Hyun-Suk Ko, 1 Hyo-Jeong Lee, 1 Hyo-Jung Lee, 1,2 Eun Jung Sohn, 1 Miyong Yun, 1 Min-Ho Lee, 3 and Sung-Hoon Kim 1. 1.

Transcription

1 Eviene-Bse Complementry n Alterntive Meiine Volume 13, Artile ID 94737, 1 pges Reserh Artile Essentil Oil of Pinus koriensis Exerts Antioesi n Hypolipiemi Ativity vi Inhiition of Peroxisome Prolifertor-Ativte Reeptors Gmm Signling Hyun-Suk Ko, 1 Hyo-Jeong Lee, 1 Hyo-Jung Lee, 1,2 Eun Jung Sohn, 1 Miyong Yun, 1 Min-Ho Lee, 3 n Sung-Hoon Kim 1 1 College of Orientl Meiine, Kyung Hee University, 1 Hoegi-ong, Dongemun-gu, Seoul 13-71, Repuli of Kore 2 Meil Genomis Reserh Center, Kore Reserh Institute of Biosiene n Biotehnology, Dejeon , Repuli of Kore 3 CollegeofLifeSienesnBiotehnology,KyungHeeUniversity,Seoul13-71,RepuliofKore Corresponene shoul e resse to Sung-Hoon Kim; sungkim7@khu..kr Reeive 16 April 13; Aepte 8 July 13 Aemi Eitor: Je Youl Cho Copyright 13 Hyun-Suk Ko et l. This is n open ess rtile istriute uner the Cretive Commons Attriution Liense, whih permits unrestrite use, istriution, n reproution in ny meium, provie the originl work is properly ite. Our group previously reporte tht essentil oil of Pinus koriensis () exerts ntihyperlipiemi effets vi upregultion of low-ensity lipoprotein reeptor n inhiition of yl-oenzyme A. In the present stuy, we investigte the ntioesity n hypolipiemi mehnism of using in vitro 3T3-L1 ells n in vivo HFD-fe rts. mrkely suppresse ft umultion n intrellulr triglyerie ssoite with ownregultion of ipogeni trnsription ftor expression, inluing PPARγ n CEBPα in the ifferentite 3T3-L1 ipoytes. Aitionlly, ttenute the expression levels of FABP n GPDH s trget genes of PPARγ uring ipoyte ifferentition. Furthermore, PPARγ inhiitor GW9662 enhne the erese expression of FABP n PPARγ n ft umultion inue y. To onfirm the in vitro tivity of, niml stuy ws performe y ministering norml iet, HFD, n/or t the ose of or mg/kg for 6 weeks. Consistently, signifintly suppresse oy weight gin, serum triglyerie, totl holesterol, LDL holesterol, n AI vlue n inrese HDL holesterol in ose-epenent mnner. Immunohistohemistry revele tht tretment rogte the expression of PPARγ in the liver tissue setions of -trete rts. Tken together, our finings suggest tht hs the ntioesi n hypolipiemi potentil vi inhiition of PPARγ-relte signling. 1. Introution Oesity s meil onition of exess oy ft use y n umultion of ipose tissue mss [1] is tightly ssoite with vrious helth isorers suh s hyperlipiemi, ietes, n riovsulr iseses [2]. In oesity, ipoytes umulte exessive ft n ipoytokine proution is isrupte [3, 4]. Aipose tissue evelopment oserve in oese iniviuls is losely relte to hypertrophy n hyperplsi, the ltter involving prolifertion n ifferentition of preipoytes to ipoytes. The peroxisome prolifertortivte reeptor (PPAR) n CCAAT/enhner-ining proteins (C/EBP) fmilies of trnsription ftors regulte this ipoyte ifferentition [5 8]. These trnsriptionl ftors regulte the expression of genes involve in the inution ipoyte phenotypes [5]. Phrmeutil ntioesity rugs re generlly evelope to loss or ontrol oy weight. Although vrious presrie or nonpresrie meitions hve een use for tretment of oesity ptients, there is limittion to pply long-term use euse of their severe sie effets. Only orlistt (Xenil) hs een pprove y the FDA for long-term use. Reently, mny stuies reporte ntioesity tivity of nturl prouts nsuggestethepotentilsntioesitygentsortheir supplements. Pinus koriensis (Phylum Pinophyt, Clss Pinopsi, Orer Pinles, Fmily Pinee) is n evergreen tree foun in Kore, Chin, fr estern Russi, n entrl Jpn. The mjor

2 2 Eviene-Bse Complementry n Alterntive Meiine iotive hemils from P. koriensis inlue mphene, D- limonene, α-pinene, orneol, β-pinene, 4-rene, iylohept-3-ene, 3-rene, β-phellnrene, n fenyl [9]. We n others emonstrte the iologil effiies n genotoxiity of essentil oil from P. koriensis see, lef, nut, n one [9 13]. However, the iologil n iohemil effets of essentil oil fromp. koriensis hve not een reporte yet. Reently, mny stuies reporte tht nturl prouts suh s Rhizom Polygonti fltum (RPF) [14], Boussingulti grilis Miers vr. pseuoselloies Biley [15], Chinese lk te (Pu-erh te) extrt, n glli i [16] showe the potentil of ntioesity tivity in vitro or in vivo. In the present stuy, we investigte the ntioesi n hypolipiemi effet of essentil oil of P. koriensis SIEB () in 3T3-L1 ells y Oil-Re O stining, mesuring triglyerie, n nlyzing expression levels of ipogeni ftors n in high-ft iet-fe rts y mesuring oy weights n fts, n lipi metolites. 2. Mterils n Methos 2.1. Preprtion of Essentil Oil of P. koriensis Leves (). ws prepre using the hyroistilltion metho [17]. Drien pulverize P. koriensis leves were immerse in istille wter n sumitte to stem istilltion using n pprtus with onenser (Hnil Lteh, Seoul, Kore). The istilltion ontinue for 3-4 h t 9 C, n then the voltile ompouns ontine in the wter-solule frtion were llowe to settle for min. The essentil oil lyer ws seprte n purifie through mirofiltrtion Cell Culture. 3T3-L1 preipoytes were purhse from Koren Cell Line Bnk (KCLB). The ells were ulture in Duleo s Moifie Egle s Meium (DMEM) with 1% FBS in humiifie tmosphere of 95% ir n 5% CO 2 t 37 C Cytotoxiity Assy. The ytotoxiity of ginst 3T3-L1 ells ws mesure y 3-(4,5-imethyl-2-thizolyl)- 2,5-iphenyl-2H-tetrzolium romie (MTT) olorimetri ssy. The ells were seee onto 96-well miropltes t ensity of ells per well n trete with vrious onentrtions of (, 12.5, 25, or 5 μg/ml) for 24 h. MTT working solution ws then e to the miropltes t 37 C for 2 h, n then MTT extrtion uffer (% SDS n 5% imethylformmie) ws e t 37 Covernight. Optil ensity (OD) ws mesure using miroplte reer (Sunrise, TECAN, Mnneorf, Switzerln) t 57 nm. Cell viility ws lulte y employing the following eqution: ell viility (%) = [OD () OD (Blnk)]/[OD (Control) OD (Blnk)] Differentition Inution n Oil-Re O Stining. The preipoyte 3T3-L1 ells were plte onto 6-well pltes on y n inute to onfluent sttus. For ipoyte ifferentition, the onfluent ells were trete with 1 μm exmethsone, 1 μg/ml insulin, n.5 mm IBMX for 2 ys, n the meium ws reple y fresh norml meium only ontining 1 μg/ml insulin. On y 8, the ifferentite ipoyte ells were ulture in the presene or sene of ( μg/ml) for 2 ys. The ells were fixe with 2% prformlehye, wshe twie with PBS, n finlly stine with Oil-Re. After issolving, ellulr-lipi retine Oil-Re O in isopropnol, n ipoyte expression ws estimte y mesuring OD using miroplte reer (Sunrise, TECAN, Mnneorf, Switzerln) t 51 nm RT-PCR Anlysis. The 3T3-L1 ells were trete with or without (5 or mg/ml) for 24 h. The totl RNA ws extrte y using TRIzol regent (Invitrogen, Crls, CA, USA) oring to the mnufturer s instrutions. DNA ws synthesize from 1 μgoftotlrnansujete to PCR retion y using SuperSript One-Step reverse trnsription-pcr (RT-PCR) kit (Invitrogen, Crls, CA, USA). The PCR onitions were 3 yles of 94 Cfor3s, 57 C for 3s, n 72 C for 3 s. The primer sequenes were s follows: PPARγ (forwr 5 -GGTGAAACTCTG- GGAGATTC-3 n reverse 5 -CAACCATTGGGTCAG- CTCTT-3 ); C/EBPα (forwr 5 -AGGTGCTGGAGTTGA- CCAGT-3 n reverse 5 -CAGCCTAGAGATCCAGCG- AC-3 ); GPDH (forwr 5 -GAACTAAGGAGCAGCTCA- AAGGTTC-3 n reverse 5 -CAGTTGACTGACTGA- GCAAACATAG-3 ); β-tin (forwr 5 -ACCGTGAAA- AGATGACCCAG-3 n reverse 5 -TACGGATGACAA- CGTCACAC-3 ).PCRproutswererunon2%grosegel n then stine with ethiium romie. Stine ns were visulize uner UV light n photogrphe Western Blotting. Whole ell lystes (25 μg) were prepre using lysis uffer ( mm Tris, ph 7.4, 25 mm NCl, 2 mm EDTA, ph 8.,.1% Triton X-,.1 mg/ml protinin,.3 mg/ml leupeptin,.4 mm phenylmethylsulfonyl fluorie(pmsf),n4mmnvo4).thelysteswerespunt 13 gfor15mintoremoveinsolulemterilnresolve on 1% SDS-PAGE gel. After eletrophoresis, the proteins were eletrotrnsferre to nitroellulose memrne, lokewith5%nonftmilk,nproewithntioies ginst PPARγ (Novus, Littleton, CO, USA), C/EBPα (Cell Signling Teh., Dnvers, MA, USA), FABP (Cell Signling Teh., Dnvers, MA, USA), n β-tin (Sigm, St. Louis, MO, USA) overnight. The lots were wshe, expose to horserish peroxise- (HRP-) onjugte seonry ntioies for 2 h, n finlly exmine y enhne hemiluminesene (ECL) (GE Helth Cre Bio-Sienes, Pistwy, NJ, USA) Animls, Diet Mnipultion, n Tretment. Mle Sprgue-Dwley rts, t 4 weeks of ge, were purhse from Hyo-Chng Siene (Degu, Kore) n mintine uner onventionl onitions. Fifty rts were ivie into four groups; norml group (low ft iet), ontrol group (high-ft iet), two -trete groups- n torvsttin (positive ontrol) trete group onsuming high-ft iets (1 rts per group). Rts were fe the norml (low ft) iet (group 1) or the high-ft iet (groups 2 5) for 6 weeks. High-ft iet omposition ws esrie in Tle 1. For tretment,

3 Eviene-Bse Complementry n Alterntive Meiine 3 Tle 1: Composition of sl n high-ft iet. Ingreient Bsl iet (%) High-ft iet (%) Csein.. DL-Methionine.3.3 Corn strh Surose Fier Corn oil 5. AIN-minerl mixture AIN-vitmin mixture Choline itrtrte.2.2 Beef tllow.5 1 Cellulose: Sigm Co. Lt., USA. 2 Minerl mixture, se on Rogers n Hper [18], ontine the following (g/kg iet): lium phosphte isi., soium hlorie 74., potssium itrte monohyrte 2., potssium sulfte 52., mgnesium oxie 24., mgnesium ronte 3.5, ferri itrte 6., zin ronte 1.6, upuri ronte.3, potssium iote.1, hromium potssium sulfte.55,surose,nfinelypoweremke1,. 3 Vitmin mixture (g/kg iet): thimine HCl.6, iotin.2, rioflvin.6, ynoolmin.1, pyrioxine HCl.7, retinyl ette.8, niotini i 3., DL-toopherol 3.8, C-pntothente 1.6, 7-ehyroholesterol.25, foli i.2, methionine.5, surose, n finely powere mke 1,. issolve in 4% tween 8/norml sline ws orlly ministere one ily to the rts t the oses of (group 3)nmg/kg(group4)for6weeksfromthe1styof high-ft iet feeing, wheres PBS ws orlly ministere tothertsinontrolgroup(group2).aspositiveontrol, torvsttin ws ministere t ose of 1 mg/kg (group 5) Preprtion of Rt Serum. Whole loo ws ollete from rts y ri punture metho, n serum ws isolte y entrifugtion t 3 rpm for 1 min Mesurement of Totl Cholesterol Level. Totl holesterol level ws mesure y using totl holesterol ssy kit (AM 2-K, Asn Phrm Co., Seoul, Kore) se on Rihmon s metho [19] Mesurement of Triglyerie Level. Triglyerie level ws mesure y triglyerie ssy kit (AM 157S-K, Asn Phrm Co., Seoul, Kore) se on MGown s metho [] Mesurement of Serum HDL n LDL Levels. The levels of high-ensity lipoprotein (HDL) n low-ensity lipoprotein (LDL) in serum were mesure using Rohe os C-111 nlyzer (Rohe-Dignostis, Ininpolis, IN, USA): AI ws lulte y employing the following eqution. AI = (totl holesterol HDL holesterol)/hdl holesterol Mesurement of Boy Weight, Retroperitonel Ft, n Epiiyml Ft. The oy weight of rts in norml (group 1), ontrol (group 2), - ( n mg/kg) trete groups (groups 3 n 4, resp.), n torvsttin (group 5) ws monitore one every week for 6 weeks. The retroperitonel n epiiyml fts were lso remove n weighe from rts trete y ( n mg/kg) or torvsttin (1 mg/kg) on the lst y of niml stuy Immunohistohemil Stining. For histopthologil exmintion, prffin setions (4 μm) from tumors issete were stine with hemtoxylin n eosin. Immunohistohemil stining PPARγ (Novus, Littleton, CO, USA) ws performe using the iniret viin/iotin-enhne horserish peroxise metho. Antigen retrievl ws performe fter ewxing n ehyrtion of the tissue setions y mirowve for 1 min in 1 mm itrte uffer. Setions were oole to room temperture, trete with 3% hyrogen peroxie in methnol for 1 min, n loke with 6% horse serum for 3 min t room temperture in humiity hmer. Setions were then inute with the primry ntioy ginst PPARγ (ilute 1 :, Novus, Littleton, CO, USA) t 4 C overnight in humiity hmer. Setions were wshe in PBS n inute with seonry ntioy (iotinylte got nti-rit (1 : 15, Vetor lortories, Burlingme, CA, USA) for 3 min in humiity hmer. After further wshes, the ntioies were etete with the Vetor ABC omplex/horserish peroxise (HRP) kit (Vetor Lortories, Burlingme, CA, USA) n olorevelope with 3,3 -iminoenziine tetrhyrohlorie. For semiquntittion, ten photomirogrphs ( x) were tken with CCD mer, voiing gross neroti res Sttistil Anlyses. All t were expresse s mens ± SD. or SE. In vitro experiment t were nlyze y Stuent s t-test. In vivo experiment t were lulte y the nlysis of vrine (ANOVA) followe y Dunn s multiple rnge test. P vlue of less thn.5 ws onsiere sttistilly signifint. Mens in the sme olumn with ifferent supersript letters (,,,, e, n f) re signifintly ifferent (P <.5) etween groups. 3. Results 3.1. Effet of on Ft Aumultion in 3T3-L1 - Like Cells. Cytotoxiity of ws etermine y MTT ssyin3t3-l1preipoytes.cellsweretretewithvrious onentrtions of (, 12.5, 25, or 5 μg/ml) for 24 h. As shown in Figure 1(), h no signifint effet on the viility of 3T3-L1 ells. To investigte whether n ffet the ellulr ifferentition into ipoytes, Oil-Re O stining ws performe in 3T3-L1 ells trete with for 8 ys. The ifferentite 3T3-L1 ipoytes signifintly inrese ft umultion n intrellulr triglyerie (Figures 1(), 1(), n 1()), when ompre to preipoytes. The ft eposits were erese y 57 n 78% t the tretment with vrious onentrtions of (25 or 5 μg/ml), respetively, ompre to untrete ontrol, ipoytes. Results inite tht 5 μg/ml of ws the most effetive ility to inhiit ipoyte ifferentition (Figures 1() n 1()). tretment reue the level of triglyerie in ell in ose-epenent mnner (37 n 6%

4 4 Eviene-Bse Complementry n Alterntive Meiine 1 Cell viility (%) 8 6 Preipoyte 25 5 (μg/ml) Conentrtion (μg/ml) () ### () Ft umultion (%) (μg/ml) Triglyerie (%) ### P 25 5 (μg/ml) P () () Figure 1: Effet of on the ifferentition of 3T3-L1 ipoytes. () Cytotoxiity of ginst 3T3-L1 ells ws etermine y MTT ssy. Cells were trete with vrious onentrtions of (, 12.5, 25, or 5 μg/ml) for 24 h. (,, n ). Confluent ells were trete with 1 μm exmethsone, 1 μg/ml insulin, n.5 mm IBMX for 2 ys, n then the meium ws reple y fresh norml meium only ontining 1 μg/ml insulin. On y 8, the ifferentite ipoyte ells were expose to for 2 ys. () The ifferentite ells were stine with Oil-Re O ye n visulize uner inverte mirosopy t mgnifitions. () After issolving n ellulr lipi retine Oil-Re O in isopropnol, ipoyte expression ws estimte y mesuring OD using miroplte reer (Sunrise, TECAN, Mnneorf, Switzerln) t 51 nm. () Level of intrellulr triglyerie. t onentrtions of 25 n 5 μg/ml, resp.), ompre to preipoytes (8.4%) (Figure 1()). 3.2.EffetofontheExpressionofC/EBP,PPARγ,GPDH, n FABP uring the Differentition of s. PPARγ, C/EBPα, GPDH, n FABP re key ftors involve in ipogenesis. In prtiulr, PPARγ ontrols ipogeni ftors s key trnsription ftor uring ipoyte ifferentition. Both mrna n protein levels of PPARγ, C/EBPα, n GPDH were ownregulte y in ose-epenent mnner (Figures 2() n 2()). To eluite the unerlying mehnism of, PPARγ ntgonist GW9662 ws use in 3T3-L1 ipoytes. PPARγ inhiitor GW9662 enhne the erese expression of FABP n PPARγ y (Figure 2()). As shown in Figures 2() n 2(e), the lipi umultion in 3T3-L1 ipoytes trete with n GW9662 ws signifintly reue ompre to untrete ontrol. Consistently, immunohistohemistry revele tht tretment ttenute the expression of PPARγ in the liver tissue setions of -trete group ( n mg/kg) ompre to untrete ontrol group (Figure 3()). Tken together, these results inite tht inhiits ipoyte ifferentition vi suppressing PPARγ tivtion.

5 Eviene-Bse Complementry n Alterntive Meiine 5 P 25 5, μg/ml PPARγ P 25 5, μg/ml C/EBPα PPARγ GPDH C/EBPα β-atin FABP P: preipoyte β-atin () () (5 μg/ml) GW9662 (1 nm) P PPARγ FABP β-atin Preipoyte () (5 μg/ml) GW9662 (1 nm) () 1 ### Ft umultion (%) 8 6 (5 μg/ml) GW9662 (1 nm) P (e) Figure 2: Effets of on ipoyte ifferentition of 3T3-L1 preipoytes. 3T3-L1 preipoytes were inute in meium ontining insulin (1.μg/mL) with or without the inite onentrtions of or GW9662. () Totl RNA ws extrte from 3T3-L1 (preipoytes or ipoytes) ells trete with n use for RT-PCR nlysis of PPARγ, C/EBPα, GPDH, n β-tin. () Totl proteins prepre from -trete 3T3-L1 (preipoytes or ipoytes) ells were sujete to western lot nlysis of PPARγ,C/EBPα FABP, n β-tin. () Totl proteins prepre from - or GW9662-trete 3T3-L1 (preipoytes or ipoytes) ells were sujete towesternlotnlysisofpparγ, FABP,nβ-tin. () Cells were fixe n stine with Oil-Re O. The Oil-Re O-stine ipoytes were photogrphe t mgnifition uner mirosope. (e) Lipis were extrte using isopropnol, n the Oil-Re O ws then nlyze t wvelength of 5 nm. Vlues re presente s mens ± SE. P <.5 versus the ontrol.

6 6 Eviene-Bse Complementry n Alterntive Meiine Boy weight (g) 3 e e f e Norml Control mg/kg mg/kg 1 mg/kg Atorvsttin () 25 (wk) Weight (mg/g) Norml Control 1 (mg/kg) Atorvsttin Retroperitonel Epiiyml () Atorvsttin Norml Control 1 (mg/kg) PPARγ () Figure 3: Effet of on oy n ominl ft weight of high-ft iet-fe rts. Rts fe high iet were orlly trete with or without ily for 6 onseutive weeks. () Boy weights of rts. () ominl ft weights n The retroperitonel n epiiyml fts from rts trete with or without ( n mg/kg) were remove n weighe. () Representtive piture of immunohistohemil stining for PPARγ in liver tissue setions. Dt were expresse s mens ± SD. Vlues with the ifferent supersript letters inite sttistil signifine (P <.5) etween groups y Dunn s multiple rnge test Effet of on Boy Weight of High-Ft Diet-Fe Rts. Theoyweightsofnorml(group1),ontrol(group2), -trete groups (groups 3 n 4), n torvsttintrete group (group 5) were monitore one every week for 6 weeks. As shown in Figure 3(), theoyweightof high-ft fe ontrol group ws signifintly inrese 2 weeks fter feeing ompre to norml low ft group. In ontrst, the oy weight gin ws ose epenently rogte in -trete groups, from the te of 3-week tretment of. Also, six weeks fter tretment, the oy weight ws signifintly gine to ± 2.8 g in high-ft fe ontrol group ompre to norml group (281.6 ± 1.7 g). However, signifintly suppresse the oy weights to 39.5 ± 1.8 gn33.7 ± 2. g, respetively, t oses of mg/kg n

7 Eviene-Bse Complementry n Alterntive Meiine 7 Serum triglyerie (mg/l) 15 5 Totl holesterol (mg/l) 8 6 f e Norml Control 1 (mg/kg) Norml Control 1 (mg/kg) Atorvsttin Atorvsttin () () Cholesterol (mg/l) Atheroslerosis inex (mg/l) Norml Control 1 (mg/kg). Norml Control 1 (mg/kg) Atorvsttin Atorvsttin HDL LDL () () Figure 4: Effets of on serum lipi n holesterol levels in high-ft iet-fe rts. () Triglyerie level ws mesure y triglyerie ssy kit (AM 157S-K, Asn Phrm Co., Seoul, Kore). () Totl holesterol level ws mesure y using totl holesterol ssy kit (AM 2-K, Asn Phrm Co., Seoul, Kore). () The levels of high-ensity lipoprotein (HDL) n low-ensity lipoprotein (LDL) in serum were mesure using os 111 nlyzer (Rohe-Dignostis, Ininpolis, IN, USA). () AI ws lulte y employing the following eqution: AI = (totl holesterol HDL holesterol)/hdl holesterol. Vlues with the ifferent supersript letters inite sttistil signifine (P <.5) etween groups y Dunn s multiple rnge test. mg/kg, lmost oming up with torvsttin s positive ontrol (296.5 ± 1.9 g). ft p weight y 1.9 ± 1.85 n 8.52 ± 1.96 mg/g t oses of n mg/kg, respetively (Figure 3()) Effets of on Aominl Ft Weight of High- Ft Diet-Fe Rts. The retroperitonel n epiiyml ft weight ws mesure to test whether the oy weight normliztion y in high-ft iet-fe rts is ssoite with reution of ft ontent in oy. In the present stuy, s expete, high-ft iet signifintly inrese the retroperitonel ft weight to 18.3 ± 2.91 mg/g from 6.21 ± 1.26 mg/kg oy weight. In ontrst, erese the retroperitonel ft weight to 15.7±1.86 n 12.7±1.43 mg/g t oses of n mg/kg, respetively, ompre to ontrol group (Figure 3()). Likewise, epiiyml ft weight ws lso inrese in the high-ft iet-fe rts ompre to norml group. Orl ministrtion of erese epiiyml 3.5. Effets of on Serum Lipi n Cholesterol Levels in High-Ft Diet-Fe Rts. The onsumption of the high-ft iet signifintly inrese triglyerie in ontrol group ompre to the norml low ft group (Figure 4()). tretment erese level of serum triglyerie from ± 18.2 to 19.3 ± 11.5 n 94.2 ± 8.43 mg/l t n mg/kg, respetively (Figure 4()). Aitionlly, the onsumption of the high-ft iet signifintly inrese serum totl holesterol ompre to the norml low ft group, while signifintly reue the level of totl holesterol in oseepenent mnner (Figure 4()). The intke of the highft iet signifintly erese level of HDL ut inrese level of LDL ompre to norml group (Figure 4() ontrol).

8 8 Eviene-Bse Complementry n Alterntive Meiine 25 Hepti triglyerie (mg/g) 3 1 e Hepti holesterol (mg/g) Norml Control 1 (mg/kg) Norml Control 1 (mg/kg) Atorvsttin Atorvsttin () () Figure 5: Effets of on hepti triglyerie n holesterol in high-ft iet-fe rts. The levels of triglyerie () n totl holesterol () in liver were mesure y Biohemistry Anlyzer. Vlues with the ifferent supersript letters inite sttistil signifine (P <.5) etween groups y Dunn s multiple rnge test. However, i not hve signifint effet on LDL while elevting HDL level in ose-epenent mnner ompre to the high-ft iet ontrol group (Figure 4()). Consistently, tretment signifintly erese the theroslerosis inex (AI) vlue in ose-epenent mnner ompre to the high-ft iet ontrol (Figure 4()) Effets of on Hepti Triglyerie n Cholesterol in High-Ft Diet-Fe Rts. The onsumption of the high-ft iet signifintly inrese hepti triglyerie (Figure 5()) n totl holesterol (Figure 5()), when ompre to the norml low ft group. Orl tretment with reue the level of triglyerie from the liver in ose-epenent mnner (24.4 ± 2.47 n 21.2 ± 1.88 mg/g t oses of n mg/kg, resp.), ompre to ontrol group (29.8 ± 3.25 mg/g tissue) (Figure 5()). In ition, ministrtion signifintly lowere totl holesterol level in liver from 16.9 ± 2.18 mg/g in the high-ft iet ontrol group to 12.6 ± 1.42 n 1.4 ± 1.59 mg/g t n mg/kg, respetively (Figure5()). 4. Disussion The Koren pine (P. koriensis) is n importnt fforesttion trees in Kore n lso istriute in Chin, Russi, Jpn, n Europe [21]. P. koriensis extrt ttenute the inrese in loo pressure in spontneously hypertensive rts [13], n its onstituent pinoleni i h holesterollowering effet vi regultion of LDL reeptor tivity in HepG2 heptom ells [22]. In ition, P. koriensis nut oil effetively regulte stiety hormones n prospetive foo intke in postmenopusl overweight women [11]. Our group lso reporte tht essentil oil from P. koriensis leves h ntihyperlipiemi effets vi upregultion of LDL reeptor n inhiition of yl-oenzyme A: holesterol yltrnsferse [9]. In the urrent stuy, we emonstrte tht hs ntioesity effets in 3T3-L1 ipoytes n high-ft ietfe rt moels. Aipogenesis is the proess of ifferentition y whih unifferentite preipoytes re onverte into ifferentite ipoytes [23] n susequently meite the synthesis n umultion of lipi [24]. 3T3-L1 ells re the est hrterize moel to stuy ipogenesis in vitro [25]. Oil-Re O stining revele tht signifintly suppresse ft umultion n intrellulr triglyerie n erese expression of PPARγ, CEBPα, FABP, n GPDH in the ifferentite 3T3-L1 ipoytes with no ytotoxiity, initing the inhiitory effet of on ipoyte ifferentition. Similrly, mny nturl ompouns, inluing EGCG, ererine, n urumin, suppresse PPARγ signling, sine PPARγ ntgonists hve een reporte to effetively inhiit ipogenesis n improve insulin sensitivity in vitro n in vivo [26]. Furthermore, here the ntioesi effet of P. koriensis ws onfirme in high-ft iet- (HFD-) trete SD rts t theosesoformg/kgvilokgeofoyweight gin. Sine loss of oy weight is minly ssoite with the erese of ft p mss s result of reution of ipoyte size or triglyerie umultion [27], our t tht signifintly reue the retroperitonel n epiiyml ft weight n lso serum triglyerie ompre to HFD fe rts suggestthtnlokoesityviinhiitionoflipi metolism inluing triglyerie. Cholesterol is lso n importnt omponent of ft omplex. In prtiulr, low level of serum HDL holesterol is tightly linke to the ourrene of oesity [28]. In our stuy, ministrtion of signifintly rogte the ontents of totl holesterol in serum. In ition, signifintly inrese level of HDL holesterol in ose-epenent mnner ompre to HFD ontrol group ut not LDL holesterol. Lipi prmeters in the loo n e ffete

9 Eviene-Bse Complementry n Alterntive Meiine 9 y the hepti metolisms. As expete, we oserve tht the hepti ontents of triglyerie n holesterol were signifintly eslte in HFD fe rts ompre to norml ontrol. In ontrst, tretment signifintly lowere the ontents of triglyerie n totl holesterol in the liver, implying tht regultes lipi metolism inluing triglyerie n holesterol ginst oesity. In summry, inhiite ft umultion, intrellulr triglyerie, n expression of PPARγ, CEBPα, FABP, n GPDH in the ifferentite 3T3-L1 ipoytes. Also, ttenute serum n hepti ontents of triglyerie ntotlholesterolinhfdfertmoels.furthermore, erese the expression of PPARγ in the liver tissue setions of -trete rts. Overll, our finings suggest the potentil of s n ntioesity gent. Conflit of Interests No potentil onflit of interests ws islose. Aknowlegments This work ws supporte y grnt from the Next- Genertion BioGreen 21 Progrm (no. PJ955) n MRC (no ), Repuli of Kore. Referenes [1] D. W. Hslm n W. P. T. 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Cristnho n M. A. Lzr, Forming funtionl ft: growing unerstning of ipoyte ifferentition, Nture Reviews Moleulr Cell Biology,vol.12,no.11,pp ,11. [25] H. Green n O. Kehine, An estlishe preipose ell line n its ifferentition in ulture. II. Ftors ffeting the ipose onversion, Cell,vol.5,no. 1,pp.19 27,1975. [26] J. Rieusset, F. Touri, L. Mihlik et l., A new seletive peroxisome prolifertor-tivte reeptor γ ntgonist with ntioesity n ntiieti tivity, Moleulr Enorinology, vol.16,no.11,pp ,2. [27] M. Rosenum, R. L. Leiel, n J. Hirsh, Oesity, The New Engln Journl of Meiine, vol. 337, no. 6, pp , 1997.

10 1 Eviene-Bse Complementry n Alterntive Meiine [28] S. C. Woos, R. J. Seeley, D. Porte Jr., n M. W. Shwrtz, Signls tht regulte foo intke n energy homeostsis, Siene,vol.28,no.5368,pp ,1998.

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