Proc. Natl. Acad. Sci. USA Vol. 93, pp. 5793-5796, June 1996 Cell Biology Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes (obesity/nuclear receptor/peroxisome proliferator-activated receptor y) CALEB B. KALLEN AND MITCHELL A. LAZAR Division 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 (receivedfor review January 11, 1996) ABSTRACT Lack of leptin (ob) protein causes obesity in We hypothesized that thiazolidinediones, which regulate the mice. The leptin gene product is important for normal regu- expression of adipocyte-specific genes via PPARy (14), might lation of appetite and metabolic rate and is produced exclu- also play a role in the regulation of leptin gene expression. We sively by adipocytes. Leptin mRNA was induced during the found that several thiazolidinediones dramatically repressed adipose conversion of 3T3-L1 cells, which are useful for leptin gene expression in differentiated 3T3-L1 adipocytes. studying adipocyte differentiation and function under con- The ED50 for inhibition of leptin expression by the thiazo- trolled conditions. We studied leptin regulation by antidia- lidinedione BRL49653 was similar to its ED50 for inducing betic thiazolidinedione compounds, which are ligands for the adipocyte differentiation and to its reported Kd for binding to adipocyte-specific nuclear receptor peroxisome proliferator- PPARy. Thus, antidiabetic thiazolidinediones down-regulate activated receptor y (PPARy) that regulates the transcription leptin expression in 3T3-L1 adipocytes by a mechanism that of other adipocyte-specific genes. Remarkably, leptin gene may involve PPARy. expression was dramatically repressed within a few hours after thiazolidinedione treatment. The EDso for inhibition of METHODS leptin expression by the thiazolidinedione BRL49653 was between 5 and 50 nM, similar to its Kd for binding to PPARy. Leptin cDNA. The published sequence of the leptin cDNA The relatively weak, nonthiazolidinedione PPAR activator WY (15) was used to design oligodeoxynucleotides (oligode- 14,643 also inhibited leptin expression, but was '1000 times oxynucleotide A: 5'-GCGGAATTCGAAGAAGATCCCA- less potent than BRL49653. These results indicate that an- GGGAGG-3'; oligodeoxynucleotide B: 5'-CGCGAATTCA- tidiabetic thiazolidinediones down-regulate leptin gene ex- CTTCAGCATTCAGGGCTAAC-3') that were employed in pression with potencies that correlate with their abilities to a polymerase chain reaction by using reverse-transcribed RNA bind and activate PPARy. from 3T3-L1 adipocytes. The resulting polymerase chain re- action product, which contained the coding region ofthe leptin The relationship between obesity and diabetes is well estab- cDNA, was subcloned into the EcoRI restriction site of lished (1). The thiazolidinedione class ofdrugs has shown great pBluescript SK (Stratagene). Sequence analysis of a selected clinical potential for the treatment of diabetes (2) because clone revealed it to be identical to the published sequence of these compounds potentiate cellular responsiveness to insulin mouse leptin. The clone did not contain a glutamine codon at (2, 3). Thiazolidinediones are also potent inducers ofadipocyte codon 49, which was also true for 30% of the leptin cDNAs differentiation (4-6). More recently, thiazolidinediones were screened by Zhang et al. (15). shown to be selective ligands for peroxisome proliferator- Cell Culture. 3T3-L1 preadipocytes (American Type Cul- activated receptor y (PPARy) (7, 8). PPARy is a member of ture Collection) were maintained in Dulbecco's modified the thyroid/retinoid/steroid receptor superfamily oftranscrip- Eagle's medium supplemented with 10% iron-supplemented tion factors (9). The relationship between the enhancement of calf serum (Intergen, Purchase, NY), penicillin (100 units/ml), insulin action by thiazolidinediones and their ability to activate and streptomycin (100 jig/ml), with a change of medium every PPARy is not known. However, expression of PPARy is fat 2-3 days. The standard method of cell differentiation was cell-specific (10, 11), and ectopic expression of PPARy pro- employed as described (25), with the exception that the cells motes adipose conversion of NIH 3T3 fibroblasts (12), espe- remained in differentiation medium for 3 days before switch- cially in the presence of thiazolidinediones (8). PPARy also ing to postdifferentiation medium. Fetal calf serum (GIBCO/ transactivates several adipocyte-specific genes including phos- BRL or HyClone) was heat-inactivated at 65°C for 30 min phoenolpyruvate carboxykinase (13) and the adipocyte fatty before use in postdifferentiation medium. Cell treatment with acid binding protein aP2 (also called 422) (12, 14) . thiazolidinediones or other compounds entailed supplement- Mutation of the murine leptin (ob) gene results in an ing medium with various concentrations of the compounds in inherited form of obesity in ob/ob mice (15). Administration dimethyl sulfoxide (thiazolidinediones) and ethanol (WY of exogenous leptin protein reverses this obesity and reduces 14,643). All cells, including control cells, were treated with the the body weight of normal mice as well (16-19). Leptin same volumes of dimethyl sulfoxide and/or ethanol. appears to be a hormone secreted by adipocytes, where the Isolation and Analyses of RNA. Northern analyses were leptin gene is specifically expressed (15). Obese human sub- performed on total cellular RNA as described (25) with cDNA jects have been found to have increased leptin mRNA in probes for leptin, aP2, glyceraldehyde-3-phosphate dehydro- adipose tissue (20-23) and increased plasma leptin levels (24), genase (GAPDH), and PPAR^y labeled with 32P using random suggesting that dysregulated leptin signaling may contribute to primers (Boehringer Mannheim). RNase protection analysis human obesity. was performed as described (26) The antisense 32P-labeled probe for mouse leptin mRNA was synthesized from pBlue- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: PPARy, peroxisome proliferator-activated receptor y, accordance with 18 U.S.C. §1734 solely to indicate this fact. GADPH, glyceraldehyde-3-phosphate dehydrogenase. 5793 Downloaded by guest on September 29, 2021 5794 Cell Biology: Kallen and Lazar Proc. Natl. Acad. Sci. USA 93 (1996) the vector's T3 and a A J cn script-leptin by using promoter yielded "4 m a (v protected fragment of the expected size (528 nucleotides) + + + + + <l <l < < < corresponding to mouse leptin cDNA bases 94-622. <. 4< 44<4 4 RESULTS . .'' - - A mouse leptin cDNA was obtained by reverse transcription- Leptin- ^ ^ .'~;.-;.: .-:*s polymerase chain reaction of 3T3-L1 adipocyte RNA. This -* cDNA was used to measure leptin mRNA levels by both Northern blot and RNase protection analyses. The Northern PPAR;.'-_ was analysis shown in Fig. 1A reveals that leptin expression -- undetected in mouse 3T3-L1 preadipocytes but was increased GAPDH 7 days after the induction of adipocyte differentiation. The aP2 -- adipocyte-specific genes PPARy and aP2 were also induced during adipogenesis, whereas GAPDH was not. Leptin expres- sion in the cultured adipocytes was markedly less than that in B o0 C) Co c mouse adipose tissue (data not shown) and varied between Zc* t en CMCV experiments, possibly due to differences in serum lots. Similar were for 3T3-L1 and 3T3-F442A o O findings recently reported az mP 0 adipocytes (27, 28). I- Leptin gene expression was first detected on day 3 of the differentiation protocol, as shown by RNase protection anal- ysis in Fig. 1B. Expression was maximal by the fourth or fifth day, consistent with the role of leptin as a hormone derived probe -_ - from mature adipocytes. By contrast, the induction of PPARy Leptin-- occurs between days 1 and 2 after exposure of the preadipo- cytes to differentiating conditions (10, 11). PPARy is known to regulate several genes that are induced late in adipocyte differentiation, including aP2 and phosphoenolpyruvate car- boxykinase (11, 13), and we therefore considered the possi- FIG. 2. Thiazolidinediones inhibit leptin gene expression. (A) bility that PPARy regulates the expression of leptin. Northern analysis showing expression of leptin, PPARy, aP2, and We tested the ability of the PPARy-activating thiazo- GAPDH in 30 tLg of total RNA from 3T3-L1 pre-adipocytes (Pre-Ad) lidinediones as well as other PPAR activators to regulate leptin and adipocytes treated for 24 h with vehicles alone (Ad), 450 -uM Wy gene expression in 3T3-L1 adipocytes. Remarkably, treatment 14,643 (Ad + Wy), 20 ,uM BRL 49653 (Ad + BRL), 20 iLM with the BRL49653 re- pioglitazone (Ad + Pio), 20 jzM troglitazone (Ad + Trog), or 20 ,iM PPARy-specific ligand dramatically CP 86,325 (Ad + CP). (B) RNase protection analysis of total RNA (30 duced leptin gene expression in the adipocytes (Fig. 2). Other j.g) from 3T3-L1 adipocytes after 24 h treatment with vehicles (No thiazolidinediones, including CP 86,325, troglitazone, and addition), Wy 14,643 (450 ,tM), BRL 49653 (20 PLM), CP 86,325 (20 pioglitazone, as well as the less specific PPAR activator Wy ,jM), or 10 nM triiodothyronine (T3). 14,643, had similar effects. In contrast, thiazolidinediones did not inhibit the expression of PPARy or aP2 (Fig. 2A). Fur-