research article essential oil of pinus koraiensis exerts...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 947037 10 pageshttpdxdoiorg1011552013947037

Research ArticleEssential Oil of Pinus koraiensis Exerts Antiobesic andHypolipidemic Activity via Inhibition of PeroxisomeProliferator-Activated Receptors Gamma Signaling

Hyun-Suk Ko1 Hyo-Jeong Lee1 Hyo-Jung Lee12 Eun Jung Sohn1 Miyong Yun1

Min-Ho Lee3 and Sung-Hoon Kim1

1 College of Oriental Medicine Kyung Hee University 1 Hoegi-dong Dongdaemun-gu Seoul 130-701 Republic of Korea2Medical Genomics Research Center Korea Research Institute of Bioscience and Biotechnology Daejeon 305-811 Republic of Korea3 College of Life Sciences and Biotechnology Kyung Hee University Seoul 130-701 Republic of Korea

Correspondence should be addressed to Sung-Hoon Kim sungkim7khuackr

Received 16 April 2013 Accepted 8 July 2013

Academic Editor Jae Youl Cho

Copyright copy 2013 Hyun-Suk Ko 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

Our group previously reported that essential oil of Pinus koraiensis (EOPK) exerts antihyperlipidemic effects via upregulationof low-density lipoprotein receptor and inhibition of acyl-coenzyme A In the present study we investigated the antiobesityand hypolipidemic mechanism of EOPK using in vitro 3T3-L1 cells and in vivo HFD-fed rats EOPK markedly suppressed fataccumulation and intracellular triglyceride associatedwith downregulation of adipogenic transcription factor expression includingPPAR120574 and CEBP120572 in the differentiated 3T3-L1 adipocytes Additionally EOPK attenuated the expression levels of FABP andGPDH as target genes of PPAR120574 during adipocyte differentiation Furthermore PPAR120574 inhibitor GW9662 enhanced the decreasedexpression of FABP and PPAR120574 and fat accumulation induced by EOPK To confirm the in vitro activity of EOPK animal studywas performed by administering normal diet HFD andor EOPK at the dose of 100 or 200mgkg for 6 weeks ConsistentlyEOPK significantly suppressed body weight gain serum triglyceride total cholesterol LDL cholesterol and AI value and increasedHDL cholesterol in a dose-dependent manner Immunohistochemistry revealed that EOPK treatment abrogated the expression ofPPAR120574 in the liver tissue sections of EOPK-treated rats Taken together our findings suggest that EOPK has the antiobesic andhypolipidemic potential via inhibition of PPAR120574-related signaling

1 Introduction

Obesity as amedical condition of excess body fat caused by anaccumulation of adipose tissue mass [1] is tightly associatedwith various health disorders such as hyperlipidemia dia-betes and cardiovascular diseases [2] In obesity adipocytesaccumulate excessive fat and adipocytokine production isdisrupted [3 4] Adipose tissue development observed inobese individuals is closely related to hypertrophy and hyper-plasia the latter involving proliferation and differentiationof preadipocytes to adipocytes The peroxisome proliferator-activated receptor (PPAR) and CCAATenhancer-bindingproteins (CEBP) families of transcription factors regulatethis adipocyte differentiation [5ndash8] These transcriptional

factors regulate the expression of genes involved in theinduction adipocyte phenotypes [5]

Pharmaceutical antiobesity drugs are generally developedto loss or control body weight Although various prescribedor nonprescribed medications have been used for treatmentof obesity patients there is a limitation to apply long-termuse because of their severe side effects Only orlistat (Xenical)has been approved by the FDA for long-term use Recentlymany studies reported antiobesity activity of natural productsand suggested the potential as antiobesity agents or theirsupplements

Pinus koraiensis (Phylum Pinophyta Class PinopsidaOrder Pinales Family Pinaceae) is an evergreen tree found inKorea China far eastern Russia and central JapanThemajor

2 Evidence-Based Complementary and Alternative Medicine

bioactive chemicals from P koraiensis include camphene D-limonene 120572-pinene borneol 120573-pinene 4-carene bicyclo-hept-3-ene 3-carene 120573-phellandrene and fencyl [9] We andothers demonstrated the biological efficacies and genotoxicityof essential oil from P koraiensis seed leaf nut and cone[9ndash13] However the biological and biochemical effects ofessential oil from P koraiensis have not been reported yet

Recently many studies reported that natural productssuch as Rhizoma Polygonati falcatum (RPF) [14] Boussin-gaultia gracilis Miers var pseudobaselloides Bailey [15] Chi-nese black tea (Pu-erh tea) extract and gallic acid [16]showed the potential of antiobesity activity in vitro or invivo In the present study we investigated the antiobesicand hypolipidemic effect of essential oil of P koraiensis SIEB(EOPK) in 3T3-L1 cells by Oil-Red O staining measuringtriglyceride and analyzing expression levels of adipogenicfactors and in high-fat diet-fed rats by measuring bodyweights and fats and lipid metabolites

2 Materials and Methods

21 Preparation of Essential Oil of P koraiensis Leaves (EOPK)EOPK was prepared using the hydrodistillation method [17]Driedand pulverized P koraiensis leaves were immersed indistilled water and submitted to steam distillation using anapparatus with a condenser (Hanil Labtech Seoul Korea)The distillation continued for 3-4 h at 90∘C and then thevolatile compounds contained in the water-soluble fractionwere allowed to settle for 20min The essential oil layer wasseparated and purified through microfiltration

22 Cell Culture 3T3-L1 preadipocytes were purchased fromKorean Cell Line Bank (KCLB) The cells were cultured inDulbeccorsquos Modified Eaglersquos Medium (DMEM) with 10 FBSin a humidified atmosphere of 95 air and 5 CO

2at 37∘C

23 Cytotoxicity Assay The cytotoxicity of EOPK against3T3-L1 cells was measured by 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2H-tetrazolium bromide (MTT) colorimetricassay The cells were seeded onto 96-well microplates at adensity of 1 times 104 cells per well and treated with variousconcentrations of EOPK (0 125 25 or 50120583gmL) for 24 hMTT working solution was then added to the microplatesat 37∘C for 2 h and then MTT extraction buffer (20 SDSand 50 dimethylformamide) was added at 37∘C overnightOptical density (OD) was measured using microplate reader(Sunrise TECAN Mannedorf Switzerland) at 570 nm Cellviability was calculated by employing the following equationcell viability () = [OD (EOPK) minus OD (Blank)][OD (Con-trol) minus OD (Blank)] times 100

24 Differentiation Induction and Oil-Red O Staining Thepreadipocyte 3T3-L1 cells were plated onto 6-well plateson day 0 and incubated to confluent status For adipocytedifferentiation the confluent cells were treated with 1120583Mdexamethasone 1 120583gmL insulin and 05mM IBMX for2 days and the medium was replaced by fresh normalmedium only containing 1 120583gmL insulin On day 8 the

differentiated adipocyte cells were cultured in the presenceor absence of EOPK (100120583gmL) for 2 days The cells werefixedwith 2 paraformaldehyde washed twice with PBS andfinally stained with Oil-Red After dissolving cellular-lipidretained Oil-Red O in isopropanol and adipocyte expressionwas estimated by measuring OD using microplate reader(Sunrise TECAN Mannedorf Switzerland) at 510 nm

25 RT-PCR Analysis The 3T3-L1 cells were treated with orwithout EOPK (50 or 100mgmL) for 24 h The total RNAwas extracted by using TRIzol reagent (Invitrogen CarlsbadCA USA) according to the manufacturerrsquos instructionscDNA was synthesized from 1120583g of total RNA and subjectedto PCR reaction by using a SuperScript One-Step reversetranscription-PCR (RT-PCR) kit (Invitrogen Carlsbad CAUSA) The PCR conditions were 30 cycles of 94∘C for 30 s57∘C for 30 s and 72∘C for 30 s The primer sequenceswere as follows PPAR120574 (forward 51015840-GGTGAAACTCTG-GGAGATTC-31015840 and reverse 51015840-CAACCATTGGGTCAG-CTCTT-31015840) CEBP120572 (forward 51015840-AGGTGCTGGAGTTGA-CCAGT-31015840 and reverse 51015840-CAGCCTAGAGATCCAGCG-AC-31015840) GPDH (forward 51015840-GAACTAAGGAGCAGCTCA-AAGGTTC-31015840 and reverse 51015840-CAGTTGACTGACTGA-GCAAACATAG-31015840) 120573-actin (forward 51015840-ACCGTGAAA-AGATGACCCAG-31015840 and reverse 51015840-TACGGATGACAA-CGTCACAC-31015840) PCR products were run on 2 agarose geland then stained with ethidium bromide Stained bands werevisualized under UV light and photographed

26 Western Blotting Whole cell lysates (25 120583g) were pre-pared using lysis buffer (20mM Tris pH 74 250mM NaCl2mM EDTA pH 80 01 Triton X-100 001mgmL apro-tinin 0003mgmL leupeptin 04mM phenylmethylsulfonylfluoride (PMSF) and 4mMNaVO4)The lysates were spun at13000timesg for 15min to remove insolublematerial and resolvedon a 10 SDS-PAGE gel After electrophoresis the pro-teins were electrotransferred to a nitrocellulose membraneblocked with 5 nonfat milk and probed with antibodiesagainst PPAR120574 (Novus Littleton CO USA) CEBP120572 (CellSignaling Tech Danvers MA USA) FABP (Cell SignalingTech Danvers MA USA) and 120573-actin (Sigma St LouisMO USA) overnight

The blots were washed exposed to horseradishperoxidase- (HRP-) conjugated secondary antibodies for2 h and finally examined by enhanced chemiluminescence(ECL) (GE Health Care Bio-Sciences Piscataway NJ USA)

27 Animals Diet Manipulation and EOPK Treatment MaleSprague-Dawley rats at 4 weeks of age were purchased fromHyo-Chang Science (Daegu Korea) and maintained underconventional conditions Fifty rats were divided into fourgroups normal group (low fat diet) control group (high-fatdiet) two EOPK-treated groups- and atorvastatin (positivecontrol) treated group consuming high-fat diets (10 rats pergroup) Rats were fed the normal (low fat) diet (group 1)or the high-fat diet (groups 2ndash5) for 6 weeks High-fat dietcomposition was described in Table 1 For EOPK treatment

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Composition of basal and high-fat diet

Ingredient Basal diet () High-fat diet ()Casein 200 200DL-Methionine 03 03Corn starch 150 150Sucrose 500 345Fiber1 50 50Corn oil 50 mdashAIN-mineral mixture2 35 35AIN-vitamin mixture3 10 10Choline bitartrate 02 02Beef tallow mdash 2051Cellulose Sigma Co Ltd USA2Mineral mixture based on Rogers and Haper [18] contained the follow-ing (gkg diet) calcium phosphate dibasic 2000 sodium chloride 740potassium citrate monohydrate 2200 potassium sulfate 520 magnesiumoxide 240 magnesium carbonate 35 ferric citrate 60 zinc carbonate 16cupuric carbonate 03 potassium iodate 001 chromium potassium sulfate055 sucrose and finely powered make 10003Vitamin mixture (gkg diet) thiamine HCl 06 biotin 002 riboflavin 06cyanocobalamin 0001 pyridoxine HCl 07 retinyl acetate 08 nicotinic acid30 DL-tocopherol 38 Ca-pantothenate 16 7-dehydrocholesterol 00025folic acid 02 methionine 0005 sucrose and finely powered make 1000

EOPK dissolved in 4 tween 80normal saline was orallyadministered once daily to the rats at the doses of 100 (group3) and 200mgkg (group 4) for 6 weeks from the 1st day ofhigh-fat diet feeding whereas PBS was orally administeredto the rats in control group (group 2) As a positive controlatorvastatin was administered at a dose of 10mgkg (group5)

28 Preparation of Rat Serum Whole blood was collectedfrom rats by cardiac puncture method and serum wasisolated by centrifugation at 3000 rpm for 10min

29 Measurement of Total Cholesterol Level Total cholesterollevel was measured by using a total cholesterol assay kit (AM202-K Asan Pharm Co Seoul Korea) based on Richmondrsquosmethod [19]

210 Measurement of Triglyceride Level Triglyceride levelwas measured by a triglyceride assay kit (AM 157S-K AsanPharm Co Seoul Korea) based on McGowanrsquos method [20]

211 Measurement of Serum HDL and LDL Levels The levelsof high-density lipoprotein (HDL) and low-density lipopro-tein (LDL) in serum were measured using Roche cobas C-111analyzer (Roche-Diagnostics Indianapolis IN USA) AI wascalculated by employing the following equation AI = (totalcholesterol minusHDL cholesterol)HDL cholesterol

212 Measurement of Body Weight Retroperitoneal Fat andEpididymal Fat The body weight of rats in normal (group1) control (group 2) EOPK- (100 and 200mgkg) treatedgroups (groups 3 and 4 resp) and atorvastatin (group 5) wasmonitored once every week for 6 weeks The retroperitoneal

and epididymal fats were also removed and weighed fromrats treated by EOPK (100 and 200mgkg) or atorvastatin(10mgkg) on the last day of animal study

213 Immunohistochemical Staining For histopathologicalexamination paraffin sections (4120583m) from tumors dissectedwere stained with hematoxylin and eosin Immunohis-tochemical staining PPAR120574 (Novus Littleton CO USA)was performed using the indirect avidinbiotin-enhancedhorseradish peroxidase method Antigen retrieval was per-formed after dewaxing and dehydration of the tissue sectionsby microwave for 10min in 10mM citrate buffer Sectionswere cooled to room temperature treated with 3 hydrogenperoxide in methanol for 10min and blocked with 6horse serum for 30min at room temperature in humiditychamber Sections were then incubated with the primaryantibody against PPAR120574 (diluted 1 200 Novus LittletonCO USA) at 4∘C overnight in humidity chamber Sectionswere washed in PBS and incubated with secondary antibody(biotinylated goat anti-rabbit (1 150 Vector laboratoriesBurlingame CA USA) for 30min in humidity chamberAfter further washes the antibodies were detected withthe Vector ABC complexhorseradish peroxidase (HRP) kit(Vector Laboratories Burlingame CA USA) and color-developed with 331015840-diaminobenzidine tetrahydrochlorideFor semiquantitation ten photomicrographs (200 x) weretaken with a CCD camera avoiding gross necrotic areas

214 Statistical Analyses All data were expressed as means plusmnSD or SE In vitro experiment data were analyzed by Studentrsquost-test In vivo experiment data were calculated by the analysisof variance (ANOVA) followed by Duncanrsquos multiple rangetest 119875 value of less than 005 was considered statistically sig-nificantMeans in the same columnwith different superscriptletters (a b c d e and f) are significantly different (119875 lt 005)between groups

3 Results

31 Effect of EOPK on Fat Accumulation in 3T3-L1 Adipocyte-Like Cells Cytotoxicity of EOPK was determined by MTTassay in 3T3-L1 preadipocytes Cells were treatedwith variousconcentrations of EOPK (0 125 25 or 50120583gmL) for 24 hAs shown in Figure 1(a) EOPK had no significant effecton the viability of 3T3-L1 cells To investigate whetherEOPK can affect the cellular differentiation into adipocytesOil-Red O staining was performed in 3T3-L1 cells treatedwith EOPK for 8 days The differentiated 3T3-L1 adipocytessignificantly increased fat accumulation and intracellulartriglyceride (Figures 1(b) 1(c) and 1(d)) when compared topreadipocytesThe fat deposits were decreased by 57 and 78at the treatment with various concentrations of EOPK (25or 50120583gmL) respectively compared to untreated controladipocytes Results indicate that 50120583gmL of EOPK wasthe most effective ability to inhibit adipocyte differentiation(Figures 1(b) and 1(c)) EOPK treatment reduced the level oftriglyceride in cell in a dose-dependent manner (37 and 60


0 10 20 30 40 50 600

20

40

60

80

100

120

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(a)

Preadipocyte 0 5025 (120583gmL)

Adipocyte

(b)

0

20

40

60

80

100

120

Fat a

ccum

ulat

ion

()

Adipocyte

25 (120583gmL)5000

lowastlowastlowast

lowastlowastlowast

P

(c)

0

20

40

60

80

100

120

Adipocyte

00 25 50 (120583gmL)P

lowastlowastlowast

lowastlowast

Trig

lyce

ride (

)

(d)

Figure 1 Effect of EOPK on the differentiation of 3T3-L1 adipocytes (a) Cytotoxicity of EOPK against 3T3-L1 cells was determined by MTTassay Cells were treated with various concentrations of EOPK (0 125 25 or 50120583gmL) for 24 h (b c and d) Confluent cells were treatedwith 1120583M dexamethasone 1 120583gmL insulin and 05mM IBMX for 2 days and then the medium was replaced by fresh normal medium onlycontaining 1 120583gmL insulin On day 8 the differentiated adipocyte cells were exposed to EOPK for 2 days (b) The differentiated cells werestained with Oil-Red O dye and visualized under inverted microscopy at times100 magnifications (c) After dissolving and cellular lipid retainedOil-Red O in isopropanol adipocyte expression was estimated by measuring OD using microplate reader (Sunrise TECAN MannedorfSwitzerland) at 510 nm (d) Level of intracellular triglyceride

at concentrations of 25 and 50 120583gmL resp) compared topreadipocytes (8004) (Figure 1(d))

32 Effect of EOPKon the Expression of CEBP PPAR120574 GPDHand FABP during the Differentiation of Adipocytes PPAR120574CEBP120572 GPDH and FABP are key factors involved in adi-pogenesis In particular PPAR120574 controls adipogenic factorsas a key transcription factor during adipocyte differentiationBoth mRNA and protein levels of PPAR120574 CEBP120572 andGPDH were downregulated by EOPK in a dose-dependentmanner (Figures 2(a) and 2(b)) To elucidate the underlyingmechanism of EOPK PPAR120574 antagonist GW9662 was used

in 3T3-L1 adipocytes PPAR120574 inhibitor GW9662 enhancedthe decreased expression of FABP and PPAR120574 by EOPK(Figure 2(c))

As shown in Figures 2(d) and 2(e) the lipid accumulationin 3T3-L1 adipocytes treated with EOPK and GW9662 wassignificantly reduced compared to untreated control Consis-tently immunohistochemistry revealed that EOPK treatmentattenuated the expression of PPAR120574 in the liver tissue sectionsof EOPK-treated group (100 and 200mgkg) compared tountreated control group (Figure 3(c)) Taken together theseresults indicate that EOPK inhibits adipocyte differentiationvia suppressing PPAR120574 activation


Adipocyte

P preadipocyte

120573-Actin

GPDH

CEBP120572

EOPK 120583gmLP 0 25 50

PPAR120574

(a)

Adipocyte

120573-Actin

CEBP120572

FABP

EOPK 120583gmLP 0 25 50

PPAR120574

(b)

Adipocyte

EOPK (50 120583gmL)GW9662 (10nM)

120573-Actin

+

+

+

+

minus

minus minus

minus

minus

minus

FABP

P

PPAR120574

(c)

AdipocytePreadipocyte

EOPK (50 120583gmL)GW9662 (10nM)

minus

minus

minus

minus minus

minus+

+

+

+

(d)

0

20

40

60

80

100

120

Adipocyte

EOPK (50 120583gmL)GW9662 (10nM)

P

Fat a

ccum

ulat

ion

()

minus

minus

minus

minus minus

minus+

+

+

+

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)

Figure 2 Effects of EOPK on adipocyte differentiation of 3T3-L1 preadipocytes 3T3-L1 preadipocytes were incubated in medium containinginsulin (10120583gmL) with or without the indicated concentrations of EOPK or GW9662 (a) Total RNA was extracted from 3T3-L1(preadipocytes or adipocytes) cells treated with EOPK and used for RT-PCR analysis of PPAR120574 CEBP120572 GPDH and 120573-actin (b) Totalproteins prepared from EOPK-treated 3T3-L1 (preadipocytes or adipocytes) cells were subjected to western blot analysis of PPAR120574 CEBP120572FABP and 120573-actin (c) Total proteins prepared from EOPK- or GW9662-treated 3T3-L1 (preadipocytes or adipocytes) cells were subjectedto western blot analysis of PPAR120574 FABP and 120573-actin (d) Cells were fixed and stained with Oil-Red O The Oil-Red O-stained adipocyteswere photographed at a times100 magnification under a microscope (e) Lipids were extracted using isopropanol and the Oil-Red O was thenanalyzed at a wavelength of 520 nm Values are presented as means plusmn SE lowast119875 lt 005 versus the control


00

100

200

300

ab ab

dd e

d

ab a

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cdc d

bc

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cdbc b

c

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Body

wei

ght (

g)

Normal Control

EOPK

1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6100mgkg 200mgkg 10mgkg

(wk)

Atorvastatin

(a)

0

5

10

15

20

25

RetroperitonealEpididymal

d

a

ab

bc

cd

a

bcd

cd

100 200 10

EOPK

Normal Control (mgkg)

Wei

ght (

mg

g)

Atorvastatin

(b)

10100 200

EOPK


PPAR120574

Atorvastatin

(c)

Figure 3 Effect of EOPK on body and abdominal fat weight of high-fat diet-fed rats Rats fed a high diet were orally treated with or withoutEOPK daily for 6 consecutive weeks (a) Body weights of rats (b) abdominal fat weights and The retroperitoneal and epididymal fats fromrats treated with or without EOPK (100 and 200mgkg) were removed and weighed (c) Representative picture of immunohistochemicalstaining for PPAR120574 in liver tissue sections Data were expressed as means plusmn SD Values with the different superscript letters indicate statisticalsignificance (119875 lt 005) between groups by Duncanrsquos multiple range test

33 Effect of EOPK on BodyWeight of High-Fat Diet-Fed RatsThe body weights of normal (group 1) control (group 2)EOPK-treated groups (groups 3 and 4) and atorvastatin-treated group (group 5) were monitored once every weekfor 6 weeks As shown in Figure 3(a) the body weight ofhigh-fat fed control groupwas significantly increased 2 weeksafter feeding compared to normal low fat group In contrast

the body weight gain was dose dependently abrogated inEOPK-treated groups from the date of 3-week treatment ofEOPK Also six weeks after treatment the body weight wassignificantly gained to 3314 plusmn 28 g in high-fat fed controlgroup compared to normal group (2816 plusmn 17 g) HoweverEOPK significantly suppressed the body weights to 3095 plusmn18 g and 3037plusmn20 g respectively at doses of 100mgkg and


0

50

100

150

d d

a

bc

c

Atorvastatin

Seru

m tr

igly

cerid

e (m

gdL

)

10Normal Control 200100 (mgkg)EOPK

(a)

Atorvastatin

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abc

d

Tota

l cho

leste

rol (

mg

dL)

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EOPK(b)

Atorvastatin

0

10

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30

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50

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a

c

bc

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cd

a a ab

b

Chol

este

rol (

mg

dL)


EOPK

(c)

00

05

10

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cd

a

b

bcAt

hero

scle

rosis

inde

x (m

gdL

)

10Normal Control 200100 (mgkg)AtorvastatinEOPK

(d)

Figure 4 Effects of EOPK on serum lipid and cholesterol levels in high-fat diet-fed rats (a) Triglyceride level was measured by a triglycerideassay kit (AM 157S-KAsanPharmCo Seoul Korea) (b) Total cholesterol level wasmeasured by using a total cholesterol assay kit (AM202-KAsan Pharm Co Seoul Korea) (c)The levels of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in serumwere measuredusing cobas c 111 analyzer (Roche-Diagnostics Indianapolis IN USA) (d) AI was calculated by employing the following equation AI =(total cholesterol minusHDL cholesterol)HDL cholesterol Values with the different superscript letters indicate statistical significance (119875 lt 005)between groups by Duncanrsquos multiple range test

200mgkg almost coming up with atorvastatin as positivecontrol (2965 plusmn 19 g)

34 Effects of EOPK on Abdominal Fat Weight of High-Fat Diet-Fed Rats The retroperitoneal and epididymal fatweight was measured to test whether the body weightnormalization by EOPK in high-fat diet-fed rats is associatedwith a reduction of fat content in body In the presentstudy as expected high-fat diet significantly increased theretroperitoneal fat weight to 183 plusmn 291mgg from 621 plusmn126mgkg body weight In contrast EOPK decreased theretroperitoneal fat weight to 157plusmn186 and 127plusmn143mgg atdoses of 100 and 200mgkg respectively compared to controlgroup (Figure 3(b)) Likewise epididymal fat weight was alsoincreased in the high-fat diet-fed rats compared to normalgroup Oral administration of EOPK decreased epididymal

fat pad weight by 109 plusmn 185 and 852 plusmn 196mgg at doses of100 and 200mgkg respectively (Figure 3(b))

35 Effects of EOPK on Serum Lipid and Cholesterol Levelsin High-Fat Diet-Fed Rats The consumption of the high-fatdiet significantly increased triglyceride in control group com-pared to the normal low fat group (Figure 4(a)) EOPK treat-ment decreased level of serum triglyceride from 1318 plusmn 182to 1093 plusmn 115 and 942 plusmn 843mgdL at 100 and 200mgkgrespectively (Figure 4(a)) Additionally the consumption ofthe high-fat diet significantly increased serum total choles-terol compared to the normal low fat group while EOPKsignificantly reduced the level of total cholesterol in a dose-dependent manner (Figure 4(b)) The intake of the high-fat diet significantly decreased level of HDL but increasedlevel of LDL compared to normal group (Figure 4(c) control)


e

010

20

30

40

d

a

bccd

Atorvastatin

Hep

atic

trig

lyce

ride (

mg

g)

EOPK

Normal Control 200100 (mgkg)

10

(a)

0

5

10

15

20

25

b

c

a

b

Hep

atic

chol

este

rol (

mg

g)

10

Atorvastatin

c

EOPK


(b)

Figure 5 Effects of EOPK on hepatic triglyceride and cholesterol in high-fat diet-fed rats The levels of triglyceride (a) and total cholesterol(b) in liver were measured by Biochemistry Analyzer Values with the different superscript letters indicate statistical significance (119875 lt 005)between groups by Duncanrsquos multiple range test

However EOPKdid not have a significant effect on LDLwhileelevating HDL level in a dose-dependent manner comparedto the high-fat diet control group (Figure 4(c)) ConsistentlyEOPK treatment significantly decreased the atherosclerosisindex (AI) value in a dose-dependent manner compared tothe high-fat diet control (Figure 4(d))

36 Effects of EOPK on Hepatic Triglyceride and Cholesterolin High-Fat Diet-Fed Rats The consumption of the high-fatdiet significantly increased hepatic triglyceride (Figure 5(a))and total cholesterol (Figure 5(b)) when compared to thenormal low fat group Oral treatment with EOPK reducedthe level of triglyceride from the liver in a dose-dependentmanner (244 plusmn 247 and 212 plusmn 188mgg at doses of 100and 200mgkg resp) compared to control group (298 plusmn325mgg tissue) (Figure 5(a)) In addition EOPK admin-istration significantly lowered total cholesterol level in liverfrom 169 plusmn 218mgg in the high-fat diet control groupto 126 plusmn 142 and 104 plusmn 159mgg at 100 and 200mgkgrespectively (Figure 5(b))

4 Discussion

The Korean pine (P koraiensis) is an important afforesta-tion trees in Korea and also distributed in China RussiaJapan and Europe [21] P koraiensis extract attenuated theincrease in blood pressure in spontaneously hypertensiverats [13] and its constituent pinolenic acid had cholesterol-lowering effect via regulation of LDL receptor activity inHepG2 hepatoma cells [22] In addition P koraiensis nut oileffectively regulated satiety hormones and prospective foodintake in postmenopausal overweight women [11] Our groupalso reported that essential oil from P koraiensis leaves hadantihyperlipidemic effects via upregulation of LDL receptor

and inhibition of acyl-coenzyme A cholesterol acyltrans-ferase [9] In the current study we demonstrate that EOPKhas antiobesity effects in 3T3-L1 adipocytes and high-fat diet-fed rat models Adipogenesis is the process of differentiationby which undifferentiated preadipocytes are converted intodifferentiated adipocytes [23] and subsequently mediate thesynthesis and accumulation of lipid [24] 3T3-L1 cells arethe best characterized model to study adipogenesis in vitro[25] Oil-Red O staining revealed that EOPK significantlysuppressed fat accumulation and intracellular triglycerideand decreased expression of PPAR120574 CEBP120572 FABP andGPDH in the differentiated 3T3-L1 adipocytes with no cyto-toxicity indicating the inhibitory effect of EOPKon adipocytedifferentiation Similarly many natural compounds includ-ing EGCG berberine and curcumin suppressed PPAR120574signaling since PPAR120574 antagonists have been reported toeffectively inhibit adipogenesis and improve insulin sensitiv-ity in vitro and in vivo [26]

Furthermore here the antiobesic effect of P koraiensiswas confirmed in high-fat diet- (HFD-) treated SD rats atthe doses of 100 or 200mgkg via blockage of body weightgain Since loss of body weight is mainly associated with thedecrease of fat pad mass as a result of reduction of adipocytesize or triglyceride accumulation [27] our data that EOPKsignificantly reduced the retroperitoneal and epididymal fatweight and also serum triglyceride compared to HFD fed ratssuggest that EOPK can block obesity via inhibition of lipidmetabolism including triglyceride

Cholesterol is also an important component of fat com-plex In particular low level of serum HDL cholesterol istightly linked to the occurrence of obesity [28] In our studyadministration of EOPK significantly abrogated the contentsof total cholesterol in serum In addition EOPK significantlyincreased level of HDL cholesterol in a dose-dependentmanner compared to HFD control group but not LDLcholesterol Lipid parameters in the blood can be affected


by the hepatic metabolisms As expected we observed thatthe hepatic contents of triglyceride and cholesterol weresignificantly escalated in HFD fed rats compared to normalcontrol In contrast EOPK treatment significantly loweredthe contents of triglyceride and total cholesterol in the liverimplying that EOPK regulates lipid metabolism includingtriglyceride and cholesterol against obesity

In summary EOPK inhibited fat accumulation intracel-lular triglyceride and expression of PPAR120574 CEBP120572 FABPand GPDH in the differentiated 3T3-L1 adipocytes AlsoEOPK attenuated serum and hepatic contents of triglycerideand total cholesterol in HFD fed rat models FurthermoreEOPK decreased the expression of PPAR120574 in the liver tissuesections of EOPK-treated rats Overall our findings suggestthe potential of EOPK as an antiobesity agent

Conflict of Interests

No potential conflict of interests was disclosed

Acknowledgments

This work was supported by a grant from the Next-Generation BioGreen 21 Program (no PJ009055) and MRC(no 2012-0005755) Republic of Korea

References

[1] DW Haslam andW P T James ldquoObesityrdquoTheLancet vol 366no 9492 pp 1197ndash1209 2005

[2] D P Schuster ldquoObesity and the development of type 2 diabetesthe effects of fatty tissue inflammationrdquo Diabetes MetabolicSyndrome and Obesity vol 3 pp 253ndash262 2010

[3] J M Friedman ldquoObesity in the new millenniumrdquo Nature vol404 no 6778 pp 632ndash634 2000

[4] J S Flier ldquoObesity wars molecular progress confronts anexpanding epidemicrdquo Cell vol 116 no 2 pp 337ndash350 2004

[5] F M Gregoire C M Smas and H S Sul ldquoUnderstandingadipocyte differentiationrdquo Physiological Reviews vol 78 no 3pp 783ndash809 1998

[6] W-C Yeh Z Cao M Classon and S L McKnight ldquoCascaderegulation of terminal adipocyte differentiation by three mem-bers of the CEBP family of leucine zipper proteinsrdquo Genes andDevelopment vol 9 no 2 pp 168ndash181 1995

[7] G J Darlington S E Ross and O AMacDougald ldquoThe role ofCEBP genes in adipocyte differentiationrdquo Journal of BiologicalChemistry vol 273 no 46 pp 30057ndash30060 1998

[8] M I Lefterova and M A Lazar ldquoNew developments inadipogenesisrdquo Trends in Endocrinology andMetabolism vol 20no 3 pp 107ndash114 2009

[9] J-H Kim H-J Lee S-J Jeong M-H Lee and S-H KimldquoEssential oil of pinus koraiensis leaves exerts antihyperlipi-demic effects via up-regulation of low-density lipoproteinreceptor and inhibition of acyl-coenzyme a cholesterol acyl-transferaserdquo Phytotherapy Research vol 26 no 9 pp 1314ndash13192012

[10] X Chen Y Zhang Z Wang and Y Zu ldquoInvivo antioxidantactivity of Pinus koraiensis nut oil obtained by optimised super-critical carbon dioxide extractionrdquo Natural Product Researchvol 25 no 19 pp 1807ndash1816 2011

[11] W J Pasman J Heimerikx C M Rubingh et al ldquoThe effect ofKorean pine nut oil on in vitro CCK release on appetite sen-sations and on gut hormones in post-menopausal overweightwomenrdquo Lipids in Health and Disease vol 7 article 10 2008

[12] G J A Speijers L H T Dederen andH Keizer ldquoA sub-chronic(13 weeks) oral toxicity study in rats and an in vitro genotoxicitystudy with Korean pine nut oil (PinnoThin TG)rdquo RegulatoryToxicology and Pharmacology vol 55 no 2 pp 158ndash165 2009

[13] M Sugano I Ikeda K Wakamatsu and T Oka ldquoInfluence ofKorean pine (Pinus koraiensis)-seed oil containing cis-5cis-9cis-12-octadecatrienoic acid on polyunsaturated fatty acidmetabolism eicosanoid production and blood pressure of ratsrdquoBritish Journal of Nutrition vol 72 no 5 pp 775ndash783 1994

[14] U H Park J C Jeong J S Jang et al ldquoNegative regulation ofadipogenesis by kaempferol a component of Rhizoma Polyg-onati falcatum in 3T3-L1 cellsrdquo Biological amp PharmaceuticalBulletin vol 35 no 9 pp 1525ndash1533 2012

[15] H Kim and S Y Choung ldquoAnti-obesity effects of Boussingaultigracilis Miers var pseudobaselloides Bailey via activation ofAMP-activated protein kinase in 3T3-L1 cellsrdquo Journal ofMedic-inal Food vol 15 no 9 pp 811ndash817 2012

[16] Y Oi I-C Hou H Fujita and K Yazawa ldquoAntiobesity effectsof Chinese black tea (Pu-erh tea) extract and gallic acidrdquoPhytotherapy Research vol 26 no 4 pp 475ndash481 2012

[17] E-J Hong K-J Na I-G Choi K-C Choi and E-B JeungldquoAntibacterial and antifungal effects of essential oils fromconiferous treesrdquoBiological and Pharmaceutical Bulletin vol 27no 6 pp 863ndash866 2004

[18] Q R Rogers and A E Harper ldquoAmino acid diets and maximalgrowth in the ratrdquo Journal of Nutrition vol 87 no 3 pp 267ndash273 1965

[19] W Richmond ldquoUse of cholesterol oxidase for assay of total andfree cholesterol in serum by continuous flow analysisrdquo ClinicalChemistry vol 22 no 10 pp 1579ndash1588 1976

[20] MWMcGowan J D Artiss D R Strandbergh and B Zak ldquoAperoxidase-coupledmethod for the colorimetric determinationof serum triglyceridesrdquo Clinical Chemistry vol 29 no 3 pp538ndash542 1983

[21] D S Choi M Kayama H O Jin C H Lee T Izutaand T Koike ldquoGrowth and photosynthetic responses of twopine species (Pinus koraiensis and Pinus rigida) in a pollutedindustrial region in Koreardquo Environmental Pollution vol 139no 3 pp 421ndash432 2006

[22] J-W Lee K-W Lee S-W Lee I-H Kim and C Rhee ldquoSelec-tive increase in pinolenic acid (all-cis-5912ndash183) in Koreanpine nut oil by crystallization and its effect on LDL-receptoractivityrdquo Lipids vol 39 no 4 pp 383ndash387 2004

[23] T C Otto and M D Lane ldquoAdipose development fromstem cell to adipocyterdquo Critical Reviews in Biochemistry andMolecular Biology vol 40 no 4 pp 229ndash242 2005

[24] A G Cristancho and M A Lazar ldquoForming functional fata growing understanding of adipocyte differentiationrdquo NatureReviews Molecular Cell Biology vol 12 no 11 pp 722ndash734 2011

[25] H Green and O Kehinde ldquoAn established preadipose cell lineand its differentiation in culture II Factors affecting the adiposeconversionrdquo Cell vol 5 no 1 pp 19ndash27 1975

[26] J Rieusset F Touri L Michalik et al ldquoA new selectiveperoxisome proliferator-activated receptor 120574 antagonist withantiobesity and antidiabetic activityrdquo Molecular Endocrinologyvol 16 no 11 pp 2628ndash2644 2002

[27] M Rosenbaum R L Leibel and J Hirsch ldquoObesityrdquo The NewEngland Journal of Medicine vol 337 no 6 pp 396ndash407 1997


[28] S C Woods R J Seeley D Porte Jr and M W SchwartzldquoSignals that regulate food intake and energy homeostasisrdquoScience vol 280 no 5368 pp 1378ndash1383 1998

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


bioactive chemicals from P koraiensis include camphene D-limonene 120572-pinene borneol 120573-pinene 4-carene bicyclo-hept-3-ene 3-carene 120573-phellandrene and fencyl [9] We andothers demonstrated the biological efficacies and genotoxicityof essential oil from P koraiensis seed leaf nut and cone[9ndash13] However the biological and biochemical effects ofessential oil from P koraiensis have not been reported yet

Recently many studies reported that natural productssuch as Rhizoma Polygonati falcatum (RPF) [14] Boussin-gaultia gracilis Miers var pseudobaselloides Bailey [15] Chi-nese black tea (Pu-erh tea) extract and gallic acid [16]showed the potential of antiobesity activity in vitro or invivo In the present study we investigated the antiobesicand hypolipidemic effect of essential oil of P koraiensis SIEB(EOPK) in 3T3-L1 cells by Oil-Red O staining measuringtriglyceride and analyzing expression levels of adipogenicfactors and in high-fat diet-fed rats by measuring bodyweights and fats and lipid metabolites

2 Materials and Methods

21 Preparation of Essential Oil of P koraiensis Leaves (EOPK)EOPK was prepared using the hydrodistillation method [17]Driedand pulverized P koraiensis leaves were immersed indistilled water and submitted to steam distillation using anapparatus with a condenser (Hanil Labtech Seoul Korea)The distillation continued for 3-4 h at 90∘C and then thevolatile compounds contained in the water-soluble fractionwere allowed to settle for 20min The essential oil layer wasseparated and purified through microfiltration

22 Cell Culture 3T3-L1 preadipocytes were purchased fromKorean Cell Line Bank (KCLB) The cells were cultured inDulbeccorsquos Modified Eaglersquos Medium (DMEM) with 10 FBSin a humidified atmosphere of 95 air and 5 CO

2at 37∘C

23 Cytotoxicity Assay The cytotoxicity of EOPK against3T3-L1 cells was measured by 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2H-tetrazolium bromide (MTT) colorimetricassay The cells were seeded onto 96-well microplates at adensity of 1 times 104 cells per well and treated with variousconcentrations of EOPK (0 125 25 or 50120583gmL) for 24 hMTT working solution was then added to the microplatesat 37∘C for 2 h and then MTT extraction buffer (20 SDSand 50 dimethylformamide) was added at 37∘C overnightOptical density (OD) was measured using microplate reader(Sunrise TECAN Mannedorf Switzerland) at 570 nm Cellviability was calculated by employing the following equationcell viability () = [OD (EOPK) minus OD (Blank)][OD (Con-trol) minus OD (Blank)] times 100

24 Differentiation Induction and Oil-Red O Staining Thepreadipocyte 3T3-L1 cells were plated onto 6-well plateson day 0 and incubated to confluent status For adipocytedifferentiation the confluent cells were treated with 1120583Mdexamethasone 1 120583gmL insulin and 05mM IBMX for2 days and the medium was replaced by fresh normalmedium only containing 1 120583gmL insulin On day 8 the

differentiated adipocyte cells were cultured in the presenceor absence of EOPK (100120583gmL) for 2 days The cells werefixedwith 2 paraformaldehyde washed twice with PBS andfinally stained with Oil-Red After dissolving cellular-lipidretained Oil-Red O in isopropanol and adipocyte expressionwas estimated by measuring OD using microplate reader(Sunrise TECAN Mannedorf Switzerland) at 510 nm

25 RT-PCR Analysis The 3T3-L1 cells were treated with orwithout EOPK (50 or 100mgmL) for 24 h The total RNAwas extracted by using TRIzol reagent (Invitrogen CarlsbadCA USA) according to the manufacturerrsquos instructionscDNA was synthesized from 1120583g of total RNA and subjectedto PCR reaction by using a SuperScript One-Step reversetranscription-PCR (RT-PCR) kit (Invitrogen Carlsbad CAUSA) The PCR conditions were 30 cycles of 94∘C for 30 s57∘C for 30 s and 72∘C for 30 s The primer sequenceswere as follows PPAR120574 (forward 51015840-GGTGAAACTCTG-GGAGATTC-31015840 and reverse 51015840-CAACCATTGGGTCAG-CTCTT-31015840) CEBP120572 (forward 51015840-AGGTGCTGGAGTTGA-CCAGT-31015840 and reverse 51015840-CAGCCTAGAGATCCAGCG-AC-31015840) GPDH (forward 51015840-GAACTAAGGAGCAGCTCA-AAGGTTC-31015840 and reverse 51015840-CAGTTGACTGACTGA-GCAAACATAG-31015840) 120573-actin (forward 51015840-ACCGTGAAA-AGATGACCCAG-31015840 and reverse 51015840-TACGGATGACAA-CGTCACAC-31015840) PCR products were run on 2 agarose geland then stained with ethidium bromide Stained bands werevisualized under UV light and photographed

26 Western Blotting Whole cell lysates (25 120583g) were pre-pared using lysis buffer (20mM Tris pH 74 250mM NaCl2mM EDTA pH 80 01 Triton X-100 001mgmL apro-tinin 0003mgmL leupeptin 04mM phenylmethylsulfonylfluoride (PMSF) and 4mMNaVO4)The lysates were spun at13000timesg for 15min to remove insolublematerial and resolvedon a 10 SDS-PAGE gel After electrophoresis the pro-teins were electrotransferred to a nitrocellulose membraneblocked with 5 nonfat milk and probed with antibodiesagainst PPAR120574 (Novus Littleton CO USA) CEBP120572 (CellSignaling Tech Danvers MA USA) FABP (Cell SignalingTech Danvers MA USA) and 120573-actin (Sigma St LouisMO USA) overnight

The blots were washed exposed to horseradishperoxidase- (HRP-) conjugated secondary antibodies for2 h and finally examined by enhanced chemiluminescence(ECL) (GE Health Care Bio-Sciences Piscataway NJ USA)

27 Animals Diet Manipulation and EOPK Treatment MaleSprague-Dawley rats at 4 weeks of age were purchased fromHyo-Chang Science (Daegu Korea) and maintained underconventional conditions Fifty rats were divided into fourgroups normal group (low fat diet) control group (high-fatdiet) two EOPK-treated groups- and atorvastatin (positivecontrol) treated group consuming high-fat diets (10 rats pergroup) Rats were fed the normal (low fat) diet (group 1)or the high-fat diet (groups 2ndash5) for 6 weeks High-fat dietcomposition was described in Table 1 For EOPK treatment













3 Results



0 10 20 30 40 50 600

20

40

60

80

100

120

Cel

l via

bilit

y (

)


(a)


Adipocyte

(b)

0

20

40

60

80

100

120

Fat a

ccum

ulat

ion

()

Adipocyte

25 (120583gmL)5000

lowastlowastlowast

lowastlowastlowast

P

(c)

0

20

40

60

80

100

120

Adipocyte

00 25 50 (120583gmL)P

lowastlowastlowast

lowastlowast

Trig

lyce

ride (

)

(d)







Adipocyte

P preadipocyte

120573-Actin

GPDH

CEBP120572

EOPK 120583gmLP 0 25 50

PPAR120574

(a)

Adipocyte

120573-Actin

CEBP120572

FABP

EOPK 120583gmLP 0 25 50

PPAR120574

(b)

Adipocyte

EOPK (50 120583gmL)GW9662 (10nM)

120573-Actin

+

+

+

+

minus

minus minus

minus

minus

minus

FABP

P

PPAR120574

(c)


EOPK (50 120583gmL)GW9662 (10nM)

minus

minus

minus

minus minus

minus+

+

+

+

(d)

0

20

40

60

80

100

120

Adipocyte

EOPK (50 120583gmL)GW9662 (10nM)

P

Fat a

ccum

ulat

ion

()

minus

minus

minus

minus minus

minus+

+

+

+

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)



00

100

200

300

ab ab

dd e

d

ab a

a

cdc d

bc

ba

ab

cdbc b

c

a aab

aa

a

a

b bc

d e f

e

Body

wei

ght (

g)

Normal Control

EOPK

1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6100mgkg 200mgkg 10mgkg

(wk)

Atorvastatin

(a)

0

5

10

15

20

25


d

a

ab

bc

cd

a

bcd

cd

100 200 10

EOPK


Wei

ght (

mg

g)

Atorvastatin

(b)

10100 200

EOPK


PPAR120574

Atorvastatin

(c)





0

50

100

150

d d

a

bc

c

Atorvastatin

Seru

m tr

igly

cerid

e (m

gdL

)


(a)

Atorvastatin

0

20

40

60

80

100

fe

abc

d

Tota

l cho

leste

rol (

mg

dL)


EOPK(b)

Atorvastatin

0

10

20

30

40

50

HDLLDL

a

c

bc

b

cd

a a ab

b

Chol

este

rol (

mg

dL)


EOPK

(c)

00

05

10

15

20

d

cd

a

b

bcAt

hero

scle

rosis

inde

x (m

gdL

)


(d)







e

010

20

30

40

d

a

bccd

Atorvastatin

Hep

atic

trig

lyce

ride (

mg

g)

EOPK


10

(a)

0

5

10

15

20

25

b

c

a

b

Hep

atic

chol

este

rol (

mg

g)

10

Atorvastatin

c

EOPK


(b)




4 Discussion










Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























3 Results



0 10 20 30 40 50 600

20

40

60

80

100

120

Cel

l via

bilit

y (

)


(a)


Adipocyte

(b)

0

20

40

60

80

100

120

Fat a

ccum

ulat

ion

()

Adipocyte

25 (120583gmL)5000

lowastlowastlowast

lowastlowastlowast

P

(c)

0

20

40

60

80

100

120

Adipocyte

00 25 50 (120583gmL)P

lowastlowastlowast

lowastlowast

Trig

lyce

ride (

)

(d)







Adipocyte

P preadipocyte

120573-Actin

GPDH

CEBP120572

EOPK 120583gmLP 0 25 50

PPAR120574

(a)

Adipocyte

120573-Actin

CEBP120572

FABP

EOPK 120583gmLP 0 25 50

PPAR120574

(b)

Adipocyte

EOPK (50 120583gmL)GW9662 (10nM)

120573-Actin

+

+

+

+

minus

minus minus

minus

minus

minus

FABP

P

PPAR120574

(c)


EOPK (50 120583gmL)GW9662 (10nM)

minus

minus

minus

minus minus

minus+

+

+

+

(d)

0

20

40

60

80

100

120

Adipocyte

EOPK (50 120583gmL)GW9662 (10nM)

P

Fat a

ccum

ulat

ion

()

minus

minus

minus

minus minus

minus+

+

+

+

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)



00

100

200

300

ab ab

dd e

d

ab a

a

cdc d

bc

ba

ab

cdbc b

c

a aab

aa

a

a

b bc

d e f

e

Body

wei

ght (

g)

Normal Control

EOPK

1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6100mgkg 200mgkg 10mgkg

(wk)

Atorvastatin

(a)

0

5

10

15

20

25


d

a

ab

bc

cd

a

bcd

cd

100 200 10

EOPK


Wei

ght (

mg

g)

Atorvastatin

(b)

10100 200

EOPK


PPAR120574

Atorvastatin

(c)





0

50

100

150

d d

a

bc

c

Atorvastatin

Seru

m tr

igly

cerid

e (m

gdL

)


(a)

Atorvastatin

0

20

40

60

80

100

fe

abc

d

Tota

l cho

leste

rol (

mg

dL)


EOPK(b)

Atorvastatin

0

10

20

30

40

50

HDLLDL

a

c

bc

b

cd

a a ab

b

Chol

este

rol (

mg

dL)


EOPK

(c)

00

05

10

15

20

d

cd

a

b

bcAt

hero

scle

rosis

inde

x (m

gdL

)


(d)







e

010

20

30

40

d

a

bccd

Atorvastatin

Hep

atic

trig

lyce

ride (

mg

g)

EOPK


10

(a)

0

5

10

15

20

25

b

c

a

b

Hep

atic

chol

este

rol (

mg

g)

10

Atorvastatin

c

EOPK


(b)




4 Discussion










Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 10 20 30 40 50 600

20

40

60

80

100

120

Cel

l via

bilit

y (

)


(a)


Adipocyte

(b)

0

20

40

60

80

100

120

Fat a

ccum

ulat

ion

()

Adipocyte

25 (120583gmL)5000

lowastlowastlowast

lowastlowastlowast

P

(c)

0

20

40

60

80

100

120

Adipocyte

00 25 50 (120583gmL)P

lowastlowastlowast

lowastlowast

Trig

lyce

ride (

)

(d)







Adipocyte

P preadipocyte

120573-Actin

GPDH

CEBP120572

EOPK 120583gmLP 0 25 50

PPAR120574

(a)

Adipocyte

120573-Actin

CEBP120572

FABP

EOPK 120583gmLP 0 25 50

PPAR120574

(b)

Adipocyte

EOPK (50 120583gmL)GW9662 (10nM)

120573-Actin

+

+

+

+

minus

minus minus

minus

minus

minus

FABP

P

PPAR120574

(c)


EOPK (50 120583gmL)GW9662 (10nM)

minus

minus

minus

minus minus

minus+

+

+

+

(d)

0

20

40

60

80

100

120

Adipocyte

EOPK (50 120583gmL)GW9662 (10nM)

P

Fat a

ccum

ulat

ion

()

minus

minus

minus

minus minus

minus+

+

+

+

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)



00

100

200

300

ab ab

dd e

d

ab a

a

cdc d

bc

ba

ab

cdbc b

c

a aab

aa

a

a

b bc

d e f

e

Body

wei

ght (

g)

Normal Control

EOPK

1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6100mgkg 200mgkg 10mgkg

(wk)

Atorvastatin

(a)

0

5

10

15

20

25


d

a

ab

bc

cd

a

bcd

cd

100 200 10

EOPK


Wei

ght (

mg

g)

Atorvastatin

(b)

10100 200

EOPK


PPAR120574

Atorvastatin

(c)





0

50

100

150

d d

a

bc

c

Atorvastatin

Seru

m tr

igly

cerid

e (m

gdL

)


(a)

Atorvastatin

0

20

40

60

80

100

fe

abc

d

Tota

l cho

leste

rol (

mg

dL)


EOPK(b)

Atorvastatin

0

10

20

30

40

50

HDLLDL

a

c

bc

b

cd

a a ab

b

Chol

este

rol (

mg

dL)


EOPK

(c)

00

05

10

15

20

d

cd

a

b

bcAt

hero

scle

rosis

inde

x (m

gdL

)


(d)







e

010

20

30

40

d

a

bccd

Atorvastatin

Hep

atic

trig

lyce

ride (

mg

g)

EOPK


10

(a)

0

5

10

15

20

25

b

c

a

b

Hep

atic

chol

este

rol (

mg

g)

10

Atorvastatin

c

EOPK


(b)




4 Discussion










Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Adipocyte

P preadipocyte

120573-Actin

GPDH

CEBP120572

EOPK 120583gmLP 0 25 50

PPAR120574

(a)

Adipocyte

120573-Actin

CEBP120572

FABP

EOPK 120583gmLP 0 25 50

PPAR120574

(b)

Adipocyte

EOPK (50 120583gmL)GW9662 (10nM)

120573-Actin

+

+

+

+

minus

minus minus

minus

minus

minus

FABP

P

PPAR120574

(c)


EOPK (50 120583gmL)GW9662 (10nM)

minus

minus

minus

minus minus

minus+

+

+

+

(d)

0

20

40

60

80

100

120

Adipocyte

EOPK (50 120583gmL)GW9662 (10nM)

P

Fat a

ccum

ulat

ion

()

minus

minus

minus

minus minus

minus+

+

+

+

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(e)



00

100

200

300

ab ab

dd e

d

ab a

a

cdc d

bc

ba

ab

cdbc b

c

a aab

aa

a

a

b bc

d e f

e

Body

wei

ght (

g)

Normal Control

EOPK

1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6100mgkg 200mgkg 10mgkg

(wk)

Atorvastatin

(a)

0

5

10

15

20

25


d

a

ab

bc

cd

a

bcd

cd

100 200 10

EOPK


Wei

ght (

mg

g)

Atorvastatin

(b)

10100 200

EOPK


PPAR120574

Atorvastatin

(c)





0

50

100

150

d d

a

bc

c

Atorvastatin

Seru

m tr

igly

cerid

e (m

gdL

)


(a)

Atorvastatin

0

20

40

60

80

100

fe

abc

d

Tota

l cho

leste

rol (

mg

dL)


EOPK(b)

Atorvastatin

0

10

20

30

40

50

HDLLDL

a

c

bc

b

cd

a a ab

b

Chol

este

rol (

mg

dL)


EOPK

(c)

00

05

10

15

20

d

cd

a

b

bcAt

hero

scle

rosis

inde

x (m

gdL

)


(d)







e

010

20

30

40

d

a

bccd

Atorvastatin

Hep

atic

trig

lyce

ride (

mg

g)

EOPK


10

(a)

0

5

10

15

20

25

b

c

a

b

Hep

atic

chol

este

rol (

mg

g)

10

Atorvastatin

c

EOPK


(b)




4 Discussion










Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

100

200

300

ab ab

dd e

d

ab a

a

cdc d

bc

ba

ab

cdbc b

c

a aab

aa

a

a

b bc

d e f

e

Body

wei

ght (

g)

Normal Control

EOPK

1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6100mgkg 200mgkg 10mgkg

(wk)

Atorvastatin

(a)

0

5

10

15

20

25


d

a

ab

bc

cd

a

bcd

cd

100 200 10

EOPK


Wei

ght (

mg

g)

Atorvastatin

(b)

10100 200

EOPK


PPAR120574

Atorvastatin

(c)





0

50

100

150

d d

a

bc

c

Atorvastatin

Seru

m tr

igly

cerid

e (m

gdL

)


(a)

Atorvastatin

0

20

40

60

80

100

fe

abc

d

Tota

l cho

leste

rol (

mg

dL)


EOPK(b)

Atorvastatin

0

10

20

30

40

50

HDLLDL

a

c

bc

b

cd

a a ab

b

Chol

este

rol (

mg

dL)


EOPK

(c)

00

05

10

15

20

d

cd

a

b

bcAt

hero

scle

rosis

inde

x (m

gdL

)


(d)







e

010

20

30

40

d

a

bccd

Atorvastatin

Hep

atic

trig

lyce

ride (

mg

g)

EOPK


10

(a)

0

5

10

15

20

25

b

c

a

b

Hep

atic

chol

este

rol (

mg

g)

10

Atorvastatin

c

EOPK


(b)




4 Discussion










Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

d d

a

bc

c

Atorvastatin

Seru

m tr

igly

cerid

e (m

gdL

)


(a)

Atorvastatin

0

20

40

60

80

100

fe

abc

d

Tota

l cho

leste

rol (

mg

dL)


EOPK(b)

Atorvastatin

0

10

20

30

40

50

HDLLDL

a

c

bc

b

cd

a a ab

b

Chol

este

rol (

mg

dL)


EOPK

(c)

00

05

10

15

20

d

cd

a

b

bcAt

hero

scle

rosis

inde

x (m

gdL

)


(d)







e

010

20

30

40

d

a

bccd

Atorvastatin

Hep

atic

trig

lyce

ride (

mg

g)

EOPK


10

(a)

0

5

10

15

20

25

b

c

a

b

Hep

atic

chol

este

rol (

mg

g)

10

Atorvastatin

c

EOPK


(b)




4 Discussion










Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















e

010

20

30

40

d

a

bccd

Atorvastatin

Hep

atic

trig

lyce

ride (

mg

g)

EOPK


10

(a)

0

5

10

15

20

25

b

c

a

b

Hep

atic

chol

este

rol (

mg

g)

10

Atorvastatin

c

EOPK


(b)




4 Discussion










Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


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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