British Journal of Nutrition

British Journl of Nutrition (2012), 108, 2166 2175 q The Authors 2012 doi:10.1017/s0007114512000347 Differentil effects of low-dose resvertrol on diposity nd heptic stetosis in diet-induced oese mice Su-Jung Cho 1,2, Un Ju Jung 1,2 nd Myung-Sook Choi 1,2 * 1 Deprtment of Food Science nd Nutrition, Kyungpook Ntionl University, 1370 Snkyuk Dong Puk-ku, 702-701 Degu, Repulic of Kore 2 Center for Food nd Nutritionl Genomics Reserch, Kyungpook Ntionl University, Degu, Repulic of Kore (Sumitted 13 June 2011 Finl revision received 2 Novemer 2011 Accepted 16 Jnury 2012 First pulished online 14 Mrch 2012) British Journl of Nutrition Astrct Consumption of high-ft diet (HFD) enriched in sturted ft induces excessive weight gin due to diposity, which cn led to metolic complictions, s well s incresed risk of ftty liver disese nd CVD. The present study investigted the underlying mechnism nd dose response effects of resvertrol (RV) on oesity, heptic stetosis nd dyslipidemi in mice fed HFD. Mle C57BL/6J mice were fed norml diet or HFD (20 % ft, w/w) comined with 0 005 or 0 02 % (w/w) RV for 10 weeks. As expected, mice fed HFD developed oesity, s shown y incresed ody weight gin, viscerl ft, heptic ft nd plsm cholesterol. RV significntly reduced viscerl ft nd plsm. In the liver of HFD-fed mice, RV significntly reduced TAG nd cholesterol, s well s lipid droplet numer nd size. A low dose of RV (0 005 %) ppered to e more effective thn higher dose of RV (0 02 %) for suppressing diposity nd heptic stetosis development with significnt decrese in ody weight gin, plsm TAG nd totl cholesterol levels. These chnges were seemingly ttriutle to suppression of the ftty cid (FA) synthse, glucose-6-phosphte dehydrogense, nd phosphtidte phosphohydrolse nd/or n ctivtion of FA oxidtion in the liver nd epididyml dipose tissue. In conclusion, dily consumption of low dose of RV is effective for protecting ginst diet-induced oesity, heptic stetosis nd dyslipidemi in HFD-fed mice. Key words: Resvertrol: High-ft diet: Adiposity: Heptic stetosis: Lipid-regulting enzymes Oesity is chrcterised y n increse in white dipose tissue (WAT) mss, which cn result from n excess of food intke reltive to energy expenditure. Oesity is lso ssocited with heptic stetosis nd hyperlipidemi, indicted y excessive ccumultion of ft in the liver nd excess ft in circultion (1). Centrl diposity due to ccumultion of viscerl dipose tissue is linked to significntly incresed risk of heptic stetosis (2). Incresed flux from viscerl ft to the liver is one of the suggested underlying mechnisms (3). Most dietry pproches for oesity prevention or tretment ttempt to limit consumption of diet rich in sturted ft (4). In contrst, n increse in fruit nd vegetle intke cn help decrese oesity, which my e ttriutle to their low-energy density, fire content or phenolic compound enrichment (5,6). Resvertrol (RV, 3,5,4 0 -trihydroxystilene) is one of the nturl polyphenolic compounds minly found in grpe skin. RV is well known for its crdioprotective (7), nti-cncer (8), nti-inflmmtory, ntioxidnt (9) nd phyto-oestrogenic (10) properties. Some studies hve suggested tht RV my lso hve eneficil effects on oesity. For exmple, RV ppers to inhiit the prolifertion of pre-dipocytes in vitro (11), nd hence my suppress the production of new ft cells. In ddition, RV ppers to inhiit lipogenesis s well s lipid ccumultion (12) in humn liver cells nd mture dipocytes (13,14). While the evidence from cellulr studies suggests tht RV my suppress the production of new ft cells nd prevent ft ccumultion in liver cells nd dipocytes, the effects of RV on oesity nd stetosis in vivo hve een fr more equivocl (15 20). Severl in vivo studies reported tht RV protected ginst diet-induced oesity (15,16) nd heptic stetosis (17) in murine models. For exmple, ody weight gin nd ft ccumultion were suppressed in mice fed high-ft diet (HFD) with high dietry RV intke (400 mg/kg ody weight per d) over 15 weeks. In these HFD-fed mice, RV up-regulted the silent Arevitions: FA, ftty cid; FAS, ftty cid synthse; G6PD, glucose-6-phosphte dehydrogense; HFD, high-ft diet; ND, norml diet; PAP, phosphtidte phosphohydrolse; RV, resvertrol; SIRT1, silent informtion regulting 2 homologue 1; WAT, white dipose tissue. * Corresponding uthor: M.-S. Choi, fx þ82 53 950 6229, emil mschoi@knu.c.kr These uthors contriuted eqully to this work. Downloded from https://www.cmridge.org/core. IP ddress: 148.251.232.83, on 07 Nov 2018 t 11:47:41, suject to the Cmridge Core terms of use, ville t https://www.cmridge.org/core/terms. https://doi.org/10.1017/s0007114512000347

Effects of resvertrol in oese mice 2167 British Journl of Nutrition informtion regulting 2 homologue 1 (SIRT1) gene, which is key regultor of energy nd metolic homeostsis (15). However, in other studies, RV ws reported to hve miniml or no effect on oesity nd heptic stetosis (18 20). In oese Zucker rts, RV supplementtion (10 mg/kg ody weight per d) over 8 weeks filed to reduce ody weight, lthough RV reduced dipose tissue mss (18). Similrly in mice, RV (22 4 mg/kg ody weight per d) supplementtion for 15 months hd no effect on ody weight, lthough RV ppered to mimic the effects of energy restriction (20). The lck of consensus on the in vivo effect of RV on diet-induced oesity nd heptic stetosis my hve een due to vrying doses, diet nd durtion in previous studies. Importntly, the potentil mechnism underlying the nti-oesity nd heptoprotective effects of RV in vivo still remins uncler. In prticulr, few studies hve determined the ctivities of enzymes involved in lipogenesis nd ftty cid (FA) oxidtion in the liver nd dipose tissue of RV-supplemented, diet-induced oese mice. Accordingly, we first determined whether RV (0 005 or 0 02 % in diet) hd ny dose-dependent effects on ody weight gin, viscerl diposity, heptic stetosis nd dyslipidemi in HFD-fed mice, well-estlished murine model of diet-induced oesity (21,22). Secondly, we exmined lipid metolic enzyme ctivity in the liver nd WAT to further elucidte potentil underlying mechnisms responsile for the effects of RV on diet-induced oesity. Experimentl methods Animls nd diets A totl of forty mle C57BL/6J mice (4 weeks old) were purchsed from the Jckson Lortory. The nimls were mintined in room with controlled temperture (20 238C) nd lighting (lternting 12 h periods of light nd drk) nd fed pelletised commercil non-purified diet for 1 week fter rrivl. The mice were then rndomly divided into four groups (n 10) nd fed the respective experimentl diets for 10 weeks, s shown in Tle 1; norml diet (ND) control (ND, Americn Institute of Nutrition (AIN)-76 semisynthetic diet), HFD control (HFD, 20 % ft sed on the AIN-76 diet plus 1 % cholesterol, w/w), 0 02 % RV (HFD þ high RV, 0 02 % RV with HFD, w/w, Sigm Chemicl Compny) nd 0 005 % RV (HFD þ low RV, w/w, 0 005 % RV with HFD, w/w). The HFD ws formulted to provide 20 % of the totl energy from ft, y replcing crohydrte energy with lrd nd mize oil, nd hd the sme mount of vitmins nd minerls per kj s the ND. The mice hd free ccess to food nd distilled wter during the experimentl period. Food intke nd ody weight were mesured dily. All niml procedures were pproved y the Ethics Committee for niml studies t Kyungpook Ntionl University, Repulic of Kore. Smpling At the end of the experimentl period, the mice were nesthetised with ketmine fter 12 h fst. Blood smples were collected in heprinised tues from the inferior ven cv nd stored t 2708C efore nlysis of plsm iomrkers. The liver nd viscerl ft depots (epididyml, perirenl, mesentery nd retroperitoneum) were removed, rinsed with physiologicl sline solution, weighed nd immeditely stored t 2708C. Lipid nlyses Plsm were mesured using commercil ssy kit (-Wko; Wko Pure Chemicl Industries). Plsm TAG Tle 1. Composition of experimentl diets (% of diet, w/w) Ingredients ND HFDþ0 RV HFDþhigh RV HFDþlow RV Csein 20 20 20 20 DL-Met 0 3 0 3 0 3 0 3 Sucrose 50 37 36 98 36 995 Cellulose 5 5 5 5 AIN-minerl* 3 5 4 2 4 2 4 2 AIN-vitmin 1 1 2 1 2 1 2 Choline itrtrte 0 2 0 2 0 2 0 2 Mize strch 15 11 1 11 1 11 1 Lrd 17 17 17 Mize oil 5 3 3 3 Cholesterol 1 1 1 RV 0 02 0 005 Totl (%) 100 100 100 100 Diet (kj/100 g) 1611 42 1893 52 1893 19 1893 44 Energy from ft (%) 11 7 39 8 39 8 39 8 ND, norml diet; HFD, high-ft diet; RV, resvertrol; HFDþ0 RV, HFD; HFDþhigh RV, HFD supplemented with 0 02 % RV; HFDþlow RV, HFD supplemented with 0 005 % RV; AIN, Americn Institute of Nutrition. * AIN-76 minerl mixture: clcium phosphte 500 g/kg, NCl 74 g/kg, potssium citrte 2220 g/kg, potssium sulphte 52 g/kg, mgnesium oxide 24 g/kg, mngnous cronte 3 5 g/kg, ferric citrte 6 g/kg, zinc cronte 1 6 g/kg, cupric cronte 0 3 g/kg, potssium iodte 0 01 g/kg, sodium selenite 0 01 g/kg, chromium potssium sulphte 0 55 g/kg, sucrose 118 03 g/kg. AIN-76 vitmin mixture: thimin HCl 0 6 g/kg, rioflvin 0 6 g/kg, pyridoxine HCl 0 7 g/kg, nicin 3 g/kg, clcium pntothente 1 6 g/kg, folic cid 0 2 g/kg, iotin 0 02 g/kg, vitmin B 12 1 g/kg, vitmin A (500 000 IU/g) 0 8 g/kg, vitmin D 3 (400 000 IU/g) 0 25 g/kg, vitmin E cette (500 IU/g) 10 g/kg, mendione sodium isulphite 0 08 g/kg, sucrose 981 15 g/kg. Downloded from https://www.cmridge.org/core. IP ddress: 148.251.232.83, on 07 Nov 2018 t 11:47:41, suject to the Cmridge Core terms of use, ville t https://www.cmridge.org/core/terms. https://doi.org/10.1017/s0007114512000347

2168 S.-J. Cho et l. British Journl of Nutrition nd totl cholesterol were mesured using commercil kits (Asn Phrmceuticl), sed on the lipse glycerol phosphte oxidse method (23) nd cholesterol oxidse method, respectively (24). Plsm pob ws lso determined using commercil kit (Nitto Boseki). Heptic lipids were extrcted using the method of Folch et l. (25). The dried lipid residues were dissolved in 1 ml of ethnol for cholesterol nd TAG ssys. Triton X-100 nd sodium cholte solution were dded to 200 ml of the dissolved lipid solution to produce finl concentrtion of 3 mm. The heptic cholesterol nd TAG levels were nlysed with the sme commercil kit s used in the plsm nlysis (Asn Phrmceuticl). Lipid-regulting enzyme ctivities To mesure lipid-regulting enzyme ctivities in the liver nd epididyml WAT, enzyme sources were prepred ccording to the method reported y Hulcher & Oleson (26). FA synthse (FAS) ctivity ws determined y spectrophotometric ssy ccording to the methods of Crl et l. (27) ; one unit of FAS ctivity represented the oxidtion of 1 nmol of NADPH per min t 308C. The glucose-6-phosphte dehydrogense (G6PD) ctivity ws determined using the method of Pitkänen et l. (28), sed on the reduction of 1 nmol NADP per min t (A) 38 Body weight (g) 35 32 29 26 23 20 17 258C mesured t 340 nm using spectrophotometer. Phosphtidte phosphohydrolse (PAP) ctivity ws mesured using the method of Wlton & Possmyer (29) nd PAP ctivity ws expressed s nmol/min per mg protein. FA -oxidtion ws mesured spectrophotometriclly y monitoring the reduction of NAD to NADH in the presence of plmitoyl- CoA s descried y Lzrow (30). The mount of protein in enzyme sources ws determined y the Brdford method (31) using ovine serum lumin s the stndrd. Histopthologicl nlysis Liver nd epididyml WAT were removed from the mice nd fixed in uffer solution of 10 % formlin. All fixed tissues were processed routinely for prffin emedding nd 4 mm sections were prepred nd stined with hemtoxylin nd eosin. Stined res were viewed using n opticl microscope (Zeiss Axioscope) with mgnifying power of 200 nd epididyml dipocyte size ws mesured y using Motic Imges Plus 2.0ML (Motic). Sttisticl nlysis All dt were presented s the men nd stndrd error. Sttisticl nlysis ws performed using softwre SPSS (version 11.0, (B) 12 10 ** c *,, 8 * *,, * *, ** 6 c 4 *** 2 ** * ** 0 0 1 2 3 4 5 6 7 8 9 10 Epididyml Perirenl Mesentery Retroperitonl Totl Feeding durtion (weeks) WAT weight (g/100 g BW) (C) 4 (D) (E) *** 0 10 70 60 0 08, 3 50 0 06 40 2 *** 0 04 30 1 20 0 02 10 0 0 00 0 ND HFD+ HFD+ HFD+ ND HFD+ HFD+ HFD+ 0RV high RV low RV 0RV high RV low RV Food intke (g/d) FER Energy intke (kj/d) ND HFD+ 0RV HFD+ HFD+ high RVlow RV Fig. 1. Effects of resvertrol (RV) supplementtion on (A) ody weight (, norml diet (ND);, high-ft diet (HFD, HFDþ0 RV);, HFD supplemented with 0 02 % RV (HFDþhigh RV);, HFD supplemented with 0 005 % RV (HFDþlow RV)), (B) viscerl white dipose tissue (WAT) (, ND;, HFDþ0 RV;, HFDþhigh RV;, HFDþlow RV), (C) food intke weight, (D) food efficiency rtio (FER) nd (E) energy intke in C57BL/6J mice fed HFD. Vlues re mens, with their stndrd errors represented y verticl rs.,,c Men vlues with unlike letters re significntly different mong groups (P,0 05). Men vlues were significntly different for ND from those of HFD: *P,0 05, ** P,0 01, ***P,0 001. Food efficiency rtio (FER) ¼ ody weight gin/ food intke. Downloded from https://www.cmridge.org/core. IP ddress: 148.251.232.83, on 07 Nov 2018 t 11:47:41, suject to the Cmridge Core terms of use, ville t https://www.cmridge.org/core/terms. https://doi.org/10.1017/s0007114512000347

Effects of resvertrol in oese mice 2169 British Journl of Nutrition SPSS, Inc.). Sttisticl differences etween ND nd HFD results were determined using Student s t test. One-wy ANOVA ws performed to compre HFD groups with or without RV nd Tukey s post hoc test ws performed when significnt differences were identified etween the groups t P,0 05. Results Effects of resvertrol on ody weight nd food intke in diet-induced oese mice Food intke ws significntly suppressed in mice fed HFD lone compred to ND-fed mice, nd dily energy intke ws not different etween the two groups (Fig. 1). However, ody weight ws significntly greter in the mice fed HFD lone compred to ND-fed mice fter 6 weeks. In HFD-fed mice, supplementtion of RV t low dose (0 005 %) significntly suppressed ody weight gin fter 3 weeks, ut the higher dose of RV (0 02 %) ws surprisingly not effective (Fig. 1). Neither food intke nor energy intke ws ffected y RV supplementtion, wheres food efficiency rtio ws significntly lower in the low-dose (0 005 %) RV group, compred to the HFD control group (Fig. 1). (A) (B) ND Effects of resvertrol on ft ccumultion in diet-induced oese mice HFD feeding resulted in significnt increse in totl viscerl WAT weight, including the epididyml, perirenl, retroperitonel nd mesentery depots compred to ND (Fig. 1). RV intke hd differentil effects on viscerl dipose depots. Perirenl nd mesentery dipose depots were significntly reduced in HFD-fed mice supplemented with either highdose or low-dose RV (Fig. 1). Only the low dose of RV (0 005 %) effectively reduced the weight of the epididyml nd retroperitonel dipose depots in HFD-fed mice (Fig. 1). Hence, overll totl viscerl dipose weight ws significntly lowered y RV supplementtion, lthough the low dose ppered to e more effective thn the higher dose (Fig. 1). Morphologicl oservtions lso indicted tht epididyml dipocyte size ws smller in the RV-supplemented mice thn in the HFD control mice (Fig. 2(B) nd (C)). Effect of resvertrol on plsm nd heptic lipid levels in diet-induced oese mice Next, we determined if there ws ny evidence of dosedependent effect of RV on plsm nd heptic lipid levels HFD+0 RV HFD+high RV HFD+low RV (C) Epididyml dipocyte size (µm) 400 350 300 250 200 150 100 50 0 *** ND HFD+0 RV HFD+ high RV c HFD+ low RV Fig. 2. Effects of resvertrol (RV) supplementtion on (A) liver nd (B) epididyml dipose tissue morphology in C57BL/6J mice fed high-ft diet (HFD). Hemtoxylin nd eosin (H&E)-stined trnsverse-section of the liver nd epididyml ft; ech liver nd epididyml ft (n 10) were removed nd wrpped with slinesoked guze fter removing the connective tissues. All were fixed in 10 % prfomldehyde/pbs, emedded in prffin, nd then stined with H&E. Originl mgnifiction 200. Effects of RV supplementtion on epididyml dipocyte size in C57BL/6J mice fed HFD. Vlues re mens, with their stndrd errors represented y verticl rs.,,c Men vlues with unlike letters were significntly different (P,0 05). *** Men vlue ws significntly different from those of the HFD groups (P,0 001). ND, norml diet; HFDþ0 RV, HFD; HFDþhigh RV, HFD supplemented with 0 02 % RV; HFDþlow RV, HFD supplemented with 0 005 % RV. (A colour version of this figure cn e found online t http://www.journls.cmridge.org/jn) Downloded from https://www.cmridge.org/core. IP ddress: 148.251.232.83, on 07 Nov 2018 t 11:47:41, suject to the Cmridge Core terms of use, ville t https://www.cmridge.org/core/terms. https://doi.org/10.1017/s0007114512000347

2170 S.-J. Cho et l. Tle 2. Effects of resvertrol (RV) supplementtion on plsm nd heptic lipid levels in C57BL/6J mice fed high-ft diet (HFD) (Men vlues with their stndrd errors) ND HFDþ0 RV HFDþhigh RV HFDþlow RV Men SE Men SE Men SE Men SE Plsm lipids (mmol/l) 1 00 0 04 1 12 0 04 0 86 0 04 0 81 0 03 TAG 0 97 0 10 1 13, 0 07 1 37 0 14 0 94 0 11 Totl cholesterol 3 47*** 0 08 4 81 0 10 4 71 0 18 4 00 0 31 HDL-cholesterol 0 83* 0 01 1 16 0 08 1 16 0 06 1 18 0 03 AI 3 13 0 20 3 18 0 01 3 03 0 01 2 75 0 07 ApoB:poA-I 0 13*** 0 001 0 21 0 002 0 19, 0 001 0 13 0 001 Heptic lipids (mmol/g) TAG 10 83*** 0 06 21 89 1 18 18 43 0 93 14 93 c 0 44 Cholesterol 2 51*** 0 15 14 64 0 58 7 73 0 64 5 74 c 0 10 ND, norml diet; HFDþ0 RV, HFD; HFDþhigh RV, HFD supplemented with 0 02 % RV; HFDþlow RV, HFD supplemented with 0 005 % RV; AI, therogenic index.,,c Men vlues with unlike superscript letters re significntly different (P,0 05). Men vlues were significntly different for ND from those of HFD: * P,0 05, *** P,0 001. AI¼ (totl cholesterol 2 HDL-cholesterol)/HDL-cholesterol. British Journl of Nutrition (Tle 2). Plsm nd TAG levels were not elevted in the mice fed the HFD lone compred to ND-fed mice. As result of concomitnt increse in oth plsm totl cholesterol nd HDL-cholesterol levels, therogenic index ws unchnged in the HFD control group compred to the ND group. However, the pob:poa-i rtio, which is more sensitive iomrker to mesure CHD thn therogenic index, ws significntly higher in the HFD control group compred to the ND group. Despite the lck of chnges in plsm lipid levels induced y the HFD, RV t oth low nd high dose (0 005 nd 0 02 %) significntly reduced plsm level compred to the HFD control group nd the ND group. Remrkly, RV ws more effective t low dose (0 005 %), ut not t higher dose (0 02 %), for reducing plsm TAG nd totl cholesterol levels in HFD-fed mice. Plsm HDL-cholesterol level ws not significntly different etween mice fed the HFD lone nd the two RV groups. However, the pob:poa-i rtio ws significntly lower in the low-dose RV group compred to the HFD control group. Heptic TAG nd cholesterol contents were incresed pproximtely 2-fold nd 6-fold, respectively, in C57BL/6 mice fed the HFD lone s previously oserved y others (32,33). In HFD-fed mice, RV significntly reduced heptic TAG nd cholesterol contents compred to the HFD control group, lthough not in dose-dependent mnner. Consistent with the oserved heptic TAG nd cholesterol contents, hemtoxylin nd eosin stining of liver sections for lipids indicted tht heptic lipid ccumultion ws more pronounced in the HFD control mice compred to ND-fed mice (Fig. 2). RV cused mrked decrese in the numer nd size of liver ft droplets, nd the low dose of RV (0 005 %) ppered to e more effective thn the higher dose of RV (0 02 %). Effect of resvertrol on lipid-regulting enzyme ctivities in liver of diet-induced oese mice To further exmine the mechnism vi which RV suppresses lipid ccumultion in the liver, we determined the ctivity of heptic lipid-regulting enzymes. The ctivity of heptic PAP, which is rte-limiting enzyme in TAG synthesis, ws elevted in mice fed the HFD lone compred to ND-fed mice. We oserved no chnges in de novo lipogenic FAS nd G6PD ctivities in the liver of mice fed the HFD lone. However, FA oxidtion ws significntly ugmented in the mice fed the HFD lone compred to ND-fed mice (Tle 3). In contrst, the ctivity of PAP ws significntly suppressed y RV in the liver of HFD-fed mice without ffecting FA oxidtion (Tle 3). Furthermore, 0 02 % RV in the diet significntly decresed heptic FAS ctivity y 43 % compred to the HFD lone (Tle 3). Effect of resvertrol on ctivities of enzymes involved in lipogenesis nd ftty cid oxidtion in epididyml white dipose tissue of diet-induced oese mice We next exmined the ctivities of enzymes tht cn regulte lipid ccumultion in epididyml WAT. C57BL/6J mice fed the HFD lone showed significnt increse of lipogenic enzyme PAP ctivity in epididyml WAT compred to NDfed mice, lthough no chnge in -oxidtion ws oserved in HFD-fed mice (Tle 3). In contrst, RV significntly decresed FAS nd PAP ctivities in epididyml WAT compred to the HFD-lone group (Tle 3). Furthermore, mice fed low dose of RV (0 005 %) showed significnt decrese in G6PD ctivity nd significnt increse in FA -oxidtion in epididyml WAT compred to the mice fed the HFD lone (Tle 3). Discussion A chronic HFD is mjor contriuting fctor underlying the development of oesity in humns. Currently, there is widespred interest in nturl compounds tht cn suppress dietinduced oesity. In the present study, we first determined whether RV exerted ny dose-dependent effect on weight gin, heptic stetosis or dyslipidemi. Next, we determined whether RV ffected lipid-regulting enzyme ctivity in the liver or dipose tissue. We used C57BL/6J mice which re prone to the development of oesity when fed HFD, Downloded from https://www.cmridge.org/core. IP ddress: 148.251.232.83, on 07 Nov 2018 t 11:47:41, suject to the Cmridge Core terms of use, ville t https://www.cmridge.org/core/terms. https://doi.org/10.1017/s0007114512000347

Effects of resvertrol in oese mice 2171 Tle 3. Effects of resvertrol (RV) supplementtion on lipid-regulting enzyme ctivities in the liver nd dipose tissue of C57BL/6J mice fed high-ft diet (HFD) (Men vlues with their stndrd errors) ND HFDþ0 RV HFDþhigh RV HFDþlow RV Men SE Men SE Men SE Men SE Liver FAS (nmol/min per mg protein) 0 96 0 05 1 22 0 12 0 92, 0 11 0 69 0 01 G6PD (nmol/min per mg protein) 4 18 0 67 3 46 0 32 3 26 0 08 1 71 0 18 PAP (nmol/min per mg protein) 17 62* 0 54 22 98 1 39 18 56 0 26 17 37 0 12 -Oxidtion (nmol/min per mg protein) 1 56* 0 07 2 43 0 28 3 03 0 02 3 13 0 43 Adipose tissue FAS (nmol/min per mg protein) 42 50 3 21 37 17 1 77 23 65 1 42 15 00 c 1 29 G6PD (nmol/min per mg protein) 27 10 1 78 32 33 1 66 26 59 1 68 20 35 0 58 PAP (nmol/min per mg protein) 251 64** 50 49 351 96 16 99 248 27 7 95 195 29 c 15 25 -Oxidtion (nmol/min per mg protein) 123 37 63 30 141 93 10 34 98 95 31 10 364 27 57 80 ND, norml diet; HFDþ0 RV, HFD; HFDþhigh RV, HFD supplemented with 0 02 % RV; HFDþlow RV, HFD supplemented with 0 005 % RV; FAS, ftty cid synthse; G6PD, glucose-6-phosphte dehydrogense; PAP, phosphtidte phosphohydrolse.,,c Men vlues with unlike superscript letters re significntly different (P,0 05). Men vlues were significntly different for ND from those of HFD: * P,0 05, ** P,0 01. British Journl of Nutrition developing similr chrcteristics s humn oesity, compred to geneticlly oese nimls (21,22,34). We found tht RV protected ginst diet-induced oesity, ut oserved unexpectedly no dose response effects. As others hve shown (21,22,34), the HFD resulted in mrked increse of ody weight gin due to incresed diposity, despite significntly decresed food intke. RV significntly suppressed diposity in HFD-fed mice, due to lower viscerl WAT weight. Consistent with these results, we oserved tht epididyml WAT size ws smller in HFD-fed mice supplemented with RV. RV supplemented t lower dose ws more effective thn the higher dose t suppressing viscerl ft ccumultion. Furthermore, RV t low dose, ut not high dose, significntly suppressed ody weight gin compred to HFD control mice. Pst niml studies hve een equivocl, with some studies reporting tht RV suppresses ody weight gin nd ft ccumultion in HFD-fed mice (15,16) nd rts (17), while severl studies hve reported tht RV hs no effect on ody weight gin (18 20). The discrepncies etween pst studies my e ttriutle to differences in diet composition, durtion of diet, dosge nd experimentl nimls used. Few pst studies hve exmined ny dose response effects of RV in HFD-fed nimls. One previous study in rts fed hyperenergetic diet contining different doses (6, 30, 60 mg/kg ody weight per d) of RV for 6 weeks reported no dose response effects on dipose weight (35). Importntly, our study compred two doses of RV, oth elow 0 02 %, equivlent to 2 mg/kg ody weight per d. Our findings suggest tht RV my e n effective ntioesity gent, reducing ody weight nd ft ccumultion in response to HFD, nd tht 0 005 % RV my e n dequte mount for suppressing ody weight gin nd diposity, lthough response study with lower doses of RV is further needed to determine the minimum effective dose. Next, we exmined potentil mechnisms underlying the ody weight nd diposity-suppressing effect of RV. RV did not led to decresed food intke, ecuse no significnt difference in food intke ws oserved in the HFD-fed mice regrdless of RV dministrtion. Mny studies hve reported tht dietry ft led to ugmenttion of lipogenic enzyme ctivity (36,37), lthough contrdictory findings hve een reported (38). WAT is mjor site of de novo FA synthesis, nd the FA synthesised in WAT cn e either re-esterified to TAG within WAT or oxidised vi mitochondril -oxidtion long with FA from the diet. FAS plys centrl role in de novo lipogenesis y ctlysing the synthesis of sturted long-chin FA from cetyl-coa, mlonyl-coa nd NADPH. G6PD is lso lipogenic enzyme involved in supplying NADPH for the iosynthesis of FA. In the present study, no chnges in FAS or G6PD ctivity s well s FA -oxidtion ctivity were oserved in epididyml WAT of ND-fed mice compred to mice fed HFD lone. However, we oserved significnt increse in PAP ctivity, rte-limiting enzyme in TAG synthesis in epididyml WAT. These findings indicte tht HFD does not promote de novo FA synthesis nd ft oxidtion in dipose tissue. The incresed epididyml WAT mss in HFD-fed mice my e ttriutle to incresed esterifiction of provided y the diet. Interestingly, in HFD-fed mice, RV dministrtion significntly inhiited the ctivity of de novo lipogenic enzymes including FAS nd G6PD s well s PAP in epididyml WAT. Agin we oserved no dose response effect, nd 0 005 % RV significntly ugmented FA -oxidtion in epididyml WAT, which my help explin the lower viscerl diposity compred to 0 02 % RV-supplemented mice. Our findings re consistent with previous study tht reported suppression of FAS gene expression nd protein level y RV in epididyml WAT of HFD-fed mice (39). Furthermore, RV hs een reported to inhiit de novo lipogenesis in prllel with the down-regultion of lipogenic gene expression in humn dipocytes (13). Tken together, the current evidence indictes tht the suppression of diposity y RV my e due to the inhiition of de novo FA synthesis or stimultion of FA oxidtion in WAT. Another possile mechnism y which dietry RV meliortes diposity my e due to the ctivtion of lipolysis. Lipolysis is one of the importnt metolic pthwys regulting TAG ccumultion in WAT nd two mjor enzymes, hormone-sensitive lipse nd dipose TAG lipse, re involved Downloded from https://www.cmridge.org/core. IP ddress: 148.251.232.83, on 07 Nov 2018 t 11:47:41, suject to the Cmridge Core terms of use, ville t https://www.cmridge.org/core/terms. https://doi.org/10.1017/s0007114512000347

2172 S.-J. Cho et l. British Journl of Nutrition in dipose TAG hydrolysis. Rylm et l. (40) hve shown tht RV down-regulted the expression of hormone-sensitive lipse s well s lipogenic gene in WAT, indicting tht RV my lter ft mss y directly ffecting the iochemicl pthwys involved in dipogenesis. In contrst, recent study reported tht RV induced the relese of ut not glycerol in 3T3 L1 cells nd up-regulted dipose TAG lipse mrna nd protein expression without ffecting hormone-sensitive lipse (41). Furthermore, relese ws incresed y RV in epididyml WAT of wild-type nd hormone-sensitive lipse knockout mice, ut no chnges were oserved in epididyml WAT of dipose TAG lipse knockout mice, suggesting tht dipose TAG lipse seems to e the min trget for the lipolytic effect of RV (41). Ft ccumultion in dipose tissue is closely ssocited with the development of heptic stetosis, which is n excessive ccumultion of ft in the liver. Severl mechnisms my led to heptic stetosis: (1) incresed supply due to incresed lipolysis from WAT nd/or incresed intke of dietry ft, (2) incresed de novo heptic lipogenesis, (3) decresed ftty oxidtion in the liver nd (4) decresed heptic VLDL TAG secretion (42). Excessive consumption of dietry ft, especilly SFA, leds to n excess ccumultion of TAG in the liver (32,33). We lso oserved tht the HFD led to significnt increse of heptic TAG content s well s heptic PAP ctivity, consistent with previous oservtion tht SFA drmticlly incresed PAP gene expression (43). However, the HFD did not led to incresed de novo lipogenic FAS nd G6PD ctivities in the liver, or led to incresed plsm β-oxidtion FA synthesis FAS G6PD Liver PAP nd TAG levels compred to ND-fed mice. In greement with the present study, plsm nd TAG levels, s well s heptic FAS ctivity nd FAS gene expression, were not incresed in HFD (38 1 % ft, w/w) -fed C57BL/6 mice fter 18 weeks (44). Furthermore, Kim et l. (45) reported tht HFD did not ffect plsm nd TAG levels in C57BL/6 mice, lthough the HFD induced heptic stetosis nd diposity. Moreover, Kim et l. (45) showed tht genes relted to FA -oxidtion were ctivted in the liver of HFD-fed mice, consistent with our dt. Accordingly, these findings suggest tht HFD cn promote heptic lipogenesis y esterifiction of provided from the diet ut not from de novo lipogenesis in the liver. Heptic TAG ccumultion my led to ugmenttion of FA oxidtion y feedck mechnism. We found tht RV significntly reduced heptic stetosis nd TAG content s well s plsm level in HFD-fed mice. The suppression of heptic lipogenesis y RV ws linked to decresed ctivities of heptic de novo lipogenic FAS s well s PAP without ffecting FA oxidtion. The low dose of RV ppered to e more effective for suppressing heptic stetosis, similr to ody weight nd ft. Tken together, these findings suggest tht the eneficil effect of RV on heptic stetosis is medited, t lest in prt, y decrese in de novo FA synthesis nd esterifiction sed on the evidence of lower heptic FAS nd PAP ctivity. Previous studies lso indicte tht RV my prevent heptic stetosis y regulting heptic lipid metolism-relted genes such s FAS nd SIRT1 (46). Also, the lowered plsm my ccount in prt for the decrese in heptic TAG content in mice supplemented with RV, since TAG synthesis TAG TAG TAG TAG Chol β-oxidtion Apo B HDL Chol Apo A 1 LDL FAP FAS G6PD FA synthesis TAG synthesis Epididyml dipose Fig. 3. Summry of the effects of resvertrol supplementtion on lipid metolism in the liver nd dipose tissue., Chnge in ctivity or concentrtion. FA, ftty cid; FAS, ftty cid synthse; G6PD, glucose-6-phosphte dehydrogense; PAP, phosphtidte phosphohydrolse; Chol, cholesterol. (A colour version of this figure cn e found online t http://www.journls.cmridge.org/jn) Downloded from https://www.cmridge.org/core. IP ddress: 148.251.232.83, on 07 Nov 2018 t 11:47:41, suject to the Cmridge Core terms of use, ville t https://www.cmridge.org/core/terms. https://doi.org/10.1017/s0007114512000347

Effects of resvertrol in oese mice 2173 British Journl of Nutrition heptic TAG ccumultion during insulin resistnce occurs s result of the increse in the supply of circulting to the liver s well s through n incresed endogenous lipogenesis (42). In the present study, we oserved tht the HFD resulted in n incresed plsm pob:poa-i rtio, s well s totl cholesterol concentrtion mostly due to incresed HDL-cholesterol concentrtion. The increse in totl cholesterol nd HDLcholesterol is common feture of most mouse strins fed HFD (47). Plsm pob or poa-i concentrtions independently or together s rtio (pob:poa-i) re reported to predict crdiovsculr risk more ccurtely thn lipid levels (48). Previously we reported tht long-term RV supplementtion over 20 weeks significntly decresed plsm totl cholesterol nd LDL-cholesterol levels in poe knockout mice fed n ND, wheres the pob:poa-i rtio ws incresed (49). Herein, we oserved tht 0 005 % RV normlised the HFD-medited increse of plsm totl cholesterol nd pob:poa-i rtio, wheres the eneficil effects were not oserved in HFD-fed mice supplemented with 0 02 % RV. Zern et l. (50) reported tht the grpe polyphenol filed to improve plsm totl cholesterol level, lthough heptic cholesterol metolism ws ltered sed on evidence of lower heptic cyl-coa: cholesterol cyltrnsferse (ACAT) ctivity, key cholesterolregulting enzyme involved in the esterifiction nd sorption of cholesterol (51). Furthermore, grpe polyphenol ws suggested to decrese the secretion of heptic LDL-cholesterol reducing cholesterol ccumultion in the rteril wll (51). Consistent with this study, we found tht oth lower nd higher RV significntly lowered heptic cholesterol content y 47 nd 61 %, respectively, s compred to the HFD-lone group. Although we did not determine heptic 3-hydroxy-3- methylglutryl (HMG)-CoA reductse ctivity nd fecl ile cid levels in this study, the inhiition of heptic HMG-CoA reductse ctivity or the increse of fecl ile cids excretion my e relted to the plsm nd heptic cholesterol-lowering effect of RV. In our previous study, we demonstrted tht RV led to decresed cholesterol level in plsm s well s the liver y suppressing the ctivity of heptic HMG-CoA reductse, rte-limiting enzyme for cholesterol synthesis, in poe-deficient mice (49). On the other hnd, Zhu et l. (52) reported tht RV significntly lowered cholesterol levels in the serum nd liver of HFD-fed rts nd these effects resulted from the enhnced excretion of ile cids into feces. Another possiility is n inhiition of pncretic ile slt-dependent lipse y RV (53). In rt pncretic AR4-2J cells, RV inhiited the secretion nd ctivity of ile slt-dependent lipse which is synthesised in pncretic cells nd involved in duodenl hydrolysis of lipid esters nd sorption of non-esterified cholesterol. Overll, our findings suggest tht lower doses of RV my hve more eneficil effects on diposity, heptic stetosis nd hyperlipidemi in HFD-fed mice compred to higher doses of RV. Currently, it is uncler why RV hs reverse dose response effect, ut it my explin why previous studies on the in vivo effects of RV hve een equivocl. One possiility suggested in recent review is tht RV t high doses cn exhiit pro-oxidnt properties (54). Oxidtive stress closely correltes with ft ccumultion in humns nd mice (55) nd rective oxygen species led to mitochondril dysfunction ssocited with oesity nd oesity-relted metolic disese (56). Considerle evidence supports tht high doses of polyphenols cn potentilly cuse dverse metolic effects through pro-oxidtive effects (57). In fct, severl recent ppers indicte tht high doses of RV in vivo cn led to dverse physiologicl effects. Dudley et l. (58) reported tht t lower doses (2 5 or 5 mg/kg ody weight per d), RV protected ginst hert ischemi y inducing survivl signl cusing the up-regultion of nti-poptotic nd redox proteins, while t higher doses (.25 mg/kg ody weight per d) RV potentited deth signl cusing the down-regultion of redox proteins nd up-regultion of pro-poptotic proteins. Consistently, RV protected the hert when dministered t reltively low doses etween 2 5 nd 10 mg/kg ody weight (59). In ddition, high doses of trns-rv in hypercholesterolemic rits hve een reported to cuse therosclerotic lesions while lower doses of RV were found to e protective (60). In conclusion, the present findings suggest tht dietry RV my e n effective nti-oesity gent nd lso protect ginst therosclerosis when supplemented t low doses. RV decreses diposity nd heptic stetosis in diet-induced oese mice nd these effects pper to e medited vi regultion of lipid metolism-relted enzyme ctivity in the liver nd dipose tissue, s summrised in Fig. 3. Low-dose RV ws more eneficil for suppressing viscerl diposity nd heptic stetosis, long with protecting ginst dyslipidemi in HFD-fed mice. In future studies, it will e importnt to estlish whether vrying doses of RV in humns consuming HFD cn provide ny protection ginst heptic stetosis, dyslipidemi nd viscerl dipose tissue ccumultion. Acknowledgements The present study ws supported y the Ntionl Reserch Foundtion of Kore grnt funded y the Kore government (no. 531-2006-1-C00064 nd no. 2011-0000912). None of the uthors hd ny conflict of interest. S.-J. C. crried out the experiments nd U. J. J. wrote the originl mnuscript with S.-J. C. M.-S. C. performed sustntil editing s well s the design of the experiment. All uthors reviewed the literture nd contriuted to the finl report. 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