A New Selective Peroxisome Proliferator-Activated Receptor γ Antagonist with Antiobesity and...

A New Selective Peroxisome Proliferator-ActivatedReceptor � Antagonist with Antiobesity andAntidiabetic Activity

JENNIFER RIEUSSET*†, FETHI TOURI*, LILIANE MICHALIK, PASCAL ESCHER‡,BEATRICE DESVERGNE, ERIC NIESOR, AND WALTER WAHLI

Institut de biologie animale (J.R., L.M., P.E., B.D., W.W.), Universite de Lausanne, CH-1015Lausanne, Switzerland; and Ilex Oncology (F.T., E.N.), CH-1290 Versoix/Geneva, Switzerland

Peroxisome proliferator-activated receptor �(PPAR-�) plays a key role in adipocyte differentia-tion and insulin sensitivity. Its synthetic ligands, thethiazolidinediones (TZD), are used as insulin sen-sitizers in the treatment of type 2 diabetes. Thesecompounds induce both adipocyte differentiationin cell culture models and promote weight gain inrodents and humans. Here, we report on the iden-tification of a new synthetic PPAR� antagonist, thephosphonophosphate SR-202, which inhibits bothTZD-stimulated recruitment of the coactivator ste-roid receptor coactivator-1 and TZD-induced tran-scriptional activity of the receptor. In cell culture,SR-202 efficiently antagonizes hormone- and TZD-induced adipocyte differentiation. In vivo, decreas-ing PPAR� activity, either by treatment with SR-202

or by invalidation of one allele of the PPAR� gene,leads to a reduction of both high fat diet-inducedadipocyte hypertrophy and insulin resistance.These effects are accompanied by a smaller size ofthe adipocytes and a reduction of TNF� and leptinsecretion. Treatment with SR-202 also dramaticallyimproves insulin sensitivity in the diabetic ob/obmice. Thus, although we cannot exclude that itsactions involve additional signaling mechanisms,SR-202 represents a new selective PPAR� antag-onist that is effective both in vitro and in vivo.Because it yields both antiobesity and antidiabeticeffects, SR-202 may be a lead for new compoundsto be used in the treatment of obesity and type 2diabetes. (Molecular Endocrinology 16: 2628–2644,2002)

THE PEROXISOME PROLIFERATOR-activated re-ceptors (PPARs, NR1C1, NR1C2, NR1C3; Ref. 1)

are members of the nuclear receptor superfamily. Theyfunction as heterodimers with the receptor of 9-cis-retinoic acid (RXR, NR2B; Ref. 1), and bind to specificperoxisome proliferator response elements (PPREs) toregulate transcription of their target genes. Three dif-ferent PPAR genes have been characterized, whichgive rise to four distinct proteins (�, �/�, �1, and �2).Although the PPARs were first cloned as orphan mem-bers of the nuclear receptor gene family, rapidprogress has been made in their functional analysis.This research contributed to a better understanding ofthe importance of fatty acids as hormones and has

established the PPARs as molecular targets for thedevelopment of drugs to treat human diseases (2).

PPAR� is expressed predominantly in adipose tis-sue, where it is known to play a critical role in adipo-cyte differentiation and fat deposition (3, 4). It can beactivated by arachidonic acid-metabolites generatedby the cyclooxygenase and lipooxygenase pathways(5–7) and by fatty acid-derived components releasedfrom oxidized low density lipoproteins (8). The antidi-abetic thiazolidinediones (TZDs), currently used as in-sulin sensitizers, are the best synthetic PPAR� ligandsin terms of specificity and affinity, although the mech-anism by which activation of PPAR� leads to an im-provement of insulin action is still debated (3, 4, 9).Moreover, deletion of one allele of PPAR� was recentlyshown to protect mice from high fat diet (HFD)-induced adipocyte hypertrophy and insulin resistance,underlying the complexity of the role of PPAR� ininsulin sensitivity (10).

The most extensively studied therapeutic utility forPPAR� has been in the treatment of type 2 diabetes.TZDs were shown to enhance the sensitivity of targettissues to insulin and to reduce plasma glucose, lipid,and insulin levels in animal models of type 2 diabetes,as well as in human (11, 12). However, due to theirability to induce gene expression in adipocytes and toenhance adipocyte differentiation (13), TZDs also havenegative side-effects. Indeed, they induce adipocytedifferentiation and weight gain in patients with already

Abbreviations: aP2, Adipocyte fatty acid binding protein;BADGE, bisphenol A diglycidyl ether; BAT, brown adiposetissue; CARLA, coactivator-dependent receptor ligand assay;CAT, chloramphenicol acetyl transferase; FAT/CD36, fattyacid translocase; FFA, free fatty acids; FXR, farnesoid Xreceptor; HFD, high fat diet; h, human; IBMX, 3-isobutyl-1-methylxanthine; LDH, lactate deshydrogenase; LPL, lipopro-tein lipase; m, murine; PPAR�, peroxisome proliferator-acti-vated receptor �; PPREs, peroxisome proliferator responseelements; RXR, receptor of 9-cis-retinoic acid; SD, standarddiet; SR-202, dimethyl �-(dimethoxyphosphinyl)-p-chloro-benzyl phosphate; SRC-1, steroid receptor coactivator-1;SREBP-1c, sterol regulatory element-binding protein 1c;TZDs, thiazolidinediones; WAT, white adipose tissue; wt,wild-type.

0888-8809/02/$15.00/0 Molecular Endocrinology 16(11):2628–2644Printed in U.S.A. Copyright © 2002 by The Endocrine Society

doi: 10.1210/me.2002-0036

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serious metabolic disorders (14, 15). For this reason,important efforts are made to identify new PPAR�modulators having antidiabetic action, without pro-moting weight increase. Recently, a novel PPAR� li-gand (GW 0072), which is a partial agonist in transac-tivation assays, was shown to inhibit adipocytedifferentiation in cell culture (16). In addition, new an-tagonists for PPAR� are being characterized: bisphe-nol A diglycidyl ether (BADGE) inhibits adipocyte dif-ferentiation (17), PD 068235 also blocks adipocytedifferentiation but does not revert the phenotype ofterminally differentiated adipocytes (18), LG 100641blocks adipocyte differentiation as well, and stimu-lates glucose uptake in 3T3-L1 adipocytes (19). So far,none of these inhibitors has been tested in vivo toverify whether they may prevent obesity and reduceinsulin resistance, which would delay the onset of type2 diabetes.

Here, we report that SR-202 [dimethyl �-(dime-thoxyphosphinyl)-p-chlorobenzyl phosphate] is anovel PPAR�-specific antagonist that blocks adipo-cyte differentiation induced either by thiazolidinedio-nes or by the combination of dexamethasone, insulin,and 3-isobutyl-1-methylxanthine (IBMX). We have in-vestigated the activity of this antagonist in vivo anddemonstrate that blocking PPAR� activity gives rise toa decrease in fat deposit and increase in insulin sen-sitivity. This new PPAR� antagonist may serve to de-velop compounds that will be more beneficial in thetreatment of obesity and type 2 diabetes than thiazo-lidinediones because it yields both antiobesity andantidiabetic effects.

RESULTS

SR-202 Is a Specific Antagonist of PPAR�

Using a transcriptional reporter assay, we have iden-tified a compound, SR-202 (Fig. 1A), that selectivelymodulates PPAR� transcriptional activity. HeLa cellswere cotransfected with full-length PPAR�, PPAR�, orPPAR� cDNA and a single PPRE-containing reporterplasmid. Alternatively, cells were also cotransfectedwith farnesoid X receptor (FXR) encoding plasmid andan FXR response element containing reporter plasmid.Cells were then treated with specific ligands of each ofthese nuclear receptors in the presence or absence ofincreasing concentrations of SR-202. As shown in Fig.1, B–E, SR-202 itself did not cause any significantchange in the basal transcriptional activity in the pres-ence of PPAR�, PPAR�, FXR, and PPAR�, but ab-sence of agonists of these receptors. Similarly, SR-202 did not inhibit specific ligand-stimulated PPAR�,PPAR�, and FXR transcriptional activities (Fig. 1, B–D).However, SR-202 showed a dose-dependent attenu-ation of troglitazone-induced PPAR� transcriptionalactivity, with an IC50 of 140 �M (Fig. 1E). Thus, SR-202shows selectivity both among the PPAR family mem-bers and other nuclear receptors because it specifi-

cally inhibits the PPAR� activity. This inhibition is notdue to cytotoxic effects because, after a 36-h expo-sure of the cells to 400 �M SR-202, the lactate dehy-drogenase (LDH) activity released in the medium neverexceeded 6% of the total cellular LDH activity (datanot shown).

We next examined whether SR-202 is a ligand ofPPAR� using the coactivator-dependent receptor li-gand assay (CARLA) described previously (7). CARLAsare based on the capacity of a ligand to stimulate theinteraction of nuclear receptors with a coactivator,here steroid receptor coactivator-1 (SRC-1). As ex-pected, the known potent ligand of PPAR�, BRL49653 (Rosiglitazone) induced interaction of PPAR�with SRC-1 in a dose-dependent manner (Fig. 2A).SR-202 alone did not promote any interaction be-tween PPAR� and SRC-1, indicating the absence ofagonist activity of the compound (Fig. 2B). However,coincubation of BRL 49653 (0.5 �M) with increasingconcentrations of SR-202 (50–400 �M) led to a dose-dependent decrease in the recruitment of SRC-1 tothe receptor (Fig. 2B). At the maximal concentration ofSR-202 (400 �M), BRL 49653-stimulated SRC-1 bind-ing to PPAR� was decreased by 75%, suggesting thatthis compound could displace the activator and al-most completely abolish the recruitment of SRC-1 tothe receptor. The association of PPAR� and PPAR�with SRC-1 was not affected by SR-202, confirmingthe specificity of SR-202 for PPAR� (data not shown).Thus, SR-202 is able to both interact specifically withPPAR� and inhibit its agonist-dependent interactionwith the coactivator SRC-1.

In conclusion, SR-202 is a selective PPAR� antag-onist, which is able to antagonize TZD-induced tran-scriptional activity of PPAR�. This effect may resultfrom its ability to inhibit TZD-stimulated recruitment ofcoactivators by PPAR�.

SR-202 Inhibits PPAR�-Dependent Differentiationof Adipocytes

A major role of PPAR� is to stimulate adipogenesis.We thus designed experiments to see whether SR-202could also antagonize PPAR� activity in a functionalassay, more particularly in adipocyte differentiation.For that purpose, preadipocyte 3T3-L1 cells were pre-treated with different concentrations of SR-202 or ve-hicle for 24 h and then induced to differentiate withmedium containing either BRL 49653 (25 nM) and in-sulin (5 �g/ml) or a mixture containing dexamethasone(1 �M), insulin (10 �g/ml), and IBMX (0.5 mM). Asshown in Fig. 3, A and B, SR-202 was able to signifi-cantly inhibit BRL 49653- and hormone-induced adi-pocyte differentiation of 3T3-L1 cells in a dose-depen-dent manner, as shown by the decrease in lipidcontent revealed by Oil red O staining. The antiadipo-genic effect of SR-202 in this experimental setting wasalso revealed by the reduced expression of an adipo-cyte differentiation marker, the adipocyte fatty acid

Rieusset et al. • SR-202, a New PPAR� Antagonist Mol Endocrinol, November 2002, 16(11):2628–2644 2629


Fig. 1. SR-202 Is a Specific PPAR� AntagonistA, Structure of SR-202. B, Effect of SR-202 on Wy14643-induced PPAR� activity. C, Effect of SR-202 on bezafibrate-

stimulated PPAR� activity. D, Effect of SR-202 on GW1358-induced FXR activity. E, Effect of SR-202 on troglitazone-inducedPPAR� activity. HeLa cells were transfected with the reporter construct (AcoA.TK.CAT or pCAT-promIBABP), and full-lengthmPPAR�, mPPAR�, mPPAR�, or hFXR expression plasmids. They were then treated for 36 h with specific ligands and increasingconcentrations of SR-202 as indicated. Each bar represents the mean � SEM of three experiments. CAT values were normalizedto the �-galactosidase activity resulting from the expression of a cotransfected vector.

2630 Mol Endocrinol, November 2002, 16(11):2628–2644 Rieusset et al. • SR-202, a New PPAR� Antagonist


binding protein (aP2) (Fig. 3C). The inhibition of adipo-genesis at high concentrations of SR-202 was not dueto a toxic effect of the antagonist because it could beattenuated by cotreating the cells with a high concen-tration of BRL 49653 (1 �M instead of 25 nM, data notshown). Furthermore, the lack of toxicity of SR-202was again confirmed by quantification of LDH activi-ties released in the culture media (reaching only 5% oftotal cellular LDH activity after a 48-h treatment with400 �M SR-202) and by the total number of cells that

remained unchanged at the end of the differentiatingprotocols (data not shown).

SR-202 Inhibits Adipose Tissue Accumulationin Vivo

Because SR-202 inhibits PPAR�-dependent adipo-cyte differentiation, we investigated the effect of SR-202 in vivo, on body and fat composition in wild-type(wt) mice under standard diet (SD) or HFD. SR-202

Fig. 2. SR-202 Inhibits BRL 49653-Mediated Recruitment of SRC-1 by PPAR�A, Representative gel and quantification by densitometry (CARLA values corresponding to SRC-1 recruitment) of radiolabeled

SRC-1 pull down assay, using the hPPAR� ligand binding domain, in the presence of increasing concentrations of BRL 49653.B, Representative gel and quantification (SRC-1 recruitment) of the effect of SR-202 on BRL 49653-stimulated recruitment ofSRC-1 by PPAR�. Each bar represents the mean � SEM of at least three experiments.



treatment given as food admixture was started at 3 wkof age, i.e. at the weaning period that corresponds tothe beginning of important white adipose tissue (WAT)accumulation (20), and prolonged during a consecu-tive 10 wk. Wt mice treated with SR-202 for 10 wkgained significantly less body weight than untreatedwt mice under both SD and HFD (Fig. 4A, left panel).Consistent with this result, the treatment of wt mice bySR-202 significantly decreased WAT mass under SDand protected them from HFD-induced increase inWAT mass (Fig. 4B, left panel, compare CTL SD micewith SR-202 HFD mice). This effect of SR-202 onadiposity was not specific of epididymal WAT becauseSR-202-treated wt mice had also a lower brown adi-pose tissue (BAT) mass than untreated wt mice, underboth SD and HFD (Fig. 4C, left panel). In addition to thedecrease in fat mass development, the size of whiteadipocytes was modulated in parallel with the WAT

mass. SR-202-treated wt mice had smaller adipocytesthan untreated wt mice on SD (Fig. 5A, a and b).Quantification of cell numbers indicated that there wasmore cells per microscopic field in WAT of SR-202-treated wt mice than in WAT of untreated wt mice,confirming the smaller size of adipocytes (Fig. 5B, aand b). Importantly, SR-202-treated wt mice were pro-tected from HFD-induced adipocyte hypertrophy (Fig.5, A and B, c and d). To assess whether the effects ofSR-202 on WAT involved the modulation of PPAR�activity, we measured the mRNA levels of PPAR� tar-get genes including lipoprotein lipase (LPL), fatty-acidtranslocase (FAT)/CD36, aP2 and sterol regulatory el-ement-binding protein 1c (SREBP-1c). As shown inFig. 6, SR-202-treated wt mice exhibited a significantdecrease in PPAR� activity as demonstrated by thereduction of LPL, aP2, CD36 and SREBP-1c mRNAlevels. Altogether, these data show that a decrease of

Fig. 3. SR-202 Is a Potent Antagonist of PPAR�-Induced Adipocyte DifferentiationA, Effect of SR-202 on BRL 49653-induced adipogenesis of 3T3-L1 cells. Cells were pretreated with SR-202 or vehicle (water)

for 24 h and then treated with BRL 49653 (25 nM) and insulin (5 �g/ml), with or without SR-202. After 6 d of treatment, cells werestained with Oil Red O. B, Effect of SR-202 on hormone-induced adipocyte differentiation of 3T3-L1 cells. Cells were pretreatedwith SR-202 and then induced with dexamethasone/IBMX/Ins with or without SR-202 for 2 d. Thereafter, the medium waschanged to insulin alone with or without SR-202 for an additional 2 d. Cells were then stained with Oil Red O. C, Effect of SR-202on the mRNA levels of aP2 adipocyte differentiation marker. Ribonuclease protection assay was performed on 10 �g of total RNAfrom 3T3-L1 cells treated as indicated above. L27, Ribosomal protein.



PPAR� activity by treatment with a specific antagonistis associated with decreased adiposity.

An alternative way to inhibit PPAR� activity is toinvalidate its own gene in mice. We have generatedPPAR� mutant mice by homologous recombination(data to be published elsewhere). Early embryonic le-thality of PPAR� null mice was observed, as also re-ported by others (10, 21). Heterozygous PPAR� mice

(PPAR� �/�) developed normally, were fertile andapparently healthy. We challenged the PPAR� �/�mice with HFD, in the presence or not of SR-202 andanalyzed the expression of PPAR� target genes. Com-pared with wt animals, untreated PPAR� �/� miceunder HFD showed, as expected, a decrease inPPAR� activity as illustrated by the reduction of LPL,CD36, aP2, and SREBP-1c mRNA levels in WAT (Fig.

Fig. 4. SR-202 Prevents Weight Gain and Adipose Tissue Deposit Content in MiceBody weight (A) and fat weight (B and C) of wt and PPAR� �/� male mice treated or not with SR-202 (400 mg/kg) under both

SD and HFD. SR-202 was given as food admixture and the treatment was started just after weaning during a consecutive 10 wk.Each bar represents the mean � SEM of at least 10 mice per group (* and $, P � 0.05; NS, no significative difference).*, Comparison between CTL and SR-202-treated mice; $, comparison between SD and HFD.



6). However, SR-202-treated PPAR� �/� mice underHFD did not show a further decrease of PPAR� activitycompared with untreated PPAR� �/� mice becauseboth groups of mice had similar levels of LPL, CD36,aP2, and SREBP-1c mRNA levels (Fig. 6). Analyses ofbody and fat composition revealed that untreatedPPAR� �/� mice had a lower body weight than un-treated wt mice, under both SD and HFD (Fig. 4A). Inaddition, untreated PPAR� �/� mice on SD, showedreduced WAT and BAT mass compared with untreatedwt mice (Fig. 4, B and C, respectively). Moreover, theywere protected from HFD-induced hypertrophy ofWAT and BAT (Fig. 4, B and C, respectively). Consis-tent with the difference in WAT mass, adipocytesof untreated PPAR� �/� mice were smaller than thoseof untreated wt mice (Fig. 5, A and B, e and a). Simi-larly, untreated PPAR� �/� mice were protected fromHFD-induced adipocyte hypertrophy (Fig. 5, A and B,g and c). Interestingly, treatment of PPAR� �/� micewith SR-202 did not further decrease body and adi-

pose tissue weight (Fig. 4, A–C, right panels), whereasit further decreased the size of white adipocytes, un-der both SD and HFD (Fig. 5B, e–h).

Thus, decreasing PPAR� activity, either by treat-ment with a specific antagonist or by invalidation ofone allele of the PPAR� gene, leads to decreasedadiposity in mice. These data indicate that the pheno-type of SR-202-treated mice is a bona fide conse-quence of the reduction of PPAR� activity in vivo.

SR-202 Decreases Adipocytokine Secretionby WAT

White adipocytes are not only forming a passive tissuemass devoted to the storage of excess energy but arealso endocrine secretory cells (22). For example, ad-ipsin, TNF�, leptin, and plasminogen activator inhibi-tor-1 are all secreted by fat cells, under control ofnutritional (feeding and fasting) and pathological (obe-sity) states (22). In particular, regulatory roles in energy

Fig. 5. SR-202 Treatment Decreases Adipocyte SizeA, Histological analysis of epididymal WAT from wt and PPAR ��/� male mice treated or not with SR-202 (400 mg/kg) under

both SD and HFD. Twenty-micrometer sections were stained with both eosin and hematoxylin and with Oil Red O. B,Quantification of adipocyte number (mean � SEM) in at least three microscopic fields of WAT sections per mouse from threedifferent mice per group.*, Comparison between CTL and SR-202-treated mice; $, comparison between SD and HFD (* and $, P � 0.05).



homeostasis and/or systemic insulin sensitivity havebeen shown for leptin and TNF� (22). In addition, theamount of circulating leptin and TNF� correlates with

body fat stores and/or hyperinsulinemia, suggesting apotential involvement of these cytokines in obesity-induced insulin resistance (23, 24). Because SR-202treatment is associated with decreased adiposity, wetested the effects of SR-202 treatment on leptin andTNF� secretion by the adipose tissue. Interestingly,SR-202-treated wt mice, under SD, had normalplasma leptin levels but lower plasma TNF� levels (Fig.7, A and B, left panels). As expected, we found astrong increase of plasma leptin and TNF� levels inuntreated wt mice under HFD (Fig. 7, A and B, leftpanels; compare CTL SD mice with CTL HFD mice).Under SR-202 treatment, these mice were protectedfrom HFD-induced hyperleptinemia and HFD-relatedincrease in TNF� levels (Fig. 7, A and B, left panels).These data provide further evidence that the secretionlevels of leptin and TNF� are closely related to the sizeof adipocytes. In addition, it suggests that inhibitingPPAR� activity by treatment with an antagonist canprevent from obesity-induced secretion of thesecytokines.

Similar analyses were performed in PPAR� �/�mice. Untreated PPAR� �/� mice, under SD, hadsimilar levels of leptin but lower plasma levels of TNF�compared with untreated wt mice (Fig. 7, A and B).Furthermore, untreated PPAR� �/� mice were pro-tected from HFD-induced increase in both leptin andTNF� levels (Fig. 7, A and B, right panels). Finally,treating PPAR� �/� mice with SR-202 further de-creased plasma levels of leptin and TNF� under HFD,whereas only leptin was further decreased under SD(Fig. 7, A and B, right panels).

In conclusion, decreasing PPAR� activity, either bytreatment with a specific antagonist or by invalidationof one of the two alleles of the PPAR� gene, leads todecreased leptin and TNF� plasma levels, which isconsistent with the smaller size of adipocytes. TreatingPPAR� �/� mice with SR-202, further decreasedplasma levels of these signaling molecules, in agree-ment with a more pronounced decrease in the size ofadipocytes (see Fig. 5).

SR-202 Protects against HFD-InducedInsulin Resistance

Both reducing the adipocyte size and modulating thesecretion of two major adipocytokines, leptin andTNF�, are known to influence insulin sensitivity (3, 25).We thus investigated whether SR-202 treatment couldincrease insulin sensitivity in mice. For that purpose,we measured glucose and insulin levels, in addition toplasma free fatty acid (FFA) levels, which are known tobe linked to insulin sensitivity. Treatment of wt mice onSD with SR-202 did not affect the plasma glucoselevels but strongly decreased the plasma insulin levels(Fig. 8, A and B, left panels). No change in FFA plasmalevels was observed in SR-202-treated wt mice underSD (Fig. 8C, left panel). As expected, HFD feeding inuntreated wt mice was associated with an increase inFFA levels (Fig. 8C, left panel), a high hyperinsulinemia

Fig. 6. SR-202 Decreases PPAR� Activity in VivoAmounts of the mRNA of the following PPAR� target genes

were determined in WAT of wt and PPAR� �/� mice underHFD treated or not with SR-202: LPL, FAT/CD36, aP2, andSREBP-1c. The mRNA level of each target was determinedby reverse transcription followed by real-time PCR using theLight-cycler-FastStart DNA Master SYBR Green I as de-scribed in Materials and Methods. Each bar represents themean � SEM (n � 5). *, P � 0.05 (comparison with CTL wtmice).



(Fig. 8B, left panel), and a slight, however statisticallynot significant, increase in glucose levels (Fig. 8A, leftpanel), indicating a compensated state of insulin re-sistance. In contrast, SR-202 treated mice were pro-tected from HFD-induced insulin resistance, as mea-sured by the maintenance of relatively low insulin andFFA plasma levels (Fig. 8, B and C, left panels). Thesedata demonstrate that decreasing PPAR� activity bytreatment with an antagonist potentiates insulin sen-sitivity and improves lipid parameters under HFD.

We next investigated insulin sensitivity in PPAR��/� mice. Consistent with the result obtained in SR-202-treated wt mice, untreated PPAR� �/� mice un-der SD showed lower plasma insulin levels than un-treated wt mice, without any significant change in FFAplasma levels (Fig. 8, B and C, right panels). However,PPAR� �/� animals were protected from HFD-induced hyperinsulinemia and HFD-related increase ofFFA plasma levels (Fig. 8, B and C, right panels).

Finally, treatment of PPAR� �/� mice with SR-202 didnot further improve insulin sensitivity, as indicated bythe lack of change in metabolic parameters (Fig. 8,A–C, right panels).

In conclusion, a partial decrease in PPAR� activityleads to increased insulin sensitivity and protects micefrom HFD-induced obesity and insulin resistance.However, a combined pharmacological and geneticaction to reduce PPAR� activity, such as in SR-202-treated PPAR� �/� mice, did not further improveinsulin sensitivity compared with untreated PPAR��/� mice.

SR-202 Increases Insulin Sensitivity inob/ob Mice

After demonstrating the ability of SR-202 to preventthe development of an insulin resistance as seen upona HFD challenge, we next tested whether SR-202

Fig. 7. SR-202 Decreased Secretion of AdipocytokinesFed levels of plasma leptin (A) and TNF� (B) in wt and PPAR ��/� male mice treated or not with SR-202 (400 mg/kg) under

both SD and HFD. Decreased PPAR� activity, by SR-202 treatment or by deletion of one allele of the PPAR� gene, results in adecrease of leptin and TNF� plasma levels. Each bar represents the mean � SEM of at least 10 mice per group (* and $, P � 0.05;** or $$, P � 0.01; NS, no significant difference). *, Comparison between untreated and treated mice; $, comparison between SDand HFD.



could improve insulin sensitivity in the context of analready developed obesity with hyperglycemia and hy-perinsulinemia. For that purpose, we used ob/ob mice,a well characterized strain of obese and diabetic mice.Treatment of the ob/ob mice with SR-202 was startedat 8 wk of age (experimental d 0), when mice are

already overtly diabetic (see glucose and insulin levels,d 0, Fig. 9, A and B). The treatment as food admixturewas maintained for 20 d, after which metabolic param-eters were measured (experimental d 20).

Although hyperglycemia and hyperinsulinemia con-tinued to increase in the untreated ob/ob mice (com-

Fig. 8. SR-202 Treatment Protects against HFD-Induced Insulin ResistanceFed levels of plasma glucose (A), insulin (B), and FFA (C) in wt and PPAR ��/� male mice treated or not with SR-202 (400

mg/kg) under both SD and HFD. Decreased PPAR� activity, by SR-202 treatment or by deletion of one allele of the PPAR� gene,was associated with increased insulin sensitivity. Each bar represents the mean � SEM of at least 10 mice per group (* and $, P �0.05; $$, 0.01; NS, no significant difference). *, Comparison between untreated and treated mice; $, comparison between SDand HFD.



pare d 0 and d 20, Fig. 9, A and B), daily treatment withSR-202 prevented the time-dependent increase in glu-cose concentrations. After 20 d of treatment, fed glu-

cose concentrations were 67% of those of untreatedanimals (P � 0.01) (Fig. 9A). This decreased hypergly-cemia was associated with decreased insulin levels to

Fig. 9. SR-202 Is an Insulin Sensitizer in Vivo, Decreasing Hyperglycemia and Hyperinsulinaemia in ob/ob MiceTreatment of 8-wk-old ob/ob mice was started at d 0 up to d 20. Serum glucose (A) and insulin (B) levels at the fed state in

ob/ob mice on d 0 (white bars) or on d 20 after the treatment with vehicle (gray bars) or SR-202 (black bars). Plasma glucose (C)and insulin (D) levels during an ip glucose tolerance test in ob/ob mice performed at experimental d 20, in untreated mice (whitecircles) or in mice treated with SR-202 (black circles). E, Intraperitoneal insulin tolerance test in ob/ob mice treated or not withSR-202. F, amounts of the mRNAs of LPL, FAT/CD36, and aP2 in WAT of ob/ob mice treated or not with SR-202 (400 mg/kg)during 20 d. Values are expressed as the mean � SEM (n � 10). *, P � 0.05, **, P � 0.01.



70% of control values (P � 0.05) (Fig. 9B). The de-crease in glucose and insulin was not a secondaryresponse to changes in body weight because all ani-mals gained weight at comparable rates (data notshown). Thus, the PPAR� antagonist SR-202 induceda coordinated decrease in both glucose and insulinconcentrations, suggesting an overall improvement inglucose homeostasis.

We then conducted glucose and insulin tolerancetests to determine whether SR-202 could improve glu-cose disposal and insulin sensitivity in ob/ob mice. Asshown in Fig. 9C, SR-202-treated ob/ob mice showedan enhanced glucose disposal compared with un-treated ob/ob mice despite a strikingly reduced insulininduction (Fig. 9D), suggesting an increase of insulinsensitivity. In agreement with these data, ip adminis-tration of insulin resulted in a stronger glucose lower-ing effect in SR-202-treated ob/ob mice comparedwith untreated mice (Fig. 9E).

Finally, SR-202 treatment directly affected the ex-pression of PPAR� target genes. 20 d of treatment ofob/ob mice with SR-202 induced a decrease ofPPAR� activity as illustrated by the decrease of LPL,CD36, and aP2 mRNA levels in WAT (Fig. 9F).

In conclusion, SR-202 functions as an insulin sen-sitizer, markedly decreasing hyperglycemia and hy-perinsulinemia in ob/ob mice, a mouse model of obe-sity and insulin resistance.

DISCUSSION

This study describes a new synthetic antagonist ofPPAR�, which has the capacity to antagonize bothTZD-stimulated recruitment of SRC-1 by PPAR� andTZD-induced transcriptional activity of this receptor.We have tested the activity of this antagonist both invitro and in vivo, in the whole animal. In vitro, we havedemonstrated that the inhibition of PPAR� activity bySR-202 indeed inhibits adipocyte differentiation of3T3-L1 cells. In vivo, treatment of mice with SR-202,from weaning onwards, prevents the full developmentof WAT and BAT. Furthermore, it protects mice fromHFD-induced adiposity. In addition, SR-202 improvesinsulin sensitivity, by decreasing HFD-induced FFA,leptin and TNF� secretion in association with a pre-vention of adipocyte hypertrophy. Finally, SR-202 is aninsulin sensitizer, markedly reducing hyperglycemiaand hyperinsulinemia in ob/ob mice. Thus, we reporton a PPAR� antagonist compound that is effective invitro and in vivo and offers the potential for an im-proved therapy for metabolic diseases.

Two Animal Models of PPAR� Decreased Activity

The strength of our study is based on the use of twomouse models in which PPAR� activity is decreased.In the first model, we use an antagonist of PPAR�,SR-202, which we have found to be specific and func-

tional in vitro. In the second model, decreased PPAR�activity was achieved by deletion of one of the twoalleles of the PPAR� gene. Comparative analyses ofboth groups of mice allows us to propose that theSR-202 effects reflect a reduced PPAR� activity.Moreover, treatment of PPAR� �/� mice with SR-202was performed to investigate the effects of severelyreduced PPAR� activity. We have demonstrated thatreduction of PPAR� activity was achieved in SR-202-treated wt mice and PPAR� �/� mice because theyshowed a similar decrease in expression levels ofknown PPAR� target genes. Moreover, they are sim-ilarly protected from HFD-induced adipocyte hypertro-phy and insulin resistance. However, treatment ofPPAR� heterozygous mice with SR-202 did not furtherreduce PPAR� activity and did not amplify the insulinsensitizing phenotype. However, a further decrease inthe size of adipocytes and plasma levels of TNF� andleptin was observed. Several explanations can be pro-posed as to why PPAR� activity in not further de-creased in SR-202-treated PPAR� �/� mice, whereassome of the SR-202 effects are amplified (see above).First, SR-202 is a low affinity compound, and it is likelythat the inability of SR-202 to further improve insulinsensitivity in PPAR� �/� mice is caused by the diffi-culty in obtaining a complete shut-down of PPAR�activity. Particularly, the reduction of PPAR� expres-sion in PPAR� �/� mice may increase this difficulty.Second, transgenic mice may use compensatorymechanisms that are not present in wt mice, maskingthe effects of SR-202. In both cases, it is possible thatSR-202 has some PPAR� independent effect, activat-ing or antagonizing other factors, and leading to ad-ditive effects in PPAR� �/� mice. Nevertheless, thefact that SR-202 amplifies some phenotypes in PPAR��/� mice (reduction of TNF� and leptin levels) and notothers (insulin sensitivity) underlines the different con-tributions of PPAR� in different processes. It indicatesthat PPAR� is incontestably an essential actor of adi-pocyte differentiation, with a participation in insulinaction that is more complex. Other pathways are likelyto be involved in insulin signaling as well. However, wecan not exclude that treatment with higher concentra-tions of SR-202 could improve insulin sensitivity inPPAR� �/� mice.

The PPAR� Antagonist SR-202 InhibitsAdipocyte Differentiation

There is now strong evidence supporting a key role ofPPAR� in adipogenesis based on both gain or loss offunction experiments. Indeed, it has been shown thatthe ectopic expression and activation of PPAR� infibroblasts are sufficient to induce an adipogenic re-sponse (26). Moreover, genetic studies using chimericmice have confirmed that PPAR� is required for adi-pogenesis in vivo (27). Finally, it was recently demon-strated that reducing PPAR� activity with a selectivePPAR� antagonist, BADGE, inhibits adipocyte differ-entiation in vitro (17). Herein, we describe a new syn-



thetic PPAR� antagonist belonging to the phosphono-phosphate family with antiadipogenic activity. In vitro,SR-202 inhibits both TZD and hormonally inducedadipocyte differentiation of 3T3-L1 cells, as shown bythe decrease in lipid accumulation and aP2 mRNAlevels. Hormone-induced aP2 expression was evenmore dramatically reduced indicating that SR-202 ismore or less effective depending on the differentiationprotocol applied. Recently, it was suggested that theremay be at least two pathways for 3T3-L1 adipocytedifferentiation (19). The first one would be regulated byPPAR� and its ligands and accordingly could beblocked by the PPAR� antagonist LG 100641. Thesecond pathway would be independent of PPAR� ac-tion. Indeed, it was insensitive to LG 100641 (19). Thefact that SR-202 antagonized both hormone- andTZD-induced adipocyte differentiation reveals thatSR-202 has effects on both pathways. In addition toantagonizing PPAR� activity, SR-202 would inhibit an-other actor of adipocyte differentiation that respondsto the IBMX/dexamethasone/insulin cocktail. A similardouble antiadipogenic effect has also been describedfor two other PPAR� antagonists, BADGE (17) andPD 068235 (18). The in vitro observations are corrob-orated by in vivo experiments where SR-202 treatedwt mice have decreased PPAR� activity, together witha decreased WAT and BAT mass, and are protectedfrom HFD-induced adipocyte hypertrophy.

Treatment of Mice with SR-202 ImprovesInsulin Sensitivity

In addition to regulating adipocyte differentiation,PPAR� plays a key role in insulin signaling and ago-nists of PPAR� are used in the treatment of type 2diabetes (11). However, the mechanism for this roleremains obscure.

We demonstrate herein that the reduction of PPAR�activity by partial gene targeting improves insulin sen-sitivity in mice, under HFD, as previously reported byothers (10). Importantly, we show that treatment of wtmice with SR-202 reproduces the insulin-sensitizingphenotype, confirming that reduction of PPAR� activ-ity is associated with an increased insulin sensitivity.This observation suggests that antagonists of PPAR�could be useful to explore the signal transductionpathways involved in PPAR�-mediated insulin sensi-tization. Although our experiments do not provide amolecular mechanism to explain the increase of insulinsensitivity associated to the reduction of PPAR� ac-tivity, they suggest that this increased sensitivity is aconsequence of the diminution of adipose tissuemass. Indeed, HFD feeding, which increases adipo-cyte size, also increases secretion of molecules caus-ing insulin resistance such as FFA and TNF�. In con-trast, SR-202-treated wt mice and untreated PPAR�heterozygous mice, which are protected from HFD-induced adipocyte hypertrophy, showed a significantdecreased FFA and TNF� levels, and a concomitantimproved insulin sensitivity. This is associated with a

decreased expression in WAT of genes involved infatty acid transport and lipogenesis, which participatein preventing adipocyte hypertrophy and thereforeobesity under HFD. We thus can conclude that reduc-tion of PPAR� activity increases insulin sensitivity, pre-sumably by preventing adipocyte hypertrophy andconsequently decreasing the secretion of FFA andTNF�. These results are in agreement with humangenetic studies. One rare mutation renders PPAR�more active, leading to increased adipocyte differen-tiation and obesity (28). An other more common mu-tation impedes PPAR� activity and is associated witha lower body mass index, improved insulin sensitivity,and higher levels of plasma high density lipoproteincholesterol (29).

The role of leptin secretion in participating to themodulation of insulin sensitivity remains unclear. In-deed, although lipoatrophic animals without leptinare insulin resistant, there is evidence that leptin caninterfere with insulin signaling in some cell types invitro (30, 31). Previous reports have shown that ac-tivation of PPAR� also results in a decrease of leptinsecretion (32–34) and that PPAR� �/� mice underHFD overexpress and hypersecrete leptin (10). Thus,at first sight, our results concerning the concomitantdecrease in PPAR� activity and plasma levels ofleptin may appear surprising. However, the fact thatwe observe a similar reduced leptin secretion in twomodels of decreased PPAR� activity (deletion of oneallele of the PPAR� gene or treatment with an an-tagonist of PPAR�) and a further decrease in SR-202treated PPAR� �/� mice are clearly consistent. Inaddition, SR-202 treatment also modulates twoother major regulators of leptin secretion, the size ofadipocytes and plasma insulin levels, in favor of adecrease in leptin plasma levels. As mentionedabove, how to relate this effect on leptin secretionand insulin sensitivity remains unclear. In particular,the fact that SR-202 increased insulin sensitivity inob/ob mice, a mouse model of obesity without lep-tin, indicates that the action of the drug is not me-diated by leptin.

PPAR� Antagonists Like SR-202 Could BeClinically Useful in the Treatment of Obesityand Type 2 Diabetes

Our data and other recent reports (10, 35) underlinethe interesting paradox that both PPAR� overactivity,due to TZD, and PPAR� underactivity, due to eitherhaploinsufficiency or treatment with a PPAR� antago-nist, protect against obesity-induced insulin resis-tance. One explanation of this paradox may reside inthe number and size of adipocytes. Indeed, TZD-mediated activation of PPAR� in rodents results in anincrease in the number of small adipocytes and adecrease in the number of large, hypertrophic fat cells(25). In parallel, expression of two cytokines that influ-ence energy homeostasis and insulin sensitivity,namely leptin and TNF�, is repressed by PPAR� ago-



nists in adipocytes (30, 32, 36). Remarkably, untreatedPPAR� �/� mice or SR-202-treated wt mice havesmaller adipocytes than untreated wt mice, and theysecrete less leptin and TNF� and release less fattyacids, contributing to enhanced insulin sensitivity. Al-though the ways of modulating PPAR� activity aredistinct, the outcome appears similar: generation ofsmall adipocytes and attenuated secretion of cyto-kines that interfere with insulin sensitivity. Conse-quently, not only agonists but also antagonists ofPPAR� could be clinically useful in the treatment ofobesity and type 2 diabetes. As a proof of concept, thetreatment of ob/ob mice clearly establishes the effi-cacy of SR-202 in improving glucose disposal andinsulin sensitivity, confirming that SR-202 is an insulinsensitizer in a model of preexisting obesity and insulinresistance. In addition, if SR-202, like BADGE, has alow affinity for PPAR�, it presents the specific advan-tages to be soluble in water and nontoxic in vivo,which is a significant bonus from a therapeutic point ofview. Furthermore, it is possible that higher affinityPPAR� antagonists can be derived based on thestructure of SR-202.

This work on SR-202, which directly acts on PPAR�,might be compared with studies on RXR, the obligatepartner of PPARs. Very recently, mice lacking RXR inadipocytes (37) or mice treated with an antagonist ofRXR, HX531 (38), have been reported to be resistant toHFD-induced obesity and insulin resistance. However,treatment of PPAR� �/� mice with HX531 severelydepleted the WAT, leading to a reemergence of insulinresistance (38). This biphasic effect may raise con-cerns for the potential clinical use of molecules suchas HX531, more particularly in patients with the variantPPAR� Ala12 allele. HX531 might worsen insulin re-sistance in patients that have lower PPAR� activity(29). The lack of effect of SR-202 in PPAR� �/� micesuggest that such an effect would not occur in thissituation. The use of SR-202 type compounds couldbe considered for patients with normal PPAR� activity(Pro12 allele) or for patients with the more active vari-ant Ala 12 PPAR�. Finally, PPAR� antagonists seemmore adapted for clinical use than RXR antagoniststhat can also act on RXR homodimers or on other RXRcontaining heterodimers, triggering undesired sideeffects.

In conclusion, we have characterized a first PPAR�antagonist compound, SR-202, which is effective bothin vitro and in vivo. We propose that, by preventingadipocyte differentiation and lipid accumulation, SR-202 protects mice from HFD-induced adipocyte hy-pertrophy and insulin resistance. SR-202 also im-proves insulin sensitivity in a model of establishedobesity, confirming that SR-202 is an insulin sensitizer.Treatment with compounds having the characteristicsof SR-202 could be extremely interesting if they be-have in human like in mouse. They would preventadipogenesis and obesity while increasing insulin sen-sitivity, in contrast to TZDs, which induce weight gain.Consequently, SR-202 offers a potential for the devel-

opment of compounds that could improve the therapyof metabolic diseases.

MATERIALS AND METHODS

Reagents

Dimethyl �-(dimethoxyphosphinyl)-p-chlorobenzyl phosphate(SR-202) was synthesized at Ilex onc. Rosiglitazone (BRL49653) was a gift from Park Davis Pharmaceuticals (AnnArbor, MI). IBMX, dexamethasone, and insulin were pur-chased from Sigma (St. Louis, MO).

CARLA

The CARLAs were performed as previously described withsmall modifications (7). Briefly, bacteria expressing GST-human (h) PPAR� LBD were incubated at 37 C until theculture reached A600 of 0.7. At that point, isopropyl �-D-1-thiogalactopyranoside was added at the final concentrationof 0.5 mM and the cells were incubated for a further 3 h at 28C. Then, bacteria were lysed by three freeze cycles in liquidnitrogen. Sepharose 4B beads equilibrated in NETN (20 mM

Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5% NonidetP-40; 1 mM dithiothreitol) were added to the cell extract (250�l for PPAR� culture). GST-PPAR� fusion protein was al-lowed to bind to the beads overnight at 4 C. SRC-1 (7) wasproduced using the TNT quick coupled transcription/transla-tion system of Promega Corp. (Madison, WI) and labeled withmethionine 35S. Beads, SRC-1, and the compounds weremixed overnight at 4 C. After three centrifugation-wash cy-cles with NETN, the beads were loaded on a 10% SDS-PAGEgel. Autoradiography was used to localize SRC-1 and theamount of this protein was determined by image analysis onan image master Amersham Pharmacia Biotech (Bucking-hamshire, UK).

Cell Culture

3T3-L1 cells were cultured in DMEM supplemented by 10%bovine calf serum. Two days after reaching confluency, dif-ferentiation was induced in DMEM supplemented with 10%fetal calf serum and dexamethasone (1 �M)/IBMX (0.5 mM)/insulin (10 �g/ml). After 48 h, the medium was changed withonly the addition of insulin for an additional 2 d. Thereafter,the cells were grown in DMEM supplemented with 10% fetalcalf serum. Alternatively, cells were also differentiated withBRL 49653 (25 nM) and insulin (5 �g/ml). SR-202 or vehicle(H2O) was added 24 h before induction of differentiation.Medium was replenished with ligands every 2 d. Adipogen-esis was determined by the staining of lipids with Oil Red Oand by measuring the expression of adipocyte markers. RNAwas isolated using the Trizol reagent (Life Technologies, Inc.).

Transfections

Human HeLa cells were transiently transfected using thephosphate calcium method with expression vectors for mu-rine (m) PPAR� (39), mPPAR� (40), or mPPAR� (41), thesingle PPRE-containing reporter plasmid AcoA.TK.CAT(chloramphenicol acetyl transferase) and a �-galactosidaseencoding plasmid as control. Cells were also transfected witha hFXR encoding plasmid and pCAT-promIBABP reporterplasmid. They were exposed to the ligands for 36 h, lysed,and assayed for CAT and �-galactosidase activity. Transfec-tions were performed in quadruplicates.



Measurement of LDH Activity

Viability of the cells was estimated by measuring the LDHactivity released in the culture medium. Because LDH is acytosolic enzyme, the activity of the enzyme found in themedium reflects cellular lysis. The LDH activity is measuredby adding 15 �l of medium samples in 1 ml of the followingreaction medium (200 mM phosphate buffer, pH 6.8; 20 mM

NADH; and 1 mM pyruvate). LDH activity was determined witha spectrophotometer. Individual values are then expressedas percentage of the total quantity of LDH present in cells,determined after sonication of the cells.

Animals

Wild-type and PPAR� heterozygous mice, on a mixed back-ground (sv129/C56Bl6), were maintained at 20 C with a 12-hlight, 12-h dark cycle. Generation of PPAR� heterozygousmice will be described elsewhere. Animal experiments wereapproved by the animal authorization committee of the can-ton of Vaud (Switzerland). At 3 wk of age, the animals wereseparated by sex, genotyped for the PPAR� gene, andstarted to be fed with SD or HFD (D12451, 45 kcal% fat,Research Diets, Inc.) supplemented or not with SR-202 (400mg/kg). All mice were weighted on a weekly basis, starting at3 wk of age. Ten weeks later, animals were killed by cervicaldislocation and tissues were removed, weighed, and frozen.Eight-week-old male ob/ob mice were purchased from Jan-vier Breeding (Le Genest Saint Isle, France). These mice werefed with a SD supplemented or not with SR-202 (400 mg/kg)for 20 d. Blood was withdrawn at the fed state on the indi-cated days from the tip of the tail for the measurement ofplasma glucose and insulin levels. Glucose and insulin toler-ance tests were performed on 6-h-fasted mice. Animals wereinjected ip with 2 mg/g body weight of glucose or 0.75 mU/gbody weight of insulin. Blood was taken by tail punctureimmediately before and 15, 30, 60, and 120 min after injectionfor measurements of plasma glucose or insulin levels.

Plasma Metabolites Measurement

Whole blood was withdrawn at the fed state, at the end of thedark cycle, from the orbital sinus by using heparinized mi-crocapillary tubes. After withdrawal, the blood samples wereimmediately centrifuged, the serum removed, placed in afresh tube and immediately frozen. Blood glucose levels weremeasured with a glucometer (Roche Molecular Biochemicals,Mannheim, Germany) on whole blood. Serum insulin, leptin,and TNF� levels were assayed using the murine ELISA kits(CrystalChem, Downers Grove, IL; or R&D Systems, Minne-apolis, MN). Serum levels of nonesterified FFA were mea-sured using a colorimetric assay (Roche MolecularBiochemicals).

Histological Analysis

Adipose tissue was removed from three animals per group,fixed in 10% paraformaldehyde/PBS and maintained at 4 Cuntil used. Fixed specimens were embedded in tissue-freez-ing medium (Leica Instruments, Nussloch, Germany) and fro-zen in dry ice and isopentane. Twenty-micrometer cryostattissue sections were mounted on slides and fixed. Afterwashing in PBS, sections were used for histological staining(hematoxylin-eosin and Oil Red O). The number of adipocyteswas manually quantified on at least three different micro-scopic fields from three different animals per group.

Measurement of mRNA

LPL, CD36/FAT, aP2, and SREBP-1c mRNA levels were de-termined by reverse transcription followed by real-time PCR

using the Light-cycler-FastStart DNA Master Sybr Green I(Roche Molecular Biochemicals). Total RNA (1 �g) was re-verse-transcribed in a 20-�l reaction containing 1� TR buffer(50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2), 10 mM dithio-threitol, 0.5 mM deoxy-NTPs, 0.25 �g of random and oli-go(deoxythymidine) primers (Promega Corp.) and 100 U ofSuperscript II enzyme (Invitrogen, Basel, Switzerland), afterthe conditions recommended by the manufacturer. PCRswere carried out in duplicate by adding 5 �l of diluted first-strand reaction in 15 �l of Hot Start reaction mix containingFastStart Taq DNA polymerase, Sybr green as dye, 2–3 mM

MgCl2 and 0,5 �M of each specific primers. The followingprimer combinations were used: 5�-CAGTGGGGCATGTT-GACATT-3� and 5�-TGAGAGCGAGTCTTCAGGTA-3� forLPL; 5�-AAGATCCAAAACTGTCTGTA-3� and 5�-GTCCTG-GCTGTGTTTGGAGG-3� for CD36/FAT; 5�-CTTGTCTCCAG-TGAAAACTT-3� and 5�-GTGGAAGTCACGCCTTTCAT-3� foraP2; and 5�-ACGGAGCCATGGATTGCACA-3� and 5�-AAG-GGTGCAGGTGTCACCTT-3� for SREBP-1c. After 10 min at95 C, the tubes were subjected to 40 cycles of amplificationincluding denaturation for 10 sec at 95 C, hybridization for5 sec at 58–60 C, and elongation for 10 sec at 72 C. Foreach target, a standard corresponding to a fragment of thecDNA, cloned in pGEM-T easy vector (Promega Corp.),was serially diluted and quantified in parallel to generate aeight-point serial standard curve for the PCR analysis.Results were expressed in fmol (10�15 mol) or amol (10�18

mol)/�g of total RNA.

Statistical Analysis

Values are reported as mean � SEM. Statistical significancewas determined by the unpaired Student’s t test.

Acknowledgments

We thank Patrick Gouait for valuable technical help inanimal handling and Hubert Vidal for the access to the IFR 62(Institut Federal de Recherche) platform.

Received January 23, 2002. Accepted July 23, 2002.Address all correspondence and requests for reprints to:

Pr. Walter Wahli, Institut de biologie animale, Universite deLausanne, Batiment de biologie, CH-1015 Lausanne, Swit-zerland. E-mail: [email protected].

This work was supported by grants to B.D. and W.W. fromthe Swiss National Science Foundation, the Etat de Vaud,l’Association Francaise de Nutrition—Societe de Nutrition etde Dietetique de Langue Francaise, the National Center ofCompetence in Research “Frontiers in Genetics,” the HumanFrontier Science Program, and by Ilex.

* J.R. and F.T. contributed equally to this work.† Present address: Institut National de la Sante et de la

Recherche Medicale Unite 449, Faculte de medecine R.T.H.Laennec, rue Guillaume Paradin, 69372 Lyon cedex 08,France.

‡ Present address: Department of Physiology/Neurobiol-ogy, University of Basel, Klingelbergstrasse 50/70, CH-4056Basel, Switzerland.

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