Original Article INTRODUCTION. Diabetes Metab J 2011;35: pissn eissn

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1 Originl rticle Dibetes Metb J 2011;35: pissn eissn D I E T E S & M E T O L I S M J O U R N L Dietry Olete Hs eneficil Effects on Every Step of Non-lcoholic Ftty Liver Disese Progression in Methionine- nd holine-deficient Diet-Fed niml Model Ji Young Lee 1,*, Je Hoon Moon 2,*, Jong Suk Prk 2, yung-wn Lee 2, Eun Seok Kng 2, hul Woo hn 2, Hyun hul Lee 2, ong Soo h 1,2 1 rin Kore 21 Project for Medicl Science, 2 Deprtment of Internl Medicine, Yonsei University ollege of Medicine, Seoul, Kore ckground: Non-lcoholic ftty liver disese (NFLD) is incresingly recognized s mjor cuse of liver-relted morbidity nd mortlity. The underlying mechnisms of disese progression remin poorly understood, nd primry therpy of NFLD is not yet estblished. We investigted the effects of dietry olete on the development nd progression of NFLD in methioninend choline-deficient () diet-fed niml model. Methods: totl of 30 57L/6J mice were rndomly divided into three groups (n=10 in ech group) nd fed vrious experimentl diets for four weeks: chow, diet, or O ( diet with olete, mg/g/dy). Liver smples were exmined for stetoheptitis nd fibrosis prmeters nd ssocited genes. Results: dditionl dietry olete drmticlly reduced diet-induced heptic stetosis. Heptic crbohydrte responsive element-binding protein ws overexpressed in diet-fed mice, nd dietry olete prevented this overexpression (P<01). Dietry olete prtilly prevented diet-induced serum level increses in sprtte minotrnsferse nd lnine minotrnsferse (P<01, respectively). The mrn expressions of heptic monocyte chemottrctnt protein 1, tumor necrosis fctor-α nd mtrix metlloproteinse-9 were incresed in diet-fed mice, nd this overexpression of inflmmtory molecules ws prevented by dietry olete (P<01). Heptic pericellulr fibrosis ws observed in diet-fed mice, nd dietry olete prevented this fibrosis. ltogether, dietry olete prevented diet-induced heptic stetosis, inflmmtion nd fibrosis. onclusion: Dietry olete hs beneficil effects in every step of NFLD development nd progression nd could be nutritionl option for NFLD prevention nd tretment. Keywords: hrep; Ftty cids, monounsturted; Non-lcoholic stetoheptitis INTRODUTION Non-lcoholic ftty liver disese (NFLD) represents spectrum of disorders chrcterized by heptic ft ccumultion rnging from simple stetosis to severe non-lcoholic stetoheptitis (NSH) with centrilobulr necroinflmmtion nd hs been incresingly recognized s mjor cuse of liver-relted morbidity nd mortlity [1]. lthough the underlying mechnisms of disese progression remin poorly understood, insulin resistnce, oxidtive stress, nd n inflmmtory cscde re believed to ply importnt roles in the development nd progression of this disese [1-3]. The tretment of NFLD orresponding uthor: ong Soo h Deprtment of Internl Medicine, Yonsei University ollege of Medicine, 250 Seongsnno, Seodemun-gu, Seoul , Kore E-mil: bsch@yuhs.c Received: Feb. 11, 2011; ccepted: pr. 11, 2011 * Ji Young Lee nd Je Hoon Moon contributed eqully to this work s first uthors. This is n Open ccess rticle distributed under the terms of the retive ommons ttribution Non-ommercil License ( which permits unrestricted non-commercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. opyright 2011 Koren Dibetes ssocition

2 Lee JY, et l. consists of modifiction of underlying risk fctors nd detection of ptients with liver cirrhosis. lthough some gents provide modest improvements in liver function tests nd histologic prmeters, primry therpy for NFLD hs not yet been estblished [2]. The methionine- nd choline-deficient () diet is wellestblished nd widely-used nutritionl model of humn NFLD. This high sucrose-contining diet lcks methionine nd choline in its composition, contins higher lipid composition thn norml chow diet, nd cuses heptic stetosis nd inflmmtion tht mimics NFLD in humns [4,5]. lthough the mechnisms responsible for the development of heptic stetosis due to the diet re not fully understood, recent studies suggest tht the diet increses heptic ftty cid uptke nd decreses very low-density lipoprotein (VLDL) secretion [4]. Oleic cid (18:1n-9), the richest source of ftty cid in olive oil, is monounsturted ftty cid (MUF). Recently, dietry olive oil ws reported to decrese heptic lipid content in rts fed n diet [6]. onsidering tht only 15% of ftty cid stored in the liver is of dietry origin in NFLD ptients [7], reduced plsm free ftty cid (FF) nd de novo lipogenesis were suggested s possible mechnisms to prevent the ccumultion of ftty cids in the liver under n olive oil-enriched diet [6]. In fct, MUF from sfflower oil ws reported to decrese lipogenesis in the rt liver [8]. dditionlly, recent studies show tht diet rich in olive oil decreses heptic injury nd poptosis in mice fed n diet [9]. However, to our knowledge, there re few studies tht hve investigted the effect of dietry MUF on ech stge of NFLD development nd progression. In this study, we investigted the effect of dietry olete on the development nd progression of NFLD in -diet mouse model. This study presents some clues to understnding the mechnisms through which the diet induces stetoheptitis nd describes how dietry MUF prevents stetoheptitis in this niml model. s result, this study suggests dietry olete s nutritionl option for NFLD prevention nd tretment. METHODS nimls, diet, nd tretment Eight-week-old 57L/6J mice (Smtko Inc., Osn, Kore) weighing 20 to 21 g were mintined t mbient temperture (22±1 ) on 12-hour light-drk cycles with free ccess to wter nd diet. Initilly, ll mice were fed chow diet during one-week qurntine nd cclimtion period. Then, t nine weeks of ge, mice displying no bnorml findings t the end of the qurntine nd cclimtion period were rndomly divided into three groups. The mice in the chow group (n=10) were fed norml chow diet; the mice in the group (n= 10) were fed n diet (Dyets Inc., ethlehem, P, US); the mice in the O group (n=10) were fed n diet with olete supplementtion ( mg/g/dy; Sigm ldrich, St. Louis, MO, US) throughout the four weeks of the experimentl period. Olete ws orlly dministrted with 10 cc syringe, nd body weights were mesured every other dy. Mice were scrificed t 13 weeks of ge. The nimls were euthnized t the end of drk cycle fter overnight fsting for tissue smpling. lood ws collected by crdic puncture, nd the livers were isolted, immeditely freeze-clmped in liquid nitrogen, nd stored t -80 until nlysis. ll experimentl procedures were performed under sterile conditions nd were pproved by the Institutionl niml re nd Use ommittee t Yonsei University ollege of Medicine. iochemicl exmintion Plsm levels of sprtte minotrnsferse (ST) nd lnine minotrnsferse (LT) were ssyed by routine utomted lbortory methods. Histopthologicl nlysis Livers were weighed to llow the clcultion of reltive liver weight (percentge of liver weight to body weight). Livers were fixed in 10% buffered formlin, embedded in prffin, nd sectioned t 3 μm. Stndrd hemtoxylin nd osin (H&E) nd trichrome stining ws performed [10]. Fresh tissue ws frozen immeditely fter ech niml ws dissected, nd the tissue ws plced in pre-lbeled bse molds filled with embedding medium used for frozen tissue to ensure the optiml cutting temperture (OT). Routine sections were cut t 7 μm, frozen nd stined with Oil-red O. For the evlution of heptic stetosis nd fibrosis, the verge res (%) of the ft droplets nd fibrosis within heptocytes were mesured with the id of n imge nlyzer in three rndomly selected fields (mgnifiction, 100) of ech section stined with Oil-red O nd trichrome [11]. 490 Dibetes Metb J 2011;35:

3 Dietry olete nd non-lcoholic ftty liver disese Immunohistochemistry Immunohistochemistry ws performed with n ntibody to crbohydrte responsive element-binding protein (hrep) (Thermo Fisher Scientific Inc., Rockford, IL, US) on prffin-embedded sections. Immunohistochemicl stining ws performed with n stining system (Snt ruz iotechnology, Snt ruz,, US) [12]. RN nd cdn preprtion Totl RN ws isolted from mouse liver tissue using Trizol regent (Invitrogen, rlsbd,, US) nd quntified using Nno Drop instrument (ND-1000; DM Science, Seoul, Kore). Following RN extrction, 4 μl RN ws treted with 1 U DNse I to remove ll contminting genomic DN. DNse-treted RN ws subsequently used for cdn synthesis using MMLV reverse trnscriptse (Promeg, Mdison, WI, US): 1 μl oligo dt primer ws dded to 4 μl RN in 5 MMLV rection buffer, 2.5 mm ech dntp, 1 U RNsin ribonuclese inhibitor nd MMLV reverse trnscriptse (200 units). cdn ws stored t -20. Quntittive RT-PR Rel-time quntittive reverse trnscription-polymerse chin rection (RT-PR) nlysis ws performed with n I 7500 instrument nd softwre (pplied iosystems, Foster ity,, US). PR rections were performed in triplicte in finl volume of 20 μl ccording to the mnufcturer s protocol. Mouse hrep mrn expression ws nlyzed using Tqmn probes (Mm _m1; pplied iosystems). For ech ssy, stndrd curve ws obtined by nlyzing dilution series of pooled cdn smples for the relevnt gene. Dt were nlyzed with Sequence Detector 1.7 softwre (pplied iosystems). Mouse glycerldehyde-3-phosphte dehydrogense (GPDH) ws used s the internl stndrd to control for vribility, nd results were expressed s rtio of the gene expression reltive to tht of GPDH. Tble 1. RT-PR primers Gene Forwrd primer (5 to 3 ) Reverse primer (3 to 5 ) PO100 cgtctcctcgccc cggtgcggtctgcttgg MP1 tggtcggcctgg ggctccgtccggtc TNF-α gccggctgtccg tttgctcgcgtgggctc MMP-9 cctgcgtgcccttgct ttgtcttcttgctcggtgc GPDH gggccgggtctct gtgtggctggctgtggt RT-PR, reverse trnscription-polymerse chin rection; PO100, polipoprotein 100; MP1, monocyte chemottrctnt protein 1; TNF-α, tumor necrosis fctor-α; MMP-9, mtrix metlloproteinse-9; GPDH, glycerldehyde-3-phosphte dehydrogense. Semi-quntittive RT-PR The heptic expressions of the mrns for polipoprotein 100 (PO100), monocyte chemottrctnt protein 1 (MP1), tumor necrosis fctor-α (TNF-α) nd mtrix metlloproteinse-9 (MMP-9) were ssessed by semi-quntittive RT-PR nlysis using GPDH s n internl control gene. RT-PR products were electrophoresed on 1% (w/v) grose gel, the gel ws stined with ethidium bromide, nd bnds were visulized by UV light. The primers used for RT-PR re listed in Tble 1. Smples from ll experimentl nimls were used for this nlysis. Sttisticl nlysis ll sttisticl nlyses were performed with SPSS softwre version 15.0 (SPSS Inc., hicgo, IL, US). Vlues were expressed s men±stndrd devition. Sttisticl nlyses were performed using the unpired Student s t-test or one-wy NO- V. Dt with P vlue less thn 5 were considered sttisticlly significnt. RESULTS n diet decresed body weight nd reltive liver weight fter the four week-experimentl period, the body weights of the diet-fed mice significntly decresed compred to those of the chow diet-fed mice (chow vs. vs. O, 25.0±0.7 g vs. 15.3±0.8 g vs. 15.6±1.2 g, P<01) (Tble 2). Reltive liver weights in the diet-fed groups were significntly incresed compred to those in the chow diet-fed group. etween the diet-fed groups, the increse in reltive liver weight in mice fed n diet only ws more prominent thn tht in mice fed n diet with olete (chow vs. vs. O, 4.8±% vs. 6.5±0.4% vs. 5.8±0.6%, P< 01) (Tble 2). Dietry olete prevented stetosis in the liver of mice fed n diet To investigte nd compre the extent of heptic stetosis mong the groups, Oil-Red-O stining of mice liver smples ws performed. In the diet-fed mice, numerous ft Dibetes Metb J 2011;35:

4 Lee JY, et l. Tble 2. linicl chrcteristics of mice fter four weeks of experimentl diets hrcteristic Dietry group O Initil body weight, g 21.8± 21.8± ±1.4 ody weight, g 25.0± ± ±1.2 b Liver weight, g 1.2± ±0.1 ±0.1 b,c Liver weight-body weight, % 4.8± 6.5± ±0.6 b,c Serum ST, IU/L 85.3± ± ±6.4 b,c Serum LT, IU/L 38.7± ± ±7.2 b,c Vlues represent men±stndrd devition for n=10 in ech group., norml chow diet;, methionine- nd choline-deficient diet; O, diet with olete ( mg/g/dy); ST, sprtte minotrnsferse; LT, lnine minotrnsferse. P<5 for D vs. chow, b P<5 for OD vs. chow, c P<5 for O vs.. hrep mrn b PO100 mrn PO100 GPDH O D O E re of ftty droplets (% of totl re) Fig. 1. The effects of the methionine- nd choline-deficient diet () diet nd dietry olete on heptic ft ccumultion. Oil-red-O stining of frozen liver sections. Scle brs, 200 μm. () diet-fed mice. () diet-fed mice. () diet with olete ( mg/g/dy for 4 weeks)-fed mice. (D) The res of ft droplets, mesured by imge nlyzer in three rndomly selected fields (mgnifiction, 100) of ech liver section., norml chow diet; O, diet with olete. P<5 vs. chow, b P<vs.., b O droplets were observed within heptocytes, nd the totl ft droplet re ws mrkedly incresed in the livers of diet-fed mice (chow vs., 5±1% vs ±0.35%, P<01) (Fig. 1). This increse in heptic lipid droplets in mice fed n diet ws ttenuted by dietry olete. In the livers of olete-treted diet-fed mice, lipid droplets were D Fig. 2. The effects of the methionine- nd choline-deficient diet () diet nd dietry olete on heptic crbohydrte responsive element-binding protein (hrep) nd polipoprotein (po ) expression. Immunohistochemicl detection of hrep in liver prffin-embedded sections. Scle brs, 50 μm. () diet-fed mice. () diet-fed mice. () diet with olete ( mg/g/dy for 4 weeks)-fed mice. (D) Reltime reverse trnscription-polymerse chin rection (RT-PR) quntifiction of hrep mrn in mouse liver smples from ech group. (E) Rel-time RT-PR quntifiction of PO100 mrn in mouse liver smples from ech group. U, rbitrry unit;, norml chow diet; O, diet with olete. P<5 vs. chow, b P<vs.. significntly decresed compred to those of the dietfed mice ( vs. O, 14.31±0.35% vs. 3.65±4%, P<01) (Fig. 1). Heptic hrep overexpression nd PO100 reduction by n diet re inhibited by dietry olete To investigte the effect of dietry MUF on heptic de novo lipogenesis, hrep expression in ech group ws evluted. Immunohistochemicl stining reveled tht heptic hrep ws overexpressed in mice fed n diet compred to tht of chow diet-fed mice (Fig. 2 nd ). Interestingly, dietry olete completely ttenuted diet-induced overexpres- 492 Dibetes Metb J 2011;35:

5 Dietry olete nd non-lcoholic ftty liver disese MPI GPDH TNF-α GPDH MPI mrn, b TN-α mrn b O O Fig. 3. Liver histology in mice fed ech experimentl diet. H&E stining of liver prffin-embedded sections. Scle brs, 200 μm. () diet-fed mice. () diet-fed mice. () diet with olete ( mg/g/dy for 4 weeks)-fed mice. rrows indicte the focl ggregtion of inflmmtory cells., norml chow diet;, methionine- nd choline-deficient diet; O, diet with olete. sion of heptic hrep (Fig. 2). The results of quntittive RT-PR demonstrte tht the mrn expression of heptic hrep ws mrkedly incresed by the diet, nd olete intke prevented this increse (chow vs. vs. O, 0±7 U vs. 1.90±0.14 U vs. 1.10±7 U, P<01) (Fig. 2D). Heptic PO100 expression ws evluted to investigte the effect of dietry MUF on heptic VLDL excretion. The mrn expression of heptic PO100 tended to be reduced in the diet-fed mice, nd dietry olete prevented this reduction (chow vs. vs. O, 0±0.25 U vs. 0.75± 0.25 U vs. 1.10±0.39 U, P=65) (Fig. 2E). Dietry olete prevented stetoheptitis nd fibrosis in the livers of diet-fed mice To investigte the heptic inflmmtory sttus of ech group, serum ST nd LT levels were mesured. s expected, the diet incresed serum ST nd LT levels (ST, chow vs., 85.3±5.9 IU/L vs ±6.8 IU/L, P<01; LT, chow vs., 38.7±9.7 IU/L vs ±8.4 IU/L, P<01) (Tble 2). Olete intke prtilly prevented these increses in serum ST nd LT levels (ST, vs. O, 465.5±6.8 IU/L vs ±6.4 IU/L, P<01; LT, vs. O, 658.3±8.4 IU/L vs ±7.2 IU/L, P<01) (Tble 2). H&E stining of mice liver smples displyed incresed inflmmtory cells nd focl ggregtes of inflmmtory cells in MMP-9 mrn MMP-9 GPDH, b O Fig. 4. The effects of the methionine- nd choline-deficient diet () diet nd dietry olete on heptic inflmmtory molecules. Semi-quntittive reverse trnscription-polymerse chin rection (RT-PR) for inflmmtory molecules in mouse liver smples from ech group. () MP1. () TNF-α. () MMP-9. MP1, monocyte chemotctic protein 1; TNF-α, tumor necrosis fctor-α; MMP-9, mtrix metlloproteinse-9; GPDH, glycerldehyde-3-phosphte dehydrogense; U, rbitrry unit;, norml chow diet;, methionine- nd choline-deficient diet; O, diet with olete ( mg/ g/dy for 4 weeks). P<5 vs. chow, b P<vs.. diet-fed mice compred to the responses in chow diet-fed or O diet-fed mice (Fig. 3). The results of semi-quntittive RT-PR demonstrte tht the mrn expressions of heptic MP1, TNF-α nd MMP-9 were incresed in the dietfed mice, nd this overexpression of inflmmtory cytokines ws prevented by dietry olete (MP1, chow vs. vs. O, 0±7 U vs. 1.95±0.14 U vs. 1±0.11 U, P<01; TNF-α, 0±8 U vs. 1.93±0.11 U vs. 1.12± 0.11 U, P<01; MMP-9, 0±7 U vs. 1.83±0.11 U vs. 1.24±0.12 U, P<01) (Fig. 4). Trichrome stining of mice liver smples ws used to evlute the heptic pericellulr fibrosis in ech group. Pericellulr fibrosis nd numerous collgen fibers were observed in livers from the diet-fed mice, but the number of liver collgen fibers ws not higher thn tht of chow diet-fed mice in olete- Dibetes Metb J 2011;35:

6 Lee JY, et l. treted mice (reltive fibrosis re, chow vs. vs. O, 0±5 U vs. 1.96±6 U vs. 1.10±5 U, P<01) (Fig. 5). DISUSSION Reltive fibrosis re Fig. 5. The effects of the methionine- nd choline-deficient diet () diet nd dietry olete on heptic fibrosis. Trichrome stining of liver prffin-embedded sections. Scle brs, 200 μm. () diet-fed mice. () diet-fed mice. () diet with olete ( mg/g/dy for 4 weeks)-fed mice. (D) The res of fibrosis, mesured by imge nlyzer in three rndomly selected fields (mgnifiction, 100) of ech liver section. U, rbitrry unit;, norml chow diet; O, diet with olete. P<5 vs. chow, b P<vs.. b O D This study demonstrted tht dietry olete prevented diet-induced stetoheptitis nd heptic fibrosis vi n increse in heptic triglyceride excretion nd decreses in heptic de novo lipogenesis nd inflmmtion. The diet is well-estblished nd widely-used nutritionl model of stetoheptitis thtcuses heptic stetosis nd inflmmtion tht mimics NFLD in humns [4,5]. In this study, s expected, heptic stetosis developed in the diet-fed mice. dditionlly, the diet tended to reduce heptic PO100. This result suggests decrese in heptic VLDL excretion nd is consistent with previous studies [4,13]. Surprisingly, our results showed tht the diet induced n importnt lipogenic trnscription fctor in the liver, hrep. hrep regultes heptic de novo lipogenesis in response to elevted glucose concentrtions by binding lipogenic enzyme genes [14,15]. Previous studies reported the opposite results for nother lipogenic trnscription fctor, sterol regultory element-binding protein-1 (SREP-1) [4,16,17]. In those studies, reduced mrn expression or nucler levels of heptic SREP-1 were observed in the diet-fed mice compred with those in the chow diet-fed mice, but no consistent reduction in ftty cid synthesis genes, such s cetyl-o crboxylse () nd ftty cid synthse (FS), ws noted [16,17]. Our results might explin this discrepncy; nd FS re regulted not only by SREP-1 but lso by hrep; thus, the overexpression of hrep due to n diet might mintin the expression of ftty cid synthesis genes. In fct, heptic de novo lipogenesis is not known to contribute to the development of heptic stetosis in the diet niml model [4,13,16,17]. lthough the diet induced heptic hrep overexpression in our study, this result does not men tht heptic stetosis is due to incresed heptic lipogenesis becuse dietinduced heptic hrep overexpression could be compenstory response for decresed heptic de novo lipogenesis. Furthermore, we did not investigte downstrem lipogenic regultion of hrep. Nevertheless, considering glucose-induced hrep ctivtion nd the up-regultion of hrep in dibetic condition, the stetogenetic potentil of this overexpression of heptic hrep by n diet might be mgnified in the condition of hyperglycemi, even if the increse in heptic de novo lipogenesis is not min mechnism of diet-induced heptic stetosis. ctully, in Otsuk Long- Evns Tokushim Ftty rts, n niml model of type 2 dibetes with insulin resistnce nd hyperglycemi, it ws reported tht the development of heptic ft ccumultion, inflmmtion nd fibrosis due to n diet ws ccelerted compred with tht in non-dibetic control rts [18]. In report with db/db mice, nother dibetic niml model, heptic fibrosis induced by n diet ws more prominent thn tht in non-dibetic db/m mice [19]. Previous studies hve reported the effect of dietry MUF ginst stetoheptitis in NFLD niml models. study with rts fed n diet show tht the extent of heptic ftty infiltrtion nd heptic triglyceride content were lower in the rts fed n diet with olive oil (0.45 mg/g rt weight) thn in the rts fed n diet only [6]. The uthors of tht study suggested MUF from olive oil might inhibit heptic triglyceride synthesis on the bsis of prior studies reporting reduced lipogenesis in the rt liver due to dietry MUF from sfflower oil [6,8]. In the present study, heptic stetosis ws reduced in mice fed n diet with olete compred with tht in 494 Dibetes Metb J 2011;35:

7 Dietry olete nd non-lcoholic ftty liver disese the mice fed n diet only. dditionlly, dietry olete lmost completely prevented the decrese in heptic PO100 expression, nd the diet induced overexpression of heptic hrep in mice. These results suggest tht dietry olete prevents heptic stetosis by mintining heptic VLDL excretion, s well s by reducing heptic de novo lipogenesis in this niml model. s mentioned bove, incresed heptic hrep does not men tht heptic de novo lipogenesis contributes to the development of stetoheptitis in this niml model. However, we cn suggest tht dietry olete prevented diet-induced heptic stetosis, nd this effect, t lest in prt, is ssocited with the reduced expression of heptic hrep nd suppression of heptic de novo lipogenesis. In ddition, our results lso suggest tht dietry olete might be more effective ginst heptic stetosis in hyperglycemic or insulin-resistnt sttus. In the present study, we cnnot confirm whether the chnge in heptic hrep expression is direct effect of the diet or dietry olete. lthough it hs been reported tht polyunsturted ftty cids (PUF) suppress hrep ctivity in primry heptocytes by incresing hrep mrn decy [20], nd high glucose concentrtion down-regultes the expression of hrep mrn in insulinom cells [21], the regultion of hrep mrn trnscription or trnsltion is not well understood [14]. The mechnisms through which the diet increses heptic hrep expression nd dietry olete prevents this overexpression need to be further investigted. H&E stining of the liver showed incresed nd foclly ggregted inflmmtory cells in the livers of diet-fed mice. Dietry olete prevented this ggregtion of heptic inflmmtory cells. These results re consistent with our results of incresed MP1 mrn expression in the liver of dietfed mice nd reduced expression by dietry olete. MP-1 is potent chemotctic fctor for monocytes nd is derived predominntly from mcrophges nd endothelil cells [22,23]. The diet nd dietry olete lso ffected the expressions of other heptic inflmmtory molecules, such s TNF-α nd MMP-9. iologiclly, TNF-α ctivtes cscde of cytokine production [24], nd MMP-9 plys crucil role for the migrtion, extrvstion, nd infiltrtion of immune cells, including monocytes [25]. TNF-α, in prticulr, is implicted in the pthogenesis of NFLD [2]. Elevted levels of TNF-α hve been detected in obese ptients with insulin resistnce nd lso in ptients with NSH [2]. In our results, the diet induced TNF-α nd MMP-9 mrn expressions in the liver, nd dietry olete prevented the up-regultion of these inflmmtory molecules. To our knowledge, this is the first study to report the effects of dietry MUF on heptic inflmmtory molecules in NFLD niml model. To explin the pthogenesis of NFLD, multi-hit (formerly double-hit ) hypothesis hs been widely ccepted; insulin resistnce nd heptic stetosis - the first hit - nd subsequent development of inflmmtion or fibrosis - the second hit [2]. With trichrome stining, it ws demonstrted tht dietry olete prevented diet-induced heptic fibrosis. This result suggests tht dietry MUF might prevent the development of cirrhosis resulting from NFLD. Further, considering the multi-hit hypothesis, our results showing the effects of dietry olete on heptic stetosis, inflmmtion nd fibrosis suggest tht dietry MUF hs beneficil effects in every step of NFLD development nd progression. In conclusion, our dt suggest tht dietry MUF cn prevent heptic stetosis by reducing heptic hrep overexpression, s well s by inducing PO100 expression. dditionlly, our dt suggest tht dietry MUF inhibits the development of heptic inflmmtion nd fibrosis by suppressing severl inflmmtory molecules. ltogether, dietry olete hs beneficil effects in every step of the development nd progression of NFLD nd my be nutritionl option for NFLD prevention nd tretment. ONFLITS OF INTEREST No potentil conflict of interest relevnt to this rticle ws reported. KNOWLEDGMENTS This work ws supported by the Kore Science nd Engineering Foundtion (KOSEF) grnt funded by the Koren government (MEST) (grnt number, ). REFERENES 1. Frrell G, Lrter Z. Nonlcoholic ftty liver disese: from stetosis to cirrhosis. Heptology 2006;43(2 Suppl 1):S Lewis JR, Mohnty SR. Nonlcoholic ftty liver disese: review nd updte. Dig Dis Sci 2010;55: heung O, Snyl J. Recent dvnces in nonlcoholic ftty Dibetes Metb J 2011;35:

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