Proc. Natl. Acad. Sci. USAVol. 93, pp. 5793-5796, June 1996Cell Biology

Antidiabetic thiazolidinediones inhibit leptin (ob) geneexpression in 3T3-L1 adipocytes

(obesity/nuclear receptor/peroxisome proliferator-activated receptor y)

CALEB B. KALLEN AND MITCHELL A. LAZARDivision of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia,PA 19104

Communicated by M. Daniel Lane, Johns Hopkins University School of Medicine, Baltimore, MD, February 20, 1996 (received for review January11, 1996)

ABSTRACT Lack of leptin (ob) protein causes obesity inmice. The leptin gene product is important for normal regu-lation of appetite and metabolic rate and is produced exclu-sively by adipocytes. Leptin mRNA was induced during theadipose conversion of 3T3-L1 cells, which are useful forstudying adipocyte differentiation and function under con-trolled conditions. We studied leptin regulation by antidia-betic thiazolidinedione compounds, which are ligands for theadipocyte-specific nuclear receptor peroxisome proliferator-activated receptor y (PPARy) that regulates the transcriptionof other adipocyte-specific genes. Remarkably, leptin geneexpression was dramatically repressed within a few hoursafter thiazolidinedione treatment. The EDso for inhibition ofleptin expression by the thiazolidinedione BRL49653 wasbetween 5 and 50 nM, similar to its Kd for binding to PPARy.The relatively weak, nonthiazolidinedione PPAR activatorWY14,643 also inhibited leptin expression, but was '1000 timesless potent than BRL49653. These results indicate that an-tidiabetic thiazolidinediones down-regulate leptin gene ex-pression with potencies that correlate with their abilities tobind and activate PPARy.

The relationship between obesity and diabetes is well estab-lished (1). The thiazolidinedione class of drugs has shown greatclinical potential for the treatment of diabetes (2) becausethese compounds potentiate cellular responsiveness to insulin(2, 3). Thiazolidinediones are also potent inducers of adipocytedifferentiation (4-6). More recently, thiazolidinediones wereshown to be selective ligands for peroxisome proliferator-activated receptor y (PPARy) (7, 8). PPARy is a member ofthe thyroid/retinoid/steroid receptor superfamily of transcrip-tion factors (9). The relationship between the enhancement ofinsulin action by thiazolidinediones and their ability to activatePPARy is not known. However, expression of PPARy is fatcell-specific (10, 11), and ectopic expression of PPARy pro-motes adipose conversion of NIH 3T3 fibroblasts (12), espe-cially in the presence of thiazolidinediones (8). PPARy alsotransactivates several adipocyte-specific genes including phos-phoenolpyruvate carboxykinase (13) and the adipocyte fattyacid binding protein aP2 (also called 422) (12, 14) .Mutation of the murine leptin (ob) gene results in an

inherited form of obesity in ob/ob mice (15). Administrationof exogenous leptin protein reverses this obesity and reducesthe body weight of normal mice as well (16-19). Leptinappears to be a hormone secreted by adipocytes, where theleptin gene is specifically expressed (15). Obese human sub-jects have been found to have increased leptin mRNA inadipose tissue (20-23) and increased plasma leptin levels (24),suggesting that dysregulated leptin signaling may contribute tohuman obesity.

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We hypothesized that thiazolidinediones, which regulate theexpression of adipocyte-specific genes via PPARy (14), mightalso play a role in the regulation of leptin gene expression. Wefound that several thiazolidinediones dramatically repressedleptin gene expression in differentiated 3T3-L1 adipocytes.The ED50 for inhibition of leptin expression by the thiazo-lidinedione BRL49653 was similar to its ED50 for inducingadipocyte differentiation and to its reported Kd for binding toPPARy. Thus, antidiabetic thiazolidinediones down-regulateleptin expression in 3T3-L1 adipocytes by a mechanism thatmay involve PPARy.

METHODS

Leptin cDNA. The published sequence of the leptin cDNA(15) was used to design oligodeoxynucleotides (oligode-oxynucleotide A: 5'-GCGGAATTCGAAGAAGATCCCA-GGGAGG-3'; oligodeoxynucleotide B: 5'-CGCGAATTCA-CTTCAGCATTCAGGGCTAAC-3') that were employed ina polymerase chain reaction by using reverse-transcribed RNAfrom 3T3-L1 adipocytes. The resulting polymerase chain re-action product, which contained the coding region of the leptincDNA, was subcloned into the EcoRI restriction site ofpBluescript SK (Stratagene). Sequence analysis of a selectedclone revealed it to be identical to the published sequence ofmouse leptin. The clone did not contain a glutamine codon atcodon 49, which was also true for 30% of the leptin cDNAsscreened by Zhang et al. (15).

Cell Culture. 3T3-L1 preadipocytes (American Type Cul-ture Collection) were maintained in Dulbecco's modifiedEagle's medium supplemented with 10% iron-supplementedcalf serum (Intergen, Purchase, NY), penicillin (100 units/ml),and streptomycin (100 jig/ml), with a change of medium every2-3 days. The standard method of cell differentiation wasemployed as described (25), with the exception that the cellsremained in differentiation medium for 3 days before switch-ing to postdifferentiation medium. Fetal calf serum (GIBCO/BRL or HyClone) was heat-inactivated at 65°C for 30 minbefore use in postdifferentiation medium. Cell treatment withthiazolidinediones or other compounds entailed supplement-ing medium with various concentrations of the compounds indimethyl sulfoxide (thiazolidinediones) and ethanol (WY14,643). All cells, including control cells, were treated with thesame volumes of dimethyl sulfoxide and/or ethanol.

Isolation and Analyses of RNA. Northern analyses wereperformed on total cellular RNA as described (25) with cDNAprobes for leptin, aP2, glyceraldehyde-3-phosphate dehydro-genase (GAPDH), and PPAR^y labeled with 32P using randomprimers (Boehringer Mannheim). RNase protection analysiswas performed as described (26) The antisense 32P-labeledprobe for mouse leptin mRNA was synthesized from pBlue-

Abbreviations: PPARy, peroxisome proliferator-activated receptor y,GADPH, glyceraldehyde-3-phosphate dehydrogenase.

5794 Cell Biology: Kallen and Lazar

script-leptin by using the vector's T3 promoter and yielded aprotected fragment of the expected size (528 nucleotides)corresponding to mouse leptin cDNA bases 94-622.

RESULTSA mouse leptin cDNA was obtained by reverse transcription-polymerase chain reaction of 3T3-L1 adipocyte RNA. ThiscDNA was used to measure leptin mRNA levels by bothNorthern blot and RNase protection analyses. The Northernanalysis shown in Fig. 1A reveals that leptin expression wasundetected in mouse 3T3-L1 preadipocytes but was increased7 days after the induction of adipocyte differentiation. Theadipocyte-specific genes PPARy and aP2 were also inducedduring adipogenesis, whereas GAPDH was not. Leptin expres-sion in the cultured adipocytes was markedly less than that inmouse adipose tissue (data not shown) and varied betweenexperiments, possibly due to differences in serum lots. Similarfindings were recently reported for 3T3-L1 and 3T3-F442Aadipocytes (27, 28).

Leptin gene expression was first detected on day 3 of thedifferentiation protocol, as shown by RNase protection anal-ysis in Fig. 1B. Expression was maximal by the fourth or fifthday, consistent with the role of leptin as a hormone derivedfrom mature adipocytes. By contrast, the induction of PPARyoccurs between days 1 and 2 after exposure of the preadipo-cytes to differentiating conditions (10, 11). PPARy is knownto regulate several genes that are induced late in adipocytedifferentiation, including aP2 and phosphoenolpyruvate car-boxykinase (11, 13), and we therefore considered the possi-bility that PPARy regulates the expression of leptin.We tested the ability of the PPARy-activating thiazo-

lidinediones as well as other PPAR activators to regulate leptingene expression in 3T3-L1 adipocytes. Remarkably, treatmentwith the PPARy-specific ligand BRL49653 dramatically re-duced leptin gene expression in the adipocytes (Fig. 2). Otherthiazolidinediones, including CP 86,325, troglitazone, andpioglitazone, as well as the less specific PPAR activator Wy14,643, had similar effects. In contrast, thiazolidinediones didnot inhibit the expression of PPARy or aP2 (Fig. 2A). Fur-

A Pre-Ad Ad

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FIG. 2. Thiazolidinediones inhibit leptin gene expression. (A)Northern analysis showing expression of leptin, PPARy, aP2, andGAPDH in 30 tLg of total RNA from 3T3-L1 pre-adipocytes (Pre-Ad)and adipocytes treated for 24 h with vehicles alone (Ad), 450 -uM Wy14,643 (Ad + Wy), 20 ,uM BRL 49653 (Ad + BRL), 20 iLMpioglitazone (Ad + Pio), 20 jzM troglitazone (Ad + Trog), or 20 ,iMCP 86,325 (Ad + CP). (B) RNase protection analysis of total RNA (30j.g) from 3T3-L1 adipocytes after 24 h treatment with vehicles (Noaddition), Wy 14,643 (450 ,tM), BRL 49653 (20 PLM), CP 86,325 (20,jM), or 10 nM triiodothyronine (T3).

thermore, the effect on leptin expression was specific forPPAR activators, as the active thyroid hormone triiodothyro-nine (T3) which works through another member of the nuclearreceptor superfamily (29) that is expressed in adipocytes (25,30) had no effect on leptin mRNA levels (Fig. 2B). The effectof BRL49653 was extremely rapid, occurring within 2-4 h ofadipocyte treatment with the compound (Fig. 3).A similar rateof decline in leptin mRNA levels was noted after inhibition oftranscription by actinomycin D (data not shown).The Kd for BRL49653 binding to PPARy is '40 nM (7).

Remarkably, the ED50 for BRL49653-induced down-regu-lation of leptin was between 5 and 50 nM (Fig. 4A). WY 14,643,a much less potent activator of PPARy (31, 32), was nearly1000-fold less potent at inhibiting leptin expression (Fig. 4A).Interestingly, the ED50 for inducing adipocyte differentiationby BRL49653 was also between 5 and 50 nM, as judged by aP2

Time of adipocyte exposureto BRL49653 (hr)

Pre-Ad 0 1 2 4 6 8 10 24

FIG. 1. Leptin gene expression in 3T3-L1 cells. (A) Northernanalysis of 30 ,ug total RNA from preadipocytes (Pre-Ad) andadipocytes (Ad). Expression of PPARy, aP2, and GAPDH are alsoshown. (B) Time course of leptin induction. RNase protection analysisof leptin mRNA in 30 ,Lg total RNA is shown.

FIG. 3. Time course of inhibition of leptin expression byBRL49653. 3T3-L1 adipocytes were treated with 20 ,iM BRL49653for the indicated times and 30 ,ug total RNA was used for RNaseprotection analysis.

Leptin- probe -_

Leptin--

Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 5795

A BRL49653 WY 14,643nM LiM

0 5 25 50 500 0 5 25 50 100 tRNA

probe -

Leptin-_ _o -l P

B BRL49653nM IuM

'110 5 50 0.5 5

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FIG. 4. Dose-responses of thiazolidinedione down-regulation ofleptin gene expression and induction of adipocyte differentiation. (A)Leptin gene expression. RNase protection analysis of 30 ,ug total RNAfrom 3T3-L1 adipocytes treated with BRL49653 or WY 14,643 at theindicated concentrations for 24 h. Control hybridization of the leptinprobe with 30 ,g yeast tRNA is also shown. (B) Adipocyte differen-tiation. Two days postconfluent 3T3-L1 preadipocytes were culturedfor 7 days in the presence of BRL49653 at the indicated concentra-tions. Total RNA (5 /xg) was subjected to Northern blot analysis foraP2 and GAPDH expression.induction (Fig. 4B) as well as by cell morphology (not shown),indicating a striking correlation between the affinity ofBRL49653 for PPARy and the effects of this compound onboth adipocyte differentiation and leptin gene expression.

DISCUSSIONWe have shown that thiazolidinedione compounds inhibitleptin gene expression in 3T3-L1 adipocytes. The ED50 forBRL49653 is in the nanomolar range and correlates well withthe ability of that compound to bind and activate PPARy.Therefore, our data are consistent with the hypothesis thatPPARy may regulate the expression of the leptin gene. Thehalf-life of the leptin mRNA after treatment with BRL49653was similar to that observed after inhibition of RNA tran-scription with actinomycin D (data not shown), suggesting thatthe thiazolidinedione inhibited transcription rather than in-creasing degradation of the leptin mRNA. Little is knownabout negative regulation by PPAR; the recent observationthat fibrates negatively regulate apolipoprotein CIII transcrip-tion may be an example of this phenomenon (33).

If the leptin gene is negatively regulated by PPARy, analysisof the leptin gene promoter will be required to determine ifthis effect is due to direct binding of PPARy to cis-regulatoryelements. Interestingly, the adipogenic transcription factorC/EBPa has recently been shown to bind and transcriptionallyactivate the leptin gene promoter (34-36). Therefore, it ispossible that liganded PPARy could negatively regulate leptinexpression by inhibiting the function of a transcriptionalactivator such as C/EBPa or AP-1 as has been demonstratedfor other nuclear receptors (37-39). It will also be importantto determine if endogenous activators of PPARy, such as fattyacids and prostaglandins (8, 32, 40), play a role in leptin generegulation.The finding that PPARy activators induce adipocyte differ-

entiation but inhibit leptin gene expression may be an indica-tion that these compounds induce a state of adipocyte differ-entiation that is subtly different from that induced by otherstimuli such as insulin, dexamethasone, and isobutylmethyx-anthine. It is also possible that PPAR-y plays different roles indifferentiation and maintenance of the adipocyte phenotype.Teleologically, the involvement of PPARy in inhibiting leptingene expression may be linked to its ability to induce adipocyte

differentiation (13), in that both would lead to an increase infat, the former via increased adipocyte number, the latter bypromoting adipocyte hypertrophy.The ability of thiazolidinediones to down-regulate leptin

gene expression in adipocytes was somewhat unexpected be-cause thiazolidinediones enhance insulin sensitivity, and insu-lin has been shown to increase leptin expression in vivo and incultured adipocytes (41, 42). Our results therefore suggest thatnegative regulation of leptin expression by thiazolidinedionesinvolves a mechanism other than enhancement of the actionsof insulin on this gene. However, insulin utilizes a number ofdistinct intracellular signaling pathways to regulate diverseeffects on cell metabolism as well as cell proliferation (43).Insulin enhancement of leptin expression may not involvePPARy and, conversely, thiazolidinediones may enhance in-sulin action by mechanisms other than activation of PPARy.For example, thiazolidinediones potentiate PI3 kinase signal-ing pathways involved in insulin action (44). Furthermore,there is evidence that much of the insulin sensitization causedby thiazolidinediones is due to actions on skeletal muscle (45,46), which expresses little PPARy (11). If thiazolidinedionesindeed utilize multiple and/or cell-specific signaling pathways,it may be possible to develop agents that selectively regulateeither insulin action or leptin expression. This may be of greatimportance given the likely role of leptin in regulating bothappetite and basal metabolic rate.

We thank E. Suh for help with RNase protection, and H. Harding,S. Ross and M. Birnbaum for helpful discussions. C.B.K. was sup-ported by the Medical Scientist Training Program. This work wassupported by National Institutes of Health Grants DK49780 andDK49210 to M.A.L.

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