CB1 antagonism exerts specific molecular effects on visceral and subcutaneous fat and reverses liver steatosis in diet-induced obese mice.

Transcription

1 CB1 ntgonism exerts speifi moleulr effets on viserl nd suutneous ft nd reverses liver stetosis in diet-indued oese mie. Tony Jourdn, Louiz Djouti, Lurent Demizieux, Joseph Gresti, Bruno Vergès, Psl Degre To ite this version: Tony Jourdn, Louiz Djouti, Lurent Demizieux, Joseph Gresti, Bruno Vergès, et l.. CB1 ntgonism exerts speifi moleulr effets on viserl nd suutneous ft nd reverses liver stetosis in diet-indued oese mie.. Dietes, Amerin Dietes Assoition, 21, 59 (4), pp <1.2337/d9-1482>. <inserm > HAL Id: inserm Sumitted on 12 Jul 216 HAL is multi-disiplinry open ess rhive for the deposit nd dissemintion of sientifi reserh douments, whether they re pulished or not. The douments my ome from tehing nd reserh institutions in Frne or rod, or from puli or privte reserh enters. L rhive ouverte pluridisiplinire HAL, est destinée u dépôt et à l diffusion de douments sientifiques de niveu reherhe, puliés ou non, émnnt des étlissements d enseignement et de reherhe frnçis ou étrngers, des lortoires pulis ou privés.

2 ORIGINAL ARTICLE CB1 Antgonism Exerts Speifi Moleulr Effets on Viserl nd Suutneous Ft nd Reverses Liver Stetosis in Diet-Indued Oese Mie Tony Jourdn, Louiz Djouti, Lurent Demizieux, Joseph Gresti, Bruno Vergès, nd Psl Degre OBJECTIVE The enefiil effets of the intivtion of endonninoid system (ECS) y dministrtion of ntgonists of the nninoid reeptor (CB) 1 on severl pthologil fetures ssoited with oesity is well demonstrted, ut the reltive ontriution of entrl versus peripherl mehnisms is unler. We exmined the impt of CB1 ntgonism on liver nd dipose tissue lipid metolism in mouse model of diet-indued oesity. RESEARCH DESIGN AND METHODS Mie were fed either with stndrd diet or high-surose high-ft () diet for 19 weeks nd then treted with the CB1-speifi ntgonist SR (1 mg kg 1 dy 1 ) for 6 weeks. RESULTS Tretment with SR redued ft mss, insulin levels, nd liver triglyerides primrily inresed y feeding. Serum diponetin levels were restored fter eing redued in mie. Gene expression of svenger reeptor lss B type I nd hepti lipse ws indued y CB1 lokde nd ssoited with n inrese in HDL-holesteryl ether uptke. Conomitntly, the expression of CB1, whih ws strongly inresed in the liver nd dipose tissue of mie, ws totlly normlized y the tretment. Interestingly, in viserl ut not suutneous ft, genes involved in trnsport, synthesis, oxidtion, nd relese of ftty ids were upregulted y feeding, while this effet ws ounterted y CB1 ntgonism. CLUSIONS A redution in the CB1-medited ECS tivity in viserl ft is ssoited with normliztion of dipoyte metolism, whih my e determining ftor in the reversion of liver stetosis indued y tretment with SR Dietes 59: , 21 Oesity results from n imlne etween energy intke nd expenditure nd is hrterized y inresed ody weight nd norml development of dipose tissue with exessive ft storge (1). Reently, evidene hs umulted for the overtivity of the endonninoid system (ECS) during onditions of unlned energy homeostsis (2). The ECS onsists of the nninoid reeptors (CBs), their endogenous lignds (the endonninoids), nd the enzyme proteins tlyzing the endonninoid formtion From the Unité Mixte de Reherhe 866 Institut Ntionl de l Snté etdel Reherhe Médile Université de Bourgogne, Tem Physiopthology of Dyslipidemi, Fulty of Sienes Griel, Dijon, Frne. Corresponding uthor: Psl Degre, psl.degre@u-ourgogne.fr. Reeived 7 Otoer 29 nd epted 15 Jnury 21. Pulished hed of print t on 28 Jnury 21. DOI: /d y the Amerin Dietes Assoition. Reders my use this rtile s long s the work is properly ited, the use is edutionl nd not for profit, nd the work is not ltered. See -n-nd/3./ for detils. The osts of pulition of this rtile were defryed in prt y the pyment of pge hrges. This rtile must therefore e herey mrked dvertisement in ordne with 18 U.S.C. Setion 1734 solely to indite this ft. nd degrdtion (3). Ativtion of entrl CB1 reeptors lerly promotes food intke nd weight gin (4 6). Aordingly, phrmologil ntgonism of CB1 hs een shown to improve severl pthologil fetures ssoited with oesity, inluding overweight, hyperinsulinemi, insulin resistne, hyperglyemi, nd dyslipidemi in oese rodents (7 9) nd humns (1,11). Even if the redution in food intke indued y entrl CB1 lokde my e the min initil use of ody weight loss nd ssoited enefiil effets, severl dt olleted from niml nd humn studies indite tht peripherl CB1 my lso diretly ontrol lipid metolism (12 14). Thus, n tivtion of ECS hs een reently reported in peripherl tissues of niml models of oesity (15,16) nd ssoited with viserl ft oesity in humns (17,18). Consequently, it hs een proposed tht the long-term effets of CB1 ntgonism re resolved y stimultion of energy expenditure nd y peripherl effets relted to dipose tissue, liver, skeletl musle, nd pnres physiology (19 21). In the present work, we tested the effets of CB1 ntgonism on the regultion of the liver nd dipose tissue lipid metolism in mouse model of diet-indued oesity. We first exmined the glol impt of CB1 ntgonism on plsm prmeters nd liver stetosis, whih were primrily ltered y long-term feeding of high-surose high-ft () diet. Next, we exmined whether CB1 intivtion ws ssoited with iohemil nd moleulr ltertions in the liver nd dipose tissue (distinguishing viserl nd suutneous ft depots) tht ould ount for n improvement of liver lipid metolism. RESEARCH DESIGN AND METHODS Offiil Frenh regultions (no ) for the use nd re of lortory nimls were followed throughout the experimentl period. The experimentl protool ws pproved y the lol ethi ommittee for niml experimenttion (no. BX622). Four-week-old C57BL/6 mle mie (Elevge Jnvier, Le Genest Sint Isle, Frne) were housed in individul plsti ges nd dpted to stndrd diet (AO4; UAR, Epiny-sur-Orge, Frne) for 1 week. A series of mie ws mintined on the stndrd diet ( group; n 5), while nother series ws sujeted to n diet ontining sein 2%, orn strh 13%, surose 29.3%, ellulose 5%, mltodextrin 2.2%, lrd 2%, soy oil 2.5%, minerl 25B SAFE 7%, vitmin 2 SAFE 1% (ref. 235HF SAFE; Augy, Frne). After 19 weeks, nimls tht were not oth overweight nd hyperinsulinemi were exluded from the study. Seleted mie were mintined on n diet nd reeived orlly either 1 mg kg 1 dy 1 of SR ( SR series; n 14) or vehile ( series; n 1). The CB1-speifi ntgonist SR (Rimonnt) ws supplied y snofiventis (Pris, Frne). Animls hd free ess to fresh food nd wter throughout the experimentl period. Mie were food deprived 4 h efore nesthesi with ketmine/xylzine (7.5 mg 1mg 1 1 g ody wt 1 ) nd tissue hndling. Epididyml nd inguinl ft were surgilly removed s representtives of viserl nd suutneous ft, respetively (22). Tissue smples were frozen in liquid nitrogen pending further nlyses. Serum nd tissue prmeters. Serum prmeters were determined using ommeril kits (gluose RTU, TG PAP15, nd holesterol RTU from 926 DIABETES, VOL. 59, APRIL 21 dietes.dietesjournls.org

3 T. JOURDAN AND ASSOCIATES SR Week FIG. 1. Evolution of ody weight during the indution period of oesity nd during the tretment with CB1 ntgonist. C57BL/6J mie were fed n diet (42.3% rohydrtes, 22.5% lipids) for 25 weeks reeiving orlly during the lst 6 weeks either 1 mg kg 1 dy 1 of the CB1-speifi ntgonist SR ( SR; n 14) or the vehile (; n 1). In prllel, series of mie ws mintined on ontrol diet (; n 5). Results re expressed s mens SE. Results of sttistil nlysis were indited t weeks 2 nd 26, vlues with different supersript letters (,, ) re sttistilly different t P <.5. BioMérieux [Mry l Etoile, Frne] for gluose, triglyerides, nd holesterol ssy, respetively; nonesterified ftty id C from Wko Pure Chemil Industries [Rihmond, VA] for free ftty id [FFA] ssy; nd mouse insulin nd diponetine enzyme-linked immunosorent ssy kits from ACys [Pris, Frne]). Liver mlonyl-coa onentrtion ws determined y highpeformne liquid hromtogrphy s previously desried (23). Liver totl lipids were extrted ording to the method of Folh et l. (24). After mixing thoroughly, 1. ml of orgni phse ws trnsferred to len tue ontining 1 ml of 1% Triton X-1 in hloroform nd dried using nitrogen. The residue ws resoluilized in.25 ml distilled wter nd used for the determintion of triglyerides nd holesterol s in serum. For determintion of diponetin ontent in dipose tissue, smples were homogenized in 1 volume of PBS. After entrifugtion (1 min t 12,g, 4 C), the superntnts were refully olleted through the ft ke, diluted to 1/4, in PBS, nd used for diponetine mesurements s in serum. Ftty id oxidtion nd polipoprotein A nd B seretion. Freshly removed livers from five nd five SR mie were slied using Brendel/Vitron slier (Tuson, AZ), nd thin slies were used to mesure [1-14 C] plmiti id oxidtion nd polipoprotein (po) A nd B seretion s previously desried (25). [ 3 H]-holesteryl ether-hdl uptke. Liver slies, prepred s desried ove, were lso intended for HDL uptke. First, n HDL frtion ws isolted from humn plsm y sequentil flottion ultrentrifugtions (26). HDL ws rdioleled with [ 3 H]-holesteryl ether (CE) omining [ 3 H]holesteryl hedyl ether with L--phosphtidylholine nd utylhydroxytoluene in 5:1:6 molr rtio nd soniting to form liposomes. HDL-[ 3 H]CE ws otined y ddition of liposomes to the HDL frtion in presene of lipoprotein-free plsm, s soure of CE trnsfer protein, fter n overnight inution t 37 C under light gittion. Leled HDL ws seprted from remining liposomes y n other sequentil flottion ultrentrifugtion nd wshed twie in solution of potssium romide (density 1.21). Finlly, HDL-[ 3 H]CE ws liquoted nd stored t 8 C until used. Mesurement of the uptke ws rried out t 37 C y inuting two liver slies in 1 ml of Willim s medium E ontining 4 g proteins (.3 mci of HDL-[ 3 H]CE) under slight gittion. After 3 h, slies were removed from medium, wshed three times, nd homogenized in 4 ml PBS with mini-edeter (BioSpe Produts, Brtlesville, OK). The rdiotivity reovered in the homogente ws finlly estimted, representing the mount of HDL uptken y the liver ells. Gene expression. Totl mrna from liver nd dipose tissue were extrted with Tri-Regent (Euromedex, Souffelweyersheim, Frne) nd reverse trnsripted using the Isript DNA kit (Bio-Rd, Mrnes-L-Coquette, Frne). Rel-time PCR ws performed s desried previously (27) using Bio-Rd icyler iq. The sequenes of forwrd nd reverse primers used for the mplifition re presented in the online supplementl Tle 1 (ville t Sttistil nlysis. Results re expressed s mens SE. Dt were nlyzed sttistilly using the Kruskl-Wllis nonprmetri test. Differenes were onsidered signifint t P.5. RESULTS Body nd orgn weights. Bseline ody weights of the three groups of mie were omprle. After 19 weeks, ody weights of nimls were signifintly higher thn tht of ontrol mie (Fig. 1). From week 2 to 26, the ody weight of SR mie rpidly deresed to eome similr to tht of mie t week 26 (Fig. 1). Consistent with tht, the msses of oth epididyml nd inguinl ft pds differed in the order SR mie (Tle 1). Conomitntly, the liver weight ws greter in nd less in SR thn in mie (Tle 1). Serum nd liver prmeters. At the end of the experiment, serum gluose onentrtion of mie ws not different from mie despite n inrese in insulin levels, inditing tht the mie were in the erly stge of developing insulin resistne (Tle 1). Likewise, FFAs nd totl holesterol levels were higher in thn in mie. Surprisingly, mie hd 5% lower plsm triglyeride levels thn mie, suggesting n inrese in triglyeride lerne y the liver nd dipose tissue. In prllel with ft mss expnsion, serum diponetin levels were less in mie fed with the diet thn in ontrol mie. Interestingly, insulin nd diponetin levels in SR mie were not different from the ontrol group (Tle 1). Serum FFA onentrtion ws less in SR thn in mie, while gluose, triglyerides, nd holesterol levels did not differ etween these groups. In the liver, dministrtion of diet indued stetosis with fivefold inrese in triglyeride dietes.dietesjournls.org DIABETES, VOL. 59, APRIL

4 CB1 ANTAGONISM IN DIET-INDUCED OBESE MICE TABLE 1 Effets of CB1 ntgonism on ody omposition, serum, nd liver prmeters group group SR group n Orgn weight Liver (g) * Epididyml ft (g).67.14* Inguinl ft (g).31.6* * Serum Gluose (mg/ml) Insulin (ng/ml).4.3* * Adiponetin ( g/ml) * * FFAs (mmol/l).3.4* Triglyerides (mg/ml).74.6* Totl holesterol (mg/ml) 1.9.4* Liver Glyogen (mg/g) * Triglyerides (mg/g) * Totl holesterol (mg/g) Mlonyl CoA (nmol/g) * Dt re mens SE. Mie were fed n diet for 25 weeks, reeiving during the lst 6 weeks either 1 mg kg 1 dy 1 of the CB1-speifi ntgonist SR ( SR) or the vehile ( diet). In prllel, series of mie ws mintined on ontrol diet ( group). Mie were food deprived 4 h efore tissue hndling. *,, Sttistilly different t P.5. ontent, while totl holesterol ontent remined unhnged. The mlonyl-coa ontent, potent inhiitor of ftty id -oxidtion, ws lso mrkedly inresed. Menwhile, glyogen stores were less in thn in mie, refleting stimultion of glyogenolysis. Interestingly, triglyeride nd mlonyl-coa umultions were prtilly reversed nd glyogen onentrtion further deresed y CB1 ntgonism (Tle 1). Adiponetin ontent in viserl nd suutneous ft. To explore whether the vritions of serum diponetin levels ould orrespond to different prodution of the A µg diponetin/g tissue Viserl ft +SR TABLE 2 Effets of CB1 ntgonism on ftty id oxidtion nd prmeters relted to lipoprotein metolism in liver explnts SR Plmiti id oxidtion (nmol h 1 g protein 1 ) ApoB seretion ( g h 1 g protein 1 ) * ApoA seretion ( g h 1 g protein 1 ) HDL-CE uptke ( 3 H -CE dpm h 1 g protein 1 ) * 1, Dt re mens SE. Thin liver slies ( 2 m) were otined from mie fed n diet nd treted either with 1 mg kg 1 dy 1 of SR ( SR; n 5) or vehile (; n 5). For ftty id oxidtion nd po seretion, slies were inuted t 37 C in oxygented Willim s medium E supplemented with l-rnitine (.5 mmol/l) in the presene of.2 mmol/l of 1-14 C plmiti id (55.5 GBq/mol) omplexed to lumin (ftty id/bsa molr rtio 2.5/1). After 4 h of inution, slies were rinsed with old PBS nd immeditely sumitted to lipid extrtion for ounting of lelled CO 2 nd id-solule produts, while the inution medium ws used for determintion of pob nd poa sereted. Mesurement of HDL uptke ws rried out t 37 C y inuting liver slies with 3 H -CE-HDL under slight gittion for 3 h. Then, slies were wshed nd homogenized in PBS. Rdiotivity reovered in the homogente represented the mount of HDL uptken y the liver ells. *, Sttistilly different t P.5. dipokine y viserl nd suutneous fts, we determined the diponetin ontent in epididyml nd inguinl ft, respetively. The diponetin ontent ws less in oth ft depots of thn mie with more mrked effet in inguinl ft. Remrkly, fter tretment with CB1 ntgonist, the diponetin ontent signifintly inresed in inguinl ft only (Fig. 2). Ftty id oxidtion nd prmeters relted to lipoprotein metolism. The ility of SR to prtilly reverse -indued liver stetosis prompted us to verify whether this effet ws relted to n inresed pity of heptoytes to -oxidize ftty id or to produe lipids nd lipoproteins. Plmiti id -oxidtion rtes mesured in liver explnts did not differ etween nd SR mie (Tle 2). ApoB seretion ws less with SR thn explnts, while poa seretion did not differ etween the two groups. This B µg diponetin/g tissue Suutneous ft +SR FIG. 2. Effet of CB1 ntgonism on diponetin onentrtion in viserl (A) nd suutneous (B) ft. Mie were fed n diet for 25 weeks reeiving during the lst 6 weeks either 1 mg kg 1 dy 1 of the CB1-speifi ntgonist SR ( SR; n 14) or the vehile (; n 1). In prllel, series of mie ws mintined on ontrol diet (; n 5). Adiponetin onentrtion ws mesured in dipose tissue homogentes prepred s desried in RESEARCH DESIGN AND METHODS. Results re expressed s mens SE. Vlues with different supersript letters (,, ) re sttistilly different t P < DIABETES, VOL. 59, APRIL 21 dietes.dietesjournls.org

5 T. JOURDAN AND ASSOCIATES model ws lso used to determine whether the lokde of CB1 ffeted HDL-CE uptke. The reovery of HDL-CE ws greter in the liver explnts from SR thn mie, suggesting interesting metoli dpttions tht ould ffet lipid nd lipoprotein metolism (Tle 2). Gene expression in liver nd dipose tissue Liver. We first tested the impt of the diet nd of the tretment with SR on CB1 gene expression s n inditor of ECS tivity (Fig. 3). CB1 mrna ws the gretest in mie nd less in SR thn in mie, refleting stimultion of ECS in our mie model of oesity nd n effetive inhiition of this pthwy fter CB1 ntgonist tretment. Then, mrna levels of phosphoenolpyruvte roxykinse (PEPCK) nd gluose-6- phosphtse (G6P) were mesured s n inditor of liver insulin resistne. The inverse reltionship existing etween insulin levels nd expression levels of this two enzymes suggested tht insulin still hd the ility to ontrol their trnsription. In line with possile impt of CB1 ntgonism on lipoprotein nd holesterol metolism, we mesured the mrna expression of hepti lipse nd svenger reeptor lss B type I (SR-BI) in the liver. The expression levels of these two genes were the lowest in mie nd did not differ from in SR mie. Besides, mrna levels of the lipogeni enzymes etyl- CoA roxylse (ACC) 1 nd 2, nd ftty id synthse (FAS) were ll mrkedly less in thn in mie. Interestingly, the expression of oth ACC isoforms ws higher in the liver of SR thn mie, while ntgonism of CB1 hd no effet on FAS expression. The expression of steroyl-coa desturse (SCD)-1, whih onverts sturted ftty ids into monounsturted ftty ids, ws higher in nd less in SR thn in mie, suggesting tht the inhiition of ECS ould hve limited the high prodution of monounsturted ftty ids primrily indued y feeding. Besides, neither the diet nor the tretment modified the trnsript levels of rnitine plmitoyltrnsferse (CPT)-I, the rte-limiting enzyme of long-hin ftty id -oxidtion. Adipose tissue. CB1 expression ws lso indued in oth viserl nd suutneous dipose tissue of ompred with mie (Fig. 4A nd B). In oese mie, SR tretment ws effiient in reduing CB1 expression in the two tissues. The expression of tumor nerosis ftor (TNF)-, n inflmmtory ytokine ssoited with insulin resistne, ws higher in oth viserl nd suutneous ft of thn mie. It is noteworthy tht the TNF- indution ws drmtilly higher in viserl thn in suutneous ft (24-fold vs. 4.8-fold, respetively). Likewise, in SR mie, TNF- mrna levels were not different from mie in suutneous ft nd were only hlf tht of mie in viserl ft. In ddition, ompred with feeding, feeding gve rise to fourfold inrese in the expression of the 2 isoform of the peroxisome prolifertor tivted reeptor (PPAR) 2 in viserl ft, while this indution ws only twofold in SR mie (Fig. 4A). In suutneous dipoytes, PPAR 2 expression ws surprisingly the highest in SR mie, while the two other groups did not differ eh other (Fig. 4B). Conomitntly, the expression of genes relted to uptke (ftty id trnslose [FAT]/ CD36), lipolysis (hormone-sensitive lipse [HSL]), -oxidtion (CPT-I), nd lipogenesis (FAS nd ACC1) ws strongly higher in viserl ft of thn mie (Fig. 4A). Conversely, in suutneous deposits of mie, the expression of these genes ws either less thn mie (FAS nd ACC1) or unhnged (FAT/CD36, HSL, nd CPT-I), suggesting different sensitivity nd funtion of suutneous versus viserl dipoytes in onditions of insulin resistne (Fig. 4B). Interestingly, in viserl ft, ll genes tht were upregulted y feeding were signifintly downregulted fter tretment with CB1 ntgonist exept FAT/CD36 (Fig. 4A). DISCUSSION In this study, the effets of CB1 ntgonism were tested on mie previously exposed to long-term diet (19 weeks) with lipid ontent nd ftty id omposition nerly similr to the humn Western diet. Administrtion of diet indued oesity, liver ft umultion nd peripherl insulin resistne s indited y the elevtion of plsm insulin nd FFA levels. A signifint numer of experimentl reports desrie enefiil effets of CB1 ntgonism on insulin resistne nd ftty liver in mie nd humns, nd these dt strongly indite tht the ECS hs mjor role in the regultion of lipid metolism not only t the entrl ut lso t the peripherl level (rev. in 28). From our mouse model of oesity, we provided further evidene tht CB1 lokde uses peripherl metoli nd moleulr hnges in liver nd dipose tissue ssoited with the reversion of ftty liver. We prtiulrly showed tht the lipid metolism of viserl nd suutneous dipoytes ws differently regulted in response to diet-indued oesity nd to CB1 ntgonism. Effets of CB1 ntgonism on liver lipid metolism. Our findings lerly indite tht the strong upregultion of liver CB1 primrily indued y n diet is fully reversed y the tretment with SR141716, suggesting tht the metoli improvements oserved ould e medited y the lokde of these reeptors. This onept is supported y other studies using CB1 / mie, demonstrting tht ECS overtivity ours in the liver of nimls fed high-ft diet nd tht hepti CB1 re required for the development of diet indued stetosis (14,29). In line with this, the normliztion of liver prmeters relted to rohydrte nd lipid metolism suh s PEPCK, G6P, ACC, nd SCD-1 mrna levels fter tretment with CB1 ntgonist strongly suggests tht these dpttions orrespond to normliztion of liver insulin responsiveness s evoked in musles of rimonnt-treted o/o mie (21). The tivtion of hepti CB1 reeptors hs een reently ssoited with n inrese in de novo lipogenesis, suggesting tht this metoli pthwy prtiiptes to stetosis development in onditions of ECS overtivity (3). Unlike this finding, we oserved no stimultion of the liver expression of ACC nd FAS in nimls, whih were quite hyperinsulinemi. In the works of Osei-Hyimn et l. (3), the stimultion of ECS onsisted of n ute injetion of CB1 gonist to ontrol nimls, while in our study, ECS tivtion ws indued for muh longer period using n diet. Sine the diet ontined high proportions of sturted ftty ids, it n e hypothesized tht the provision of diet rih in preformed sturted ftty ids led to the redued expression of mrna for lipogeni genes. The inhiitory effet of plmitoyl-coa on ACC demonstrted y Ogiwr et l. (31) supports this onept. The indution of the SCD-1 gene nd the inrese in monounsturted ftty id ontent in the liver of mie (dt not shown) indite tht sturted ftty id delivery to the liver ws inresed. Indeed, in mie, dietes.dietesjournls.org DIABETES, VOL. 59, APRIL

6 CB1 ANTAGONISM IN DIET-INDUCED OBESE MICE RT-PCR produts (ritrry units) CB1 RT-PCR produts (ritrry units) PEPCK RT-PCR produts (ritrry units) G6P RT-PCR produts (ritrry units) +SR HL SR-B1 RT-PCR produts (ritrry units) CPT-I +SR RT-PCR produts (ritrry units) ACC1 RT-PCR produts (ritrry units) ACC FAS +SR +SR +SR +SR +SR +SR +SR RT-PCR produts (ritrry units) RT-PCR produts (ritrry units) RT-PCR produts (ritrry units) SCD-1 +SR FIG. 3. Effet of CB1 ntgonism on the mrna expression of CB1 nd genes involved in rohydrte nd lipid metolism in the liver. Mie were fed n diet for 25 weeks reeiving during the lst 6 weeks either 1 mg kg 1 dy 1 of the CB1-speifi ntgonist SR ( SR) or the vehile (). In prllel, series of mie ws mintined on ontrol diet (). For eh gene, stndrd urve ws estlished from four DNA dilutions (1/1 1/1,) nd used to determine the reltive gene expression fter normliztion with geometri verge of 18S nd TATA ox inding protein expression. Results re expressed s mens SE (n 5 per group). Vlues with different supersript letters (,, ) re sttistilly different t P <.5. the liver stetosis ppers to e minly due to n enhned delivery of FFAs to the liver rther to n inrese in de novo lipogenesis. Aside from diret effets on the liver, stetosis might hve lso een redued indiretly y the limittion of the influx of ftty ids originting from dipose tissue. The gene expression profile of viserl dipose tissue is onsistent with n hypertivtion of lipid metolism s suggested y the strong upregultion 93 DIABETES, VOL. 59, APRIL 21 dietes.dietesjournls.org

7 T. JOURDAN AND ASSOCIATES A RT-PCR produts (ritrry units) Viserl ft CB1 TNF-α PPARγ FAT/CD SR RT-PCR produts (ritrry units) +SR RT-PCR produts (ritrry units) +SR RT-PCR produts (ritrry units) +SR RT-PCR produts (ritrry units) HSL +SR RT-PCR produts (ritrry units) FAS ACC1 CPT-I SR RT-PCR produts (ritrry units) +SR RT-PCR produts (ritrry units) +SR B RT-PCR produts (ritrry units) Suutneous ft CB1 +SR RT-PCR produts (ritrry units) TNF-α +SR RT-PCR produts (ritrry units) PPARγ +SR RT-PCR produts (ritrry units) FAT/CD3 6 +SR RT-PCR produts (ritrry units) HSL +SR RT-PCR produts (ritrry units) FAS +SR RT-PCR produts (ritrry units) ACC1 +SR RT-PCR produts (ritrry units) CPT-I +SR FIG. 4. Effet of CB1 ntgonism tretment on the mrna expression of CB1 nd genes involved in dipoyte metolism in epididyml (A) nd inguinl (B) ft. Mie were fed n diet for 25 weeks reeiving during the lst 6 weeks either 1 mg kg 1 dy 1 of the CB1-speifi ntgonist SR ( SR) or the vehile (). In prllel, series of mie ws mintined on ontrol diet (). For eh gene, stndrd urve ws estlished from four DNA dilutions (1/1 1/1,) nd used to determine the reltive gene expression fter normliztion with geometri verge of 18S nd TATA ox inding protein expression. Results re expressed s mens SE (n 5 per group). Vlues with different supersript letters (,, ) re sttistilly different t P <.5. dietes.dietesjournls.org DIABETES, VOL. 59, APRIL

8 CB1 ANTAGONISM IN DIET-INDUCED OBESE MICE of genes involved in trnsport, synthesis, oxidtion, nd relese of ftty ids. Altogether, these dt suggest tht the reversion of liver stetosis indued y the tretment with CB1 ntgonist ws ssoited with n improvement of dipose tissue metolism. In line with n improvement of rdiovsulr risk in type 2 dieti ptients treted with rimonnt (11,32), our findings support the possiility tht CB1 ntgonism is ssoited with n ltertion of liver HDL tolism. Previous studies (33) showed tht overexpression of SR-BI in the liver, while reduing plsm HDL holesterol levels, redued theroslerosis in mie, suggesting tht hepti SR-BI overexpression my promote reverse holesterol trnsport. Aordingly, the inrese in SR-BI nd hepti lipse expression indued y CB1 ntgonism my e ssoited with modifition of HDL size nd kinetis (34) nd therey explin the inrese in HDL-CE uptke oserved in our model of liver slies. Additionl studies re urrently under investigtion to lerly identify the diret effets of CB1 ntgonism on liver lipid metolism. Effets of CB1 ntgonism on viserl ft. Reently, evidene hs umulted from niml nd humn studies (17,35 37) tht oesity is lso ssoited with overtivtion of ECS in viserl ft. Conordnt findings from this study nd from literture support the view tht CB1 lokde exerts speifi effets on viserl ft metolism tht ould e ssoited with the redution of liver triglyeride ontent. Hene, the oordinted upregultion of genes ting t different levels of the lipogeni pthwy nd tht of the nuler tivtor PPAR strongly suggested tht n diet fvored triglyeride synthesis nd therey formtion of enlrged viserl ft deposits. Adipoyte hypertrophy in oesity is onseutive to defiit in dipogenesis (38), nd the limittion of ft stores would promote etopi lipid deposition in liver nd skeletl musle, leding to deresed insulin tion in these tissues (39). Remrkly, the ft tht CB1 ntgonism totlly or prtilly normlized the expression levels of lipogeni genes in dipoytes my limit the umultion of intrellulr lipid droplets nd give rise to smller ells nd redution of viserl ft mss s lso suggested in (4). The derese in HSL expression onseutive to CB1 ntgonism is of prtiulr importne sine exessive HSL-dependent ft lipolysis leds to n inresed relese of FFAs into the irultion, whih in turn hs deleterious effets on insulin sensitivity (41). It hs een suggested reently tht oesity-indued inflmmtion of dipose tissue my diretly tivte ECS (42). This ould result in protetive response ginst inflmmtion s desried in olon (43). From our reserh, it ppers tht ECS tivtion indued y n diet is lso ssoited with n inrese in TNF- in dipose tissue. This intertion etween inflmmtion nd ECS needs to e further explored to determine whether inflmmtion uses ECS tivtion or vie vers. However, the onentrtion-dependent stimultion of lipolysis y TNF- demonstrted in rodent nd humn ft ells is onsidered to e n importnt pthogeneti ftor in the development of insulin resistne nd type 2 dietes (44). Therefore, it is resonle to suggest tht the redution of TNF- expression in viserl dipose tissue of mie treted with CB1 ntgonist is linked to the normliztion of dipoyte metolism nd to underlying effets on lipid nd rohydrte metolism. Effets of n diet nd CB1 ntgonism in suutneous versus viserl ft. This study lso provides new informtion regrding the impt of diet nd susequent CB1 ntgonism on the regultion of lipid metolism in suutneous ompred with viserl dipose tissue. Tken together, our findings give moleulr evidene tht 1) n diet uses deleterious effets in viserl dipose tissue tht were not oserved in suutneous ft nd 2) CB1 lokde is le to reverse the moleulr hnges primrily indued y n diet in viserl dipose tissue nd to exert speifi effets on suutneous dipoytes. These disrepnies in gene regultion etween viserl nd suutneous dipoytes in response to high-ft diet nd CB1 ntgonism re onsistent with different degree of ECS tivtion in these tissues. This onsidertion is supported y severl reent findings (36,37,45,46) inditing differenes in endonninoid levels etween epididyml nd suutneous ft. In ddition, the overexpression of PPAR 2, FAS, nd ACC gene in epididyml ft of oese mie is lso in fvor of the tivtion of ECS in this tissue sine it hs een reported tht CB1 tivtion stimultes lipogenesis y inresing PPAR nd lipogeni enzyme expression in dipoytes nd liver (3,36,47). Colletively, dt suggest tht ECS is more tivted in epididyml thn in suutneous ft in our mie model of oesity nd it n e predited tht ntgonism of CB1 ws more effetive in the tissue presenting elevted levels of endonninoids (36,45). Interestingly, n diet or CB1 ntgonist tretment indued nerly similr effets on the mounts of epididyml nd suutneous ft, suggesting tht moleulr nd metoli differenes oserved re not solely relted to the modifition of the ft depot size. In ontrst, the indution of TNF- expression y n diet ws fr less importnt in suutneous thn in viserl ft, nd the tretment of oese mie with CB1 ntgonist indued the omplete normliztion of TNF- expression only in suutneous ft, wheres inflmmtion remins high in viserl dipoytes. Conomitntly, the normliztion of TNF- mrna levels in suutneous ft is ssoited with n inrese in diponetin ontent in this tissue. An inverse reltionship etween irulting diponetin nd TNF- hs lredy een evoked (48), suggesting tht dipose tissue inflmmtion ould lter diponetin prodution. The inresed expression of PPAR 2 indued y CB1 ntgonism in suutneous ft my lso orrespond to n tivtion of dipoyte differentition (49) nd therey of diponetin seretion (5). In ddition, our findings regrding the diponetin ontent in viserl nd suutneous ft suggest tht the normliztion of diponetin plsm levels indued y CB1 ntgonism my e exlusively ssoited with n inresed prodution of this dipokine y suutneous dipoytes. In onlusion, this study indites tht treting oese mie with CB1 ntgonist exerts enefiil effets on liver stetosis nd vrious lipid prmeters, providing supportive evidene tht the hypertivity of ECS ssoited with oesity ws djusted y the ntgonism of CB1. This notion is further supported y dt from n ongoing study inditing tht CB1 ntgonism exerts no effets on ody weight, ft mss, nd liver triglyeride ontent in ontrol mie (T.J., L.Dj., L.De., J.G., B.V., nd P.D.; personl dt). Our findings re lso onsistent with ontriution of peripherl CB1 nd suggest different degrees of ECS tivity in viserl nd suutneous ft. In this wy, 932 DIABETES, VOL. 59, APRIL 21 dietes.dietesjournls.org

9 T. JOURDAN AND ASSOCIATES the improvement of viserl dipose tissue metolism ppers to e determining ftor for the normliztion of plsm prmeters nd the reversion of liver stetosis. Therefore, future studies should investigte the diret effets of CB1 ntgonism on the liver to preise the respetive implition of ECS nd produts sereted y dipose tissue in the regultion of lipid metolism. ACKNOWLEDGMENTS This work ws supported y grnts from snofi-ventis nd the Région Bourgogne. No other potentil onflits of interest relevnt to this rtile were reported. REFERENCES 1. Tryhurn P, Bettie JH. Physiologil role of dipose tissue: white dipose tissue s n endorine nd seretory orgn. Pro Nutr So 21;6: Mtis I, Di Mrzo V. Endonninoids nd the ontrol of energy lne. Trends Endorinol Met 27;18: Di Mrzo V, Mtis I. Endonninoid ontrol of food intke nd energy lne. Nt Neurosi 25;8: Di Mrzo V, Goprju SK, Wng L, Liu J, Btki S, Jri Z, Fezz F, Miur GI, Plmiter RD, Sugiur T, Kunos G. Leptin-regulted endonninoids re involved in mintining food intke. Nture 21;41: Jmshidi N, Tylor DA. Anndmide dministrtion into the ventromedil hypothlmus stimultes ppetite in rts. Br J Phrmol 21;134: Willims CM, Kirkhm TC. Anndmide indues overeting: medition y entrl nninoid (CB1) reeptors. Psyhophrmology (Berl) 1999; 143: Gry-Boo M, Elhouri G, Glls JF, Jnik P, Mrini P, Rvinet-Trillou C, Chert M, Cruioli N, Pfersdorff C, Roque C, Arnone M, Croi T, Sourie P, Oury-Dont F, Mffrnd JP, Stton B, Lheretz F, Le Fur G, Herert JM, Bensid M. Rimonnt redues oesity-ssoited hepti stetosis nd fetures of metoli syndrome in oese Zuker f/f rts. Heptology 27;46: Poirier B, Bidourd JP, Cdrouvele C, Mrniquet X, Stels B, O Connor SE, Jnik P, Herert JM. The nti-oesity effet of rimonnt is ssoited with n improved serum lipid profile. Dietes Oes Met 25;7: Rvinet Trillou C, Arnone M, Delgorge C, Gonlons N, Kene P, Mffrnd JP, Sourie P. Anti-oesity effet of SR141716, CB1 reeptor ntgonist, in diet-indued oese mie. Am J Physiol Regul Integr Comp Physiol 23;284:R345 R Despres JP, Goly A, Sjostrom L. Effets of rimonnt on metoli risk ftors in overweight ptients with dyslipidemi. N Engl J Med 25;353: Vn Gl LF, Rissnen AM, Sheen AJ, Ziegler O, Rossner S. Effets of the nninoid-1 reeptor loker rimonnt on weight redution nd rdiovsulr risk ftors in overweight ptients: 1-yer experiene from the RIO-Europe study. Lnet 25;365: Engeli S, Jordn J. The endonninoid system: ody weight nd metoli regultion. Clin Cornerstone 26;8(Suppl. 4):S24 S Nogueirs R, Veyrt-Dureex C, Suhnek PM, Klein M, Tshop J, Cldwell C, Woods SC, Wittmnn G, Wtne M, Liposits Z, Fekete C, Reizes O, Rohner-Jenrenud F, Tshop MH. Peripherl, ut not entrl, CB1 ntgonism provides food intke-independent metoli enefits in dietindued oese rts. Dietes 28;57: Osei-Hyimn D, Liu J, Zhou L, Godlewski G, Hrvey-White J, Jeong WI, Btki S, Mrsino G, Lutz B, Buettner C, Kunos G. Hepti CB1 reeptor is required for development of diet-indued stetosis, dyslipidemi, nd insulin nd leptin resistne in mie. J Clin Invest 28;118: Bensid M, Gry-Boo M, Eslngon A, Mffrnd JP, Le Fur G, Oury-Dont F, Sourie P. The nninoid CB1 reeptor ntgonist SR inreses Arp3 mrna expression in dipose tissue of oese f/f rts nd in ultured dipoyte ells. Mol Phrmol 23;63: Mtis I, Petrosino S, Rioppi A, Cpsso R, Izzo AA, Di Mrzo V. Dysregultion of peripherl endonninoid levels in hyperglyemi nd oesity: effet of high ft diets. Mol Cell Endorinol 28;286:S66 S Cote M, Mtis I, Lemieux I, Petrosino S, Almers N, Despres JP, Di Mrzo V. Cirulting endonninoid levels, dominl diposity nd relted rdiometoli risk ftors in oese men. Int J Oes (Lond) 27;31: Di Mrzo V, Cote M, Mtis I, Lemieux I, Arsenult BJ, Crtier A, Pisitelli F, Petrosino S, Almers N, Despres JP. Chnges in plsm endonninoid levels in viserlly oese men following 1 yer lifestyle modifition progrmme nd wist irumferene redution: ssoitions with hnges in metoli risk ftors. Dietologi 29;52: Duvivier VF, Delfoy-Plsse L, Delion V, Lehevlier P, Le Bil JC, Guillot E, Pruniux MP, Glzin AM. Benefiil effet of hroni tretment with rimonnt on pnreti funtion nd et-ell morphology in Zuker Ftty rts. Eur J Phrmol 29;616: Getty-Kushik L, Rihrd AM, Deeney JT, Krwzyk S, Shirihi O, Corkey BE. The CB1 ntgonist rimonnt dereses insulin hyperseretion in rt pnreti islets. Oesity (Silver Spring) 29;17: Liu YL, Connoley IP, Wilson CA, Stok MJ. Effets of the nninoid CB1 reeptor ntgonist SR on oxygen onsumption nd soleus musle gluose uptke in Lep(o)/Lep(o) mie. Int J Oes (Lond) 25;29: Kim JY, vn de Wll E, Lplnte M, Azzr A, Trujillo ME, Hofmnn SM, Shrw T, Durnd JL, Li H, Li G, Jeliks LA, Mehler MF, Hui DY, Deshies Y, Shulmn GI, Shwrtz GJ, Sherer PE. Oesity-ssoited improvements in metoli profile through expnsion of dipose tissue. J Clin Invest 27;117: Degre P, Demizieux L, Gresti J, Chrdigny JM, Seedio JL, Clouet P. Hepti stetosis is not due to impired ftty id oxidtion pities in C57BL/6J mie fed the onjugted trns-1,is-12-isomer of linolei id. J Nutr 24;134: Folh J, Lees M, Slone Stnley GH. A simple method for the isoltion nd purifition of totl lipides from niml tissues. J Biol Chem 1957;226: Jourdn T, Djouti L, Demizieux L, Gresti J, Verges B, Degre P. Liver rohydrte nd lipid metolism of insulin-defiient mie is ltered y trns-1, is-12 onjugted linolei id. J Nutr Persegol L, Verges B, Foiss M, Gmert P, Duvillrd L. Inility of HDL from type 2 dieti ptients to ountert the inhiitory effet of oxidised LDL on endothelium-dependent vsorelxtion. Dietologi 26;49: Degre P, Moindrot B, Mohmed I, Gresti J, Du ZY, Chrdigny JM, Seedio JL, Clouet P. Upregultion of liver VLDL reeptor nd FAT/CD36 expression in LDLR-/- pob1/1 mie fed trns-1,is-12 onjugted linolei id. J Lipid Res 26;47: Vettor R, Pgno C. The role of the endonninoid system in lipogenesis nd ftty id metolism. Best Prt Res Clin Endorinol Met 29;23: Rvinet Trillou C, Delgorge C, Menet C, Arnone M, Sourie P. CB1 nninoid reeptor knokout in mie leds to lenness, resistne to diet-indued oesity nd enhned leptin sensitivity. Int J Oes Relt Met Disord 24;28: Osei-Hyimn D, DePetrillo M, Pher P, Liu J, Rdev S, Btki S, Hrvey-White J, Mkie K, Offertler L, Wng L, Kunos G. Endonninoid tivtion t hepti CB1 reeptors stimultes ftty id synthesis nd ontriutes to diet-indued oesity. J Clin Invest 25;115: Ogiwr H, Tne T, Nikw J, Num S. Inhiition of rt-liver etyloenzyme-a roxylse y plmitoyl-oenzyme A: formtion of equimolr enzyme-inhiitor omplex. Eur J Biohem 1978;89: Pi-Sunyer FX, Aronne LJ, Heshmti HM, Devin J, Rosenstok J. Effet of rimonnt, nninoid-1 reeptor loker, on weight nd rdiometoli risk ftors in overweight or oese ptients: RIO-North Ameri: rndomized ontrolled tril. JAMA 26;295: Kozrsky KF, Donhee MH, Glik JM, Krieger M, Rder DJ. Gene trnsfer nd hepti overexpression of the HDL reeptor SR-BI redues theroslerosis in the holesterol-fed LDL reeptor-defiient mouse. Arteriosler Throm Vs Biol 2;2: Nijstd N, Wiersm H, Gutier T, vn der Giet M, Mugeis C, Tietge UJ. Svenger reeptor BI-medited seletive uptke is required for the remodeling of high density lipoprotein y endothelil lipse. J Biol Chem 29;284: Bluher M, Engeli S, Kloting N, Berndt J, Fsshuer M, Btki S, Pher P, Shon MR, Jordn J, Stumvoll M. Dysregultion of the peripherl nd dipose tissue endonninoid system in humn dominl oesity. Dietes 26;55: Mtis I, Gonthier MP, Orlndo P, Mrtidis V, De Petroellis L, Cervino C, Petrosino S, Horeu L, Festy F, Psquli R, Rohe R, Mj M, Pgotto U, Monteleone P, Di Mrzo V. Regultion, funtion, nd dysregultion of endonninoids in models of dipose nd et-pnreti ells nd in oesity nd hyperglyemi. J Clin Endorinol Met 26;91: Strowiz KM, Cristino L, Mtis I, Cpsso R, Rioppi A, Izzo AA, Di Mrzo V. Endonninoid dysregultion in the pnres nd dipose tissue of mie fed with high-ft diet. Oesity (Silver Spring) 28;16: Ndler ST, Stoehr JP, Shueler KL, Tnimoto G, Yndell BS, Attie AD. The dietes.dietesjournls.org DIABETES, VOL. 59, APRIL

10 CB1 ANTAGONISM IN DIET-INDUCED OBESE MICE expression of dipogeni genes is deresed in oesity nd dietes mellitus. Pro Ntl Ad Si U S A 2;97: Heilronn L, Smith SR, Rvussin E. Filure of ft ell prolifertion, mitohondril funtion nd ft oxidtion results in etopi ft storge, insulin resistne nd type II dietes mellitus. Int J Oes Relt Met Disord 24;28(Suppl. 4):S12 S21 4. Jilo O, Rvinet-Trillou C, Arnone M, Buisson I, Bries E, Pelerux A, Penrier G, Sourie P, Le Fur G, Gliegue S, Csells P. The CB1 reeptor ntgonist rimonnt reverses the diet-indued oesity phenotype through the regultion of lipolysis nd energy lne. Fse J 25;19: Arner P. Insulin resistne in type 2 dietes: role of ftty ids. Dietes Met Res Rev 22;18(Suppl. 2):S5 S9 42. Kempf K, Hetor J, Strte T, Shwrzloh B, Rose B, Herder C, Mrtin S, Algenstedt P. Immune-medited tivtion of the endonninoid system in viserl dipose tissue in oesity. Horm Met Res 27;39: Mss F, Mrsino G, Hermnn H, Cnnih A, Monory K, Crvtt BF, Ferri GL, Siev A, Storr M, Lutz B. The endogenous nninoid system protets ginst oloni inflmmtion. J Clin Invest 24;113: Run H, Lodish HF. Insulin resistne in dipose tissue: diret nd indiret effets of tumor nerosis ftor-lph. Cytokine Growth Ftor Rev 23;14: D Eon TM, Piere KA, Roix JJ, Tyler A, Chen H, Teixeir SR. The role of dipoyte insulin resistne in the pthogenesis of oesity-relted elevtions in endonninoids. Dietes 28;57: Izzo AA, Pisitelli F, Cpsso R, Aviello G, Romno B, Borrelli F, Petrosino S, Di Mrzo V. Peripherl endonninoid dysregultion in oesity: reltion to intestinl motility nd energy proessing indued y food deprivtion nd re-feeding. Br J Phrmol 29;158: Pgno C, Pilon C, Clgno A, Urnet R, Rossto M, Miln G, Binhi K, Rizzuto R, Bernnte P, Federspil G, Vettor R. The endogenous nninoid system stimultes gluose uptke in humn ft ells vi phosphtidylinositol 3-kinse nd lium-dependent mehnisms. J Clin Endorinol Met 27;92: Lu DC, Dhillon B, Yn H, Szmitko PE, Verm S. Adipokines: moleulr links etween oesity nd theroslerosis. Am J Physiol Hert Cir Physiol 25;288:H231 H Shoonjns K, Stels B, Auwerx J. The peroxisome prolifertor tivted reeptors (PPARS) nd their effets on lipid metolism nd dipoyte differentition. Biohim Biophys At 1996;132: Gustfson B, Jk MM, Cushmn SW, Smith U. Adiponetin gene tivtion y thizolidinediones requires PPAR gmm 2, ut not C/EBP lph-evidene for differentil regultion of the P2 nd diponetin genes. Biohem Biophys Res Commun 23;38: DIABETES, VOL. 59, APRIL 21 dietes.dietesjournls.org