation of WNT10b inhibitor SFRP5 andupregulation of the ligand WNT10b may be related to theincreased cytosolic accumulation of 120573-catenin and thus thereduction in adipogenesis and visceral adiposity

The galanin-mediated signaling pathway is also involvedin the regulation of adipogenesis Galanin a 2930-residueneuropeptide is expressed and widely distributed in thecentral nervous system [42] However recent studies haverevealed that galanin expression has also been observed inperipheral tissues such as visceral adipose tissue and stomach[5] Our previous study demonstrated that the expression ofgalanin its receptors and other genes involved in galanin-mediated signaling pathway was significantly elevated inthe epididymal adipose tissues of HFD-fed mice [5] UponprolongedHFDconsumptionGalR1 stimulated themitogen-activated protein kinase activity in a PKC-independent man-ner and activated 12 ERKs Activation of GalR2 increasedthe expression of phospholipase C (PLC) which catalyzesthe cleavage of phosphatidylinositol 45-bisphosphate intoinositol 145-triphosphate and diacyl glycerol a PKC acti-vator Subsequently PKC120575 induced the activation of ERKwhich upregulated the expression of PPAR120574 and CEBP120572 Inthe present study OLE significantly reversed HFD-inducedelevation of the expression of genes (GalR1 GalR2 PKC120575and ERK) in the epididymal adipose tissue ofmiceThereforethe effect of OLE on the reduction of visceral fat-pad weightsis presumed to be associated with the downregulation ofgenes related to galanin-mediated adipogenesis in HFD-fedmice

In addition to the inhibition of fat accumulation inWAT improvement of the capacity of thermogenesis can alsoaccount for the body fat-lowering effect of OLE Adaptivethermogenesis is defined as heat production in response toenvironmental temperature or diet which protects the organ-ism from exposure to cold or regulates the energy balanceafter changes in the diet [1] Mitochondria the organellesthat convert food to carbon dioxide water and ATP areindispensable for energy dissipation Activation of UCP1a mitochondrial proton transporter enhances uncoupledrespiration and thereby results in dissipation of oxidationenergy as heat [1] Galanin receptors [5] and SIRT1 [43]are known to reverse obesity-induced downregulation ofUCP1 expression in WAT Our previous study demonstratedthat the HFD-induced overexpression of GalR1 and GalR2inhibited cAMP which in turn reduced protein kinase A(PKA) in the epididymal and subcutaneous adipose tissuesof mice Decreased PKA downregulated the phosphorylationof CREB leading to decreased gene expression of PGC-1120572 [5]SIRT1 which is responsible for deacetylating and activatingPGC-1120572 at multiple lysine sites was downregulated by HFDin the adipose tissue of mice [44] Consequently HFD-induced downregulation of PGC-1120572 decreased the expressionof UCP1 and eventually suppressed uncoupled respirationIn the present study OLE simultaneously reversed the HFD-induced upregulation of GalR1 and GalR2 expression anddownregulation of SIRT1 PGC-1120572 and UCP1 expression inthe epididymal adipose tissue of mice Therefore we suggestthat the visceral fat-pad weight-lowering effects of OLEcan be associated with the elevation of the gene expressioninvolved in uncoupled respiration in the visceral adiposetissue of HFD-fed mice

The coordination of mitochondrial biogenesis withuncoupled respiration is crucial in increasing the capacity


of thermogenesis [1] PGC-1120572 is a potent inducer of mito-chondrial biogenesis Shadel and Clayton demonstrated thatPGC-1120572 led to an activation of nuclear respiratory factor-1(NRF-1) which binds to and activates the gene promoter ofthe mitochondrial electron transport system such as COX2Another target of NRF-1 is TFAM which translocates intomitochondria and initiates the transcription and replicationof the mitochondrial DNA [45] OLE significantly increasedthe mRNA expression of genes involved in mitochondrialbiogenesis (PGC-1120572 NRF-1 COX2 and TFAM) in theepididymal adipose tissue of HFD-fed mice Accordinglywe presume that the visceral adiposity-suppressing effectsof OLE may be linked to the increased mitochondrialbiogenesis-related genes in the epididymal adipose tissue ofmice

In conclusion OLE significantly decreased body weightgain visceral fat-padweights and plasma lipid levels inHFD-fed mice These beneficial effects against obesity in miceappear to be mediated at least in part through downregu-lating the expression of molecules involved in adipogenesisand upregulating the expression of molecules involved inthermogenesis in the visceral adipose tissue ofHFD-fedmiceTaken together our results suggest that supplementationwithOLE might be helpful to combat or prevent obesity

Abbreviations

CEBP120572 CCAATenhancer-binding protein alphaCD Chow dietCD36 Cluster of differentiation 36CREB cAMP responsive element-binding proteinCyc-D Cyclin DCOX2 Cytochrome C oxidase subunit 2ERK Extracellular signal-regulated kinase 12FAS Fatty acid synthaseFFA Free fatty acidsGalR1 Galanin receptor 1GalR2 Galanin receptor 2GAPDH Glyceraldehyde-3-phosphate

dehydrogenaseHFD High-fat dietNRF-1 Nuclear respiratory factor-1OLE Olive leaf extractPGC1120572 Peroxisome proliferator-activated receptor

gamma coactivator 1alphaPPAR120574 Peroxisome proliferator-activated receptor

gammaPKC120575 Protein kinase C deltaSFRP5 Secreted frizzled-related protein 5SIRT1 Sirtuin 1TFAM Mitochondrial transcription factor AUCP1 Uncoupling protein 1WNT10b Wingless-type MMTV integration site

family member 10b

Conflict of Interests

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

Acknowledgments

This research was supported by the MOTIE (The Ministry ofTrade Industry and Energy) Republic of Korea under theResearch and Business Development (RampBD) support pro-gram (Grant no N0000869) supervised by the KIAT (KoreaInstitute for Advancement of Technology) and by the SRCprogram (Center for Food and Nutritional Genomics Grantno 2008-0062618) of the National Research Foundation ofKorea funded by the Ministry of Education Science andTechnology

References

[1] B B Lowell and B M Spiegelman ldquoTowards a molecularunderstanding of adaptive thermogenesisrdquoNature vol 404 no6778 pp 652ndash660 2000

[2] W P Cawthorn E L Scheller andO AMacDougald ldquoAdiposetissue stem cells the great WAT hoperdquo Trends in Endocrinologyamp Metabolism vol 23 no 6 pp 270ndash277 2012

[3] T C Prestwich and O A MacDougald ldquoWnt120573-catenin signal-ing in adipogenesis and metabolismrdquo Current Opinion in CellBiology vol 19 no 6 pp 612ndash617 2007

[4] W S Wright K A Longo V W Dolinsky et al ldquoWnt10binhibits obesity in obob and agouti micerdquo Diabetes vol 56 no2 pp 295ndash303 2007

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

[6] C Christodoulides C Lagathu J K Sethi and A Vidal-PuigldquoAdipogenesis and WNT signallingrdquo Trends in Endocrinologyand Metabolism vol 20 no 1 pp 16ndash24 2009

[7] J Boucher M A Mori K Y Lee et al ldquoImpaired ther-mogenesis and adipose tissue development in mice with fat-specific disruption of insulin and IGF-1 signallingrdquo NatureCommunications vol 3 article 902 2012

[8] P Puigserver ZWuCW Park RGravesMWright andBMSpiegelman ldquoA cold-inducible coactivator of nuclear receptorslinked to adaptive thermogenesisrdquo Cell vol 92 no 6 pp 829ndash839 1998

[9] P Seale H M Conroe J Estall et al ldquoPrdm16 determines thethermogenic program of subcutaneous white adipose tissue inmicerdquoThe Journal of Clinical Investigation vol 121 no 1 pp 96ndash105 2011

[10] H Wang Y Zhang E Yehuda-Shnaidman et al ldquoLiver Xreceptor 120572 is a transcriptional repressor of the uncouplingprotein 1 gene and the brown fat phenotyperdquo Molecular andCellular Biology vol 28 no 7 pp 2187ndash2200 2008

[11] V M Rodrıguez M P Portillo C Pico M Teresa Macarullaand A Palou ldquoOlive oil feeding up-regulates uncoupling pro-tein genes in rat brown adipose tissue and skeletal musclerdquoTheAmerican Journal of Clinical Nutrition vol 75 no 2 pp 213ndash220 2002

[12] S Samec J Seydoux and A G Dulloo ldquoPost-starvationgene expression of skeletal muscle uncoupling protein 2 anduncoupling protein 3 in response to dietary fat levels and fattyacid composition a link with insulin resistancerdquo Diabetes vol48 no 2 pp 436ndash441 1999


[13] G Alberdi V M Rodriguez J Miranda M T Macarulla IChurruca andM P Portillo ldquoThermogenesis is involved in thebody-fat lowering effects of resveratrol in ratsrdquo Food Chemistryvol 141 no 2 pp 1530ndash1535 2013

[14] A G Dulloo C Duret D Rohrer et al ldquoEfficacy of a green teaextract rich in catechin polyphenols and caffeine in increasing24-h energy expenditure and fat oxidation in humansrdquo TheAmerican Journal of Clinical Nutrition vol 70 no 6 pp 1040ndash1045 1999

[15] S N El and S Karakaya ldquoOlive tree (Olea europaea) leavespotential beneficial effects on human healthrdquoNutrition Reviewsvol 67 no 11 pp 632ndash638 2009

[16] M T Khayyal M A El-Ghazaly D M Abdallah N N NassarS N Okpanyi and M H Kreuter ldquoBlood pressure loweringeffect of an olive leaf extract (Olea europaea) in L-NAMEinduced hypertension in ratsrdquo Arzneimittel-Forschung vol 52no 11 pp 797ndash802 2002

[17] L Wang C Geng L Jiang et al ldquoThe anti-atheroscleroticeffect of olive leaf extract is related to suppressed inflammatoryresponse in rabbits with experimental atherosclerosisrdquo Euro-pean Journal of Nutrition vol 47 no 5 pp 235ndash243 2008

[18] L Abaza T P N Talorete P Yamada Y KuritaM Zarrouk andH Isoda ldquoInduction of growth inhibition and differentiation ofhuman leukemia HL-60 cells by a Tunisian gerboui olive leafextractrdquo Bioscience Biotechnology and Biochemistry vol 71 no5 pp 1306ndash1312 2007

[19] A Eidi M Eidi and R Darzi ldquoAntidiabetic effect of Oleaeuropaea L in normal and diabetic ratsrdquo Phytotherapy Researchvol 23 no 3 pp 347ndash350 2009

[20] H Poudyal F Campbell and L Brown ldquoOlive leaf extractattenuates cardiac hepatic and metabolic changes in highcarbohydrate- high fat-fed ratsrdquo The Journal of Nutrition vol140 no 5 pp 946ndash953 2010

[21] T Perrinjaquet-Moccetti A Busjahn C Schmidlin ASchmidt B Bradl and C Aydogan ldquoFood supplementationwith an olive (Olea europaeaL) leaf extract reduces bloodpressure in borderline hypertensive monozygotic twinsrdquoPhytotherapy Research vol 22 no 9 pp 1239ndash1242 2008

[22] R Japon-Lujan J M Luque-Rodrıguez andM D L de CastroldquoDynamic ultrasound-assisted extraction of oleuropein andrelated biophenols from olive leavesrdquo Journal of Chromatogra-phy A vol 1108 no 1 pp 76ndash82 2006

[23] Y Kim Y Choi and T Park ldquoHepatoprotective effect ofoleuropein in mice mechanisms uncovered by gene expressionprofilingrdquoBiotechnology Journal vol 5 no 9 pp 950ndash960 2010

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

[25] C Gundlah K F Martin D J Heal and S B Auerbach ldquoInvivo criteria to differentiate monoamine reuptake inhibitorsfrom releasing agents sibutramine is a reuptake inhibitorrdquoTheJournal of Pharmacology and Experimental Therapeutics vol283 no 2 pp 581ndash591 1997

[26] R Drira S Chen and K Sakamoto ldquoOleuropein and hydrox-ytyrosol inhibit adipocyte differentiation in 3 T3-L1 cellsrdquo LifeSciences vol 89 no 19-20 pp 708ndash716 2011

[27] J Hao W Shen G Yu et al ldquoHydroxytyrosol promotesmitochondrial biogenesis and mitochondrial function in 3T3-L1 adipocytesrdquo The Journal of Nutritional Biochemistry vol 21no 7 pp 634ndash644 2010

[28] H S Park S H Kim Y S Kim et al ldquoLuteolin inhibitsadipogenic differentiation by regulating PPAR120574 activationrdquoBioFactors vol 35 no 4 pp 373ndash379 2009

[29] H J Myoung G Kim and K W Nam ldquoApigenin isolated fromthe seeds of Perilla frutescens britton var crispa (Benth) inhibitsfood intake in C57BL6J micerdquo Archives of Pharmacal Researchvol 33 no 11 pp 1741ndash1746 2010

[30] M Ono and K Fujimori ldquoAntiadipogenic effect of dietaryapigenin through activation of AMPK in 3T3-L1 cellsrdquo Journalof Agricultural and Food Chemistry vol 59 no 24 pp 13346ndash13352 2011

[31] C L Hsu C H Wu S L Huang and G C Yen ldquoPhenoliccompounds rutin and o-coumaric acid ameliorate obesityinduced byhigh-fat diet in ratsrdquo Journal of Agricultural andFoodChemistry vol 57 no 2 pp 425ndash431 2009

[32] A S Cho SM JeonM J Kim et al ldquoChlorogenic acid exhibitsanti-obesity property and improves lipidmetabolism in high-fatdiet-induced-obese micerdquo Food and Chemical Toxicology vol48 no 3 pp 937ndash943 2010

[33] E P Rafferty A R Wylie C T Elliott O P Chevallier DJ Grieve and B D Green ldquoIn vitro and in vivo effects ofnatural putative secretagogues of glucagon-like peptide-1 (GLP-1)rdquo Scientia Pharmaceutica vol 79 no 3 pp 615ndash621 2011

[34] M Sumiyoshi and Y Kimura ldquoEffects of olive leaf extractand its main component oleuroepin on acute ultraviolet Birradiation-induced skin changes in C57BL6Jmicerdquo Phytother-apy Research vol 24 no 7 pp 995ndash1003 2010

[35] U Koca I Suntar E K Akkol D Yilmazer and M AlperldquoWound repair potential of Olea europaea L leaf extractsrevealed by in vivo experimental models and comparative eval-uation of the extractsrsquo antioxidant activityrdquo Journal of MedicinalFood vol 14 no 1-2 pp 140ndash146 2011

[36] E Valassi M Scacchi and F Cavagnini ldquoNeuroendocrine con-trol of food intakerdquo Nutrition Metabolism and CardiovascularDiseases vol 18 no 2 pp 158ndash168 2008

[37] O Benavente-Garcıa J Castillo J Lorente A Ortuno and J ADel Rio ldquoAntioxidant activity of phenolics extracted from Oleaeuropaea L leavesrdquo Food Chemistry vol 68 no 4 pp 457ndash4622000

[38] Z Wu E D Rosen R Brun et al ldquoCross-regulation ofCEBP120572 and PPAR120574 controls the transcriptional pathway ofadipogenesis and insulin sensitivityrdquo Molecular Cell vol 3 no2 pp 151ndash158 1999

[39] C Lagathu C Christodoulides S Virtue et al ldquoDact1 a nutri-tionally regulated preadipocyte gene controls adipogenesis bycoordinating the wnt120573-catenin signaling networkrdquo Diabetesvol 58 no 3 pp 609ndash619 2009

[40] R T Moon A D Kohn G V de Ferrari and A KaykasldquoWNT and 120573-catenin signalling diseases and therapiesrdquoNatureReviews Genetics vol 5 no 9 pp 691ndash701 2004

[41] R A Koza L Nikonova J Hogan et al ldquoChanges in geneexpression foreshadowdiet-induced obesity in genetically iden-tical micerdquo PLoS Genetics vol 2 no 5 article e81 2006

[42] 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

[43] J T Rodgers C Lerin W Haas S P Gygi B M Spiegelmanand P Puigserver ldquoNutrient control of glucose homeostasisthrough a complex of PGC-1120572 and SIRT1rdquo Nature vol 434 no7029 pp 113ndash118 2005


[44] M Lagouge C Argmann Z Gerhart-Hines et al ldquoResver-atrol improves mitochondrial function and protects againstmetabolic disease by activating SIRT1 and PGC-1120572rdquo Cell vol127 no 6 pp 1109ndash1122 2006

[45] G S Shadel and D A Clayton ldquoMitochondrial transcriptioninitiation Variation and conservationrdquoThe Journal of BiologicalChemistry vol 268 no 22 pp 16083ndash16086 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


increase energy expenditure and thermogenesis in animalsand humans Therefore it is possible that novel therapeuticagents activating or elevating the expression of PGC-1120572 andUCP1 would have crucial impacts on increasing the energyexpenditure to ultimately prevent obesity

Olive (Olea europaea) leaf has been widely used intraditional remedies as well as in human diet as an extractin herbal tea and in the powder form in the European andMediterranean countries [15] Olive leaf extract (OLE) ismarketed as a natural nutraceutical with wide-ranging healthbenefits In recent years research has mainly focused on theeffects of extracts from olive leaves related to the preventionof hypertension [16] atherosclerosis [17] cancer [18] diabetes[19] and cardiovascular diseases [20 21] Olive leaves containseveral different compounds collectively termed as olivebiophenols which impart the therapeutic properties Themost abundant biophenol is oleuropein followed by otherbiophenols such as verbascoside luteolin rutin catechinand hydroxytyrosol in lower quantities [22] Our previousstudy demonstrated that the addition of oleuropein to HFDdecreased body weight gain and improved the lipid pro-files in the plasma of mice [23] Although several studieshave investigated the biochemical roles of olive leaves andolive leaves preparations their protective effect against diet-induced obesity has never been reported Therefore theobjective of this study was to investigate the weight-reducingeffect of a well-defined OLE by supplementation in HFD-induced mice and to investigate the underlying mechanismsof this effect with a particular focus on the expression ofmolecules involved in adipogenesis and thermogenesis

2 Materials and Method

21 Materials Dry OLE was obtained from FrutaromSwitzerland Ltd (Wadenswil Switzerland) under the tradename of Benolea The extract was manufactured from thedried leaves of Olea europaea L applying an ethanol (80mm) extraction procedure After a patented filtration pro-cess (EFLA Hyperpure) the crude extract was dried Finally15 (mm) Acaciae Gummi Ph Eur was added as a carriertogether with lt05 (mm) of silica colloidalis anhydrica PhEur The extract is characterized by a DER of 3ndash7 = 1 (5 =1) Characteristic component is 16ndash24 (mm) oleuropeinSibutramine was purchased from Sigma-Aldrich (MA USA)

22 High-Performance Liquid Chromatography (HPLC) Anal-ysis of OLE HPLC analysis was conducted using the Nova-Pak C18 column (150mm times 39mm 60AWaters AssociatesHarrow UK) at 25∘C at a flow rate of 1mLmin by using agradient mobile phase (water methanol in 600 400 ratio)as the initial condition of the chromatography The sampleinjection volume used was 10 120583L The absorption signal ofoleuropein was examined at 233 nm by using the UV detector(Agilent Technologies DE USA)

23 Animal and Diets Five-week-old male C57BL6N mice(Orient Bio Gyeonggi-do Korea)were housed in a pathogen-free facility at 21plusmn 20∘C with 50plusmn 5 relative humidity and a

Table 1 Composition of the experimental diets

Ingredients HFD(gkg diet)

OLD(gkg diet)

SD(gkg diet)

Casein 200 200 200DL-methionine 3 3 3Corn starch 111 1095 1109Sucrose 370 370 370Cellulose 50 50 50Corn oil 30 30 30Lard 170 170 170Mineral mixture1 42 42 42Vitamin mixture2 12 12 12Choline bitartrate 2 2 2Cholesterol 10 10 10tert-Butylhydroquinone3 004 004 004

Olive leaf extract mdash 15 mdashSibutramine mdash mdash 01Total (g) 1000 1000 1000Fat ( calories) 40 40 40Total energy(kcalkg diet) 4616 4616 46161AIN-76A mineral mix2AIN-76A vitamin mix3Antioxidant agent 001 g50 g lipids

12 h lightdark cycle the mice were provided access to rodentchow and tap water ad libitum for 1 weekThereafter themicewere divided into the following 4 groups matched for bodyweight (119899 = 8 in each group) the chow diet (CD) high-fatdiet (HFD) 015 (ww)OLE-supplemented diet (OLD) and001 (ww) sibutramine-supplemented diet (SD) groupsThe HFD consisted of 200 g fatkg body weight (170 g of lardand 30 g of corn oil) and 1 (ww) cholesterol The OLD wasidentical to the HFD and contained 015 OLE The SD wasalso identical to the HFD except that it was supplementedwith 001 sibutramineThe composition of the experimentaldiets including total energy (kcalkg diet) is shown in Table 1All the diets were provided in the form of pellets for 8 weeks

The food intake was monitored daily and body weightswere recorded on a weekly basis At the end of the experi-mental period the mice were anesthetized with diethyl etherafter they were fasted for 16 h Blood samples were drawnfrom the inferior vena cava into an ethylene-diamine-tetra-acetic acid (EDTA)-coated tube and the plasma sampleswere obtained by centrifuging the blood at 4000timesg for15min at 4∘C The epididymal retroperitoneal mesentericand perirenal fat-pads were dissected removed weighedand immediately snap-frozen in liquid nitrogen and storedat minus80∘C until further use All animal experiments wereperformed in accordance with the Korea Food and DrugAdministration (KFDA) guidelines All the experimentalprotocols were reviewed and approved by the InstitutionalAnimal Care and Use Committee (IACUC) of the YonseiLaboratory Animal Research Center (YLARC)


80

60

40

20

0

0

2 4 6 8 10 12 14 16 18

(min)

(mAu

)

Ole

urop

ein









3 Results






(∘C) Size (bp)

























b b c c cc c c c

a aa

aa

aa a

a

b b bb

bc d d d

bb b

bb

b b bb

20

25

30

35

40

0 4 7 14 21 28 35 42 49 56

CDHFD

OLDSD


Days

(a)

c

a

d

b

0

5

10

15

20

CD HFD OLD SD


(b)

CD HFD OLD SD

ab

cb

0

1

2

3

4


(c)

c

a

c

b

000002004006008010

CD HFDOLD SD

FER

(d)

cc c c

c

a

a a a

a

cc c c

cb

b b b

b

0

1

2

3

4


CDHFD

OLDSD


(e)

CD

SDOLD

HFD

100120583m 100120583m

100120583m100120583m

b

a

c

b

0

20

40

60

80

100

CD HFD OLD SD


(f)





b

a

c

b

00

03

06

09

12

15

CD HFD OLD SD


(a)

d

a

cb

0

2

4

6

CD HFD OLD SD


(b)

c

a

c

b

0

1

2

3

CD HFD OLD SD


(c)

CD HFD OLD SD

ab

a

b b

0

200

400

600


(d)

CD HFD OLD SD

c

a

b

c

0

5

10

15Glucose (mmolL)

(e)

b

a

bb

0

6

12

18

24

30

CD HFD OLD SD

Leptin (ngmL)

(f)





4 Discussion



b a a

a

CD

SFRP5

WNT10b

LRP5

GAPDH

HFD OLD CD HFD OLD

b cca

b

00

05

10

15

20

25

SFRP5 WNT10b LRP5

CDHFDOLD

Relat

ive g

ene e

xpre

ssio

n

(a)

CD HFD OLD

a

b

a

00

05

10

15

20

CD HFD OLD

Relat

ive p

rote

in ex

pres

sion

120573-Catenin

120573-Actin

(b)

b b b b b b

a a a aa

a

b b bc

b

b

00051015202530

Galanin

Galanin

GalR1

GalR1

GalR2

GalR2

Cyc-D

Cyc-D

E2F1

E2F1

CD

CD

HFD

GAPDH

HFD

OLD

OLD CD HFD OLD

Rela

tive g

ene

expr

essio

n

PKC120575

PKC120575

(c)

CD HFD OLD

b

a

b

00

05

10

15

20

CD HFD OLD

Total ERK

(Thr202Tyr204)

Rela

tive p

rote

inex

pres

sion

p-ERK

(d)

b b b b ba a a

a a

b b b cb

00

05

10

15

20

25

CD36

CD36

FAS

FAS

Leptin

Leptin

GAPDH

CDHFDOLD

CD HFD OLD

PPAR120574

PPAR120574

CEBP120572

CEBP120572

Relat

ive g

ene e

xpre

ssio

n

(e)



a a ab b a a

c cb c

c bb b

aa

ba

00

05

10

15

COX2

COX2

TFAM

TFAM

NRF-1

NRF-1

SIRT1

SIRT1

UCP1

GAPDH

UCP1Re

lativ

e gen

eex

pres

sion

PGC-1120572

PGC-1120572

CDHFDOLD


(a)

a

b

a

00

05

10

15

20

CD HFD OLD

CD HFD OLD

Rela

tive p

rote

inex

pres

sion

CREB

p-CREB

(b)


















Abbreviations





family member 10b



Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















80

60

40

20

0

0

2 4 6 8 10 12 14 16 18

(min)

(mAu

)

Ole

urop

ein









3 Results






(∘C) Size (bp)

























b b c c cc c c c

a aa

aa

aa a

a

b b bb

bc d d d

bb b

bb

b b bb

20

25

30

35

40

0 4 7 14 21 28 35 42 49 56

CDHFD

OLDSD


Days

(a)

c

a

d

b

0

5

10

15

20

CD HFD OLD SD


(b)

CD HFD OLD SD

ab

cb

0

1

2

3

4


(c)

c

a

c

b

000002004006008010

CD HFDOLD SD

FER

(d)

cc c c

c

a

a a a

a

cc c c

cb

b b b

b

0

1

2

3

4


CDHFD

OLDSD


(e)

CD

SDOLD

HFD

100120583m 100120583m

100120583m100120583m

b

a

c

b

0

20

40

60

80

100

CD HFD OLD SD


(f)





b

a

c

b

00

03

06

09

12

15

CD HFD OLD SD


(a)

d

a

cb

0

2

4

6

CD HFD OLD SD


(b)

c

a

c

b

0

1

2

3

CD HFD OLD SD


(c)

CD HFD OLD SD

ab

a

b b

0

200

400

600


(d)

CD HFD OLD SD

c

a

b

c

0

5

10

15Glucose (mmolL)

(e)

b

a

bb

0

6

12

18

24

30

CD HFD OLD SD

Leptin (ngmL)

(f)





4 Discussion



b a a

a

CD

SFRP5

WNT10b

LRP5

GAPDH

HFD OLD CD HFD OLD

b cca

b

00

05

10

15

20

25

SFRP5 WNT10b LRP5

CDHFDOLD

Relat

ive g

ene e

xpre

ssio

n

(a)

CD HFD OLD

a

b

a

00

05

10

15

20

CD HFD OLD

Relat

ive p

rote

in ex

pres

sion

120573-Catenin

120573-Actin

(b)

b b b b b b

a a a aa

a

b b bc

b

b

00051015202530

Galanin

Galanin

GalR1

GalR1

GalR2

GalR2

Cyc-D

Cyc-D

E2F1

E2F1

CD

CD

HFD

GAPDH

HFD

OLD

OLD CD HFD OLD

Rela

tive g

ene

expr

essio

n

PKC120575

PKC120575

(c)

CD HFD OLD

b

a

b

00

05

10

15

20

CD HFD OLD

Total ERK

(Thr202Tyr204)

Rela

tive p

rote

inex

pres

sion

p-ERK

(d)

b b b b ba a a

a a

b b b cb

00

05

10

15

20

25

CD36

CD36

FAS

FAS

Leptin

Leptin

GAPDH

CDHFDOLD

CD HFD OLD

PPAR120574

PPAR120574

CEBP120572

CEBP120572

Relat

ive g

ene e

xpre

ssio

n

(e)



a a ab b a a

c cb c

c bb b

aa

ba

00

05

10

15

COX2

COX2

TFAM

TFAM

NRF-1

NRF-1

SIRT1

SIRT1

UCP1

GAPDH

UCP1Re

lativ

e gen

eex

pres

sion

PGC-1120572

PGC-1120572

CDHFDOLD


(a)

a

b

a

00

05

10

15

20

CD HFD OLD

CD HFD OLD

Rela

tive p

rote

inex

pres

sion

CREB

p-CREB

(b)


















Abbreviations





family member 10b



Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(∘C) Size (bp)

























b b c c cc c c c

a aa

aa

aa a

a

b b bb

bc d d d

bb b

bb

b b bb

20

25

30

35

40

0 4 7 14 21 28 35 42 49 56

CDHFD

OLDSD


Days

(a)

c

a

d

b

0

5

10

15

20

CD HFD OLD SD


(b)

CD HFD OLD SD

ab

cb

0

1

2

3

4


(c)

c

a

c

b

000002004006008010

CD HFDOLD SD

FER

(d)

cc c c

c

a

a a a

a

cc c c

cb

b b b

b

0

1

2

3

4


CDHFD

OLDSD


(e)

CD

SDOLD

HFD

100120583m 100120583m

100120583m100120583m

b

a

c

b

0

20

40

60

80

100

CD HFD OLD SD


(f)





b

a

c

b

00

03

06

09

12

15

CD HFD OLD SD


(a)

d

a

cb

0

2

4

6

CD HFD OLD SD


(b)

c

a

c

b

0

1

2

3

CD HFD OLD SD


(c)

CD HFD OLD SD

ab

a

b b

0

200

400

600


(d)

CD HFD OLD SD

c

a

b

c

0

5

10

15Glucose (mmolL)

(e)

b

a

bb

0

6

12

18

24

30

CD HFD OLD SD

Leptin (ngmL)

(f)





4 Discussion



b a a

a

CD

SFRP5

WNT10b

LRP5

GAPDH

HFD OLD CD HFD OLD

b cca

b

00

05

10

15

20

25

SFRP5 WNT10b LRP5

CDHFDOLD

Relat

ive g

ene e

xpre

ssio

n

(a)

CD HFD OLD

a

b

a

00

05

10

15

20

CD HFD OLD

Relat

ive p

rote

in ex

pres

sion

120573-Catenin

120573-Actin

(b)

b b b b b b

a a a aa

a

b b bc

b

b

00051015202530

Galanin

Galanin

GalR1

GalR1

GalR2

GalR2

Cyc-D

Cyc-D

E2F1

E2F1

CD

CD

HFD

GAPDH

HFD

OLD

OLD CD HFD OLD

Rela

tive g

ene

expr

essio

n

PKC120575

PKC120575

(c)

CD HFD OLD

b

a

b

00

05

10

15

20

CD HFD OLD

Total ERK

(Thr202Tyr204)

Rela

tive p

rote

inex

pres

sion

p-ERK

(d)

b b b b ba a a

a a

b b b cb

00

05

10

15

20

25

CD36

CD36

FAS

FAS

Leptin

Leptin

GAPDH

CDHFDOLD

CD HFD OLD

PPAR120574

PPAR120574

CEBP120572

CEBP120572

Relat

ive g

ene e

xpre

ssio

n

(e)



a a ab b a a

c cb c

c bb b

aa

ba

00

05

10

15

COX2

COX2

TFAM

TFAM

NRF-1

NRF-1

SIRT1

SIRT1

UCP1

GAPDH

UCP1Re

lativ

e gen

eex

pres

sion

PGC-1120572

PGC-1120572

CDHFDOLD


(a)

a

b

a

00

05

10

15

20

CD HFD OLD

CD HFD OLD

Rela

tive p

rote

inex

pres

sion

CREB

p-CREB

(b)


















Abbreviations





family member 10b



Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















b b c c cc c c c

a aa

aa

aa a

a

b b bb

bc d d d

bb b

bb

b b bb

20

25

30

35

40

0 4 7 14 21 28 35 42 49 56

CDHFD

OLDSD


Days

(a)

c

a

d

b

0

5

10

15

20

CD HFD OLD SD


(b)

CD HFD OLD SD

ab

cb

0

1

2

3

4


(c)

c

a

c

b

000002004006008010

CD HFDOLD SD

FER

(d)

cc c c

c

a

a a a

a

cc c c

cb

b b b

b

0

1

2

3

4


CDHFD

OLDSD


(e)

CD

SDOLD

HFD

100120583m 100120583m

100120583m100120583m

b

a

c

b

0

20

40

60

80

100

CD HFD OLD SD


(f)





b

a

c

b

00

03

06

09

12

15

CD HFD OLD SD


(a)

d

a

cb

0

2

4

6

CD HFD OLD SD


(b)

c

a

c

b

0

1

2

3

CD HFD OLD SD


(c)

CD HFD OLD SD

ab

a

b b

0

200

400

600


(d)

CD HFD OLD SD

c

a

b

c

0

5

10

15Glucose (mmolL)

(e)

b

a

bb

0

6

12

18

24

30

CD HFD OLD SD

Leptin (ngmL)

(f)





4 Discussion



b a a

a

CD

SFRP5

WNT10b

LRP5

GAPDH

HFD OLD CD HFD OLD

b cca

b

00

05

10

15

20

25

SFRP5 WNT10b LRP5

CDHFDOLD

Relat

ive g

ene e

xpre

ssio

n

(a)

CD HFD OLD

a

b

a

00

05

10

15

20

CD HFD OLD

Relat

ive p

rote

in ex

pres

sion

120573-Catenin

120573-Actin

(b)

b b b b b b

a a a aa

a

b b bc

b

b

00051015202530

Galanin

Galanin

GalR1

GalR1

GalR2

GalR2

Cyc-D

Cyc-D

E2F1

E2F1

CD

CD

HFD

GAPDH

HFD

OLD

OLD CD HFD OLD

Rela

tive g

ene

expr

essio

n

PKC120575

PKC120575

(c)

CD HFD OLD

b

a

b

00

05

10

15

20

CD HFD OLD

Total ERK

(Thr202Tyr204)

Rela

tive p

rote

inex

pres

sion

p-ERK

(d)

b b b b ba a a

a a

b b b cb

00

05

10

15

20

25

CD36

CD36

FAS

FAS

Leptin

Leptin

GAPDH

CDHFDOLD

CD HFD OLD

PPAR120574

PPAR120574

CEBP120572

CEBP120572

Relat

ive g

ene e

xpre

ssio

n

(e)



a a ab b a a

c cb c

c bb b

aa

ba

00

05

10

15

COX2

COX2

TFAM

TFAM

NRF-1

NRF-1

SIRT1

SIRT1

UCP1

GAPDH

UCP1Re

lativ

e gen

eex

pres

sion

PGC-1120572

PGC-1120572

CDHFDOLD


(a)

a

b

a

00

05

10

15

20

CD HFD OLD

CD HFD OLD

Rela

tive p

rote

inex

pres

sion

CREB

p-CREB

(b)


















Abbreviations





family member 10b



Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















b

a

c

b

00

03

06

09

12

15

CD HFD OLD SD


(a)

d

a

cb

0

2

4

6

CD HFD OLD SD


(b)

c

a

c

b

0

1

2

3

CD HFD OLD SD


(c)

CD HFD OLD SD

ab

a

b b

0

200

400

600


(d)

CD HFD OLD SD

c

a

b

c

0

5

10

15Glucose (mmolL)

(e)

b

a

bb

0

6

12

18

24

30

CD HFD OLD SD

Leptin (ngmL)

(f)





4 Discussion



b a a

a

CD

SFRP5

WNT10b

LRP5

GAPDH

HFD OLD CD HFD OLD

b cca

b

00

05

10

15

20

25

SFRP5 WNT10b LRP5

CDHFDOLD

Relat

ive g

ene e

xpre

ssio

n

(a)

CD HFD OLD

a

b

a

00

05

10

15

20

CD HFD OLD

Relat

ive p

rote

in ex

pres

sion

120573-Catenin

120573-Actin

(b)

b b b b b b

a a a aa

a

b b bc

b

b

00051015202530

Galanin

Galanin

GalR1

GalR1

GalR2

GalR2

Cyc-D

Cyc-D

E2F1

E2F1

CD

CD

HFD

GAPDH

HFD

OLD

OLD CD HFD OLD

Rela

tive g

ene

expr

essio

n

PKC120575

PKC120575

(c)

CD HFD OLD

b

a

b

00

05

10

15

20

CD HFD OLD

Total ERK

(Thr202Tyr204)

Rela

tive p

rote

inex

pres

sion

p-ERK

(d)

b b b b ba a a

a a

b b b cb

00

05

10

15

20

25

CD36

CD36

FAS

FAS

Leptin

Leptin

GAPDH

CDHFDOLD

CD HFD OLD

PPAR120574

PPAR120574

CEBP120572

CEBP120572

Relat

ive g

ene e

xpre

ssio

n

(e)



a a ab b a a

c cb c

c bb b

aa

ba

00

05

10

15

COX2

COX2

TFAM

TFAM

NRF-1

NRF-1

SIRT1

SIRT1

UCP1

GAPDH

UCP1Re

lativ

e gen

eex

pres

sion

PGC-1120572

PGC-1120572

CDHFDOLD


(a)

a

b

a

00

05

10

15

20

CD HFD OLD

CD HFD OLD

Rela

tive p

rote

inex

pres

sion

CREB

p-CREB

(b)


















Abbreviations





family member 10b



Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















b a a

a

CD

SFRP5

WNT10b

LRP5

GAPDH

HFD OLD CD HFD OLD

b cca

b

00

05

10

15

20

25

SFRP5 WNT10b LRP5

CDHFDOLD

Relat

ive g

ene e

xpre

ssio

n

(a)

CD HFD OLD

a

b

a

00

05

10

15

20

CD HFD OLD

Relat

ive p

rote

in ex

pres

sion

120573-Catenin

120573-Actin

(b)

b b b b b b

a a a aa

a

b b bc

b

b

00051015202530

Galanin

Galanin

GalR1

GalR1

GalR2

GalR2

Cyc-D

Cyc-D

E2F1

E2F1

CD

CD

HFD

GAPDH

HFD

OLD

OLD CD HFD OLD

Rela

tive g

ene

expr

essio

n

PKC120575

PKC120575

(c)

CD HFD OLD

b

a

b

00

05

10

15

20

CD HFD OLD

Total ERK

(Thr202Tyr204)

Rela

tive p

rote

inex

pres

sion

p-ERK

(d)

b b b b ba a a

a a

b b b cb

00

05

10

15

20

25

CD36

CD36

FAS

FAS

Leptin

Leptin

GAPDH

CDHFDOLD

CD HFD OLD

PPAR120574

PPAR120574

CEBP120572

CEBP120572

Relat

ive g

ene e

xpre

ssio

n

(e)



a a ab b a a

c cb c

c bb b

aa

ba

00

05

10

15

COX2

COX2

TFAM

TFAM

NRF-1

NRF-1

SIRT1

SIRT1

UCP1

GAPDH

UCP1Re

lativ

e gen

eex

pres

sion

PGC-1120572

PGC-1120572

CDHFDOLD


(a)

a

b

a

00

05

10

15

20

CD HFD OLD

CD HFD OLD

Rela

tive p

rote

inex

pres

sion

CREB

p-CREB

(b)


















Abbreviations





family member 10b



Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















a a ab b a a

c cb c

c bb b

aa

ba

00

05

10

15

COX2

COX2

TFAM

TFAM

NRF-1

NRF-1

SIRT1

SIRT1

UCP1

GAPDH

UCP1Re

lativ

e gen

eex

pres

sion

PGC-1120572

PGC-1120572

CDHFDOLD


(a)

a

b

a

00

05

10

15

20

CD HFD OLD

CD HFD OLD

Rela

tive p

rote

inex

pres

sion

CREB

p-CREB

(b)


















Abbreviations





family member 10b



Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Abbreviations





family member 10b



Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article olive leaf extract attenuates obesity in...

Documents