A Selective Peroxisome Proliferator-Activated Receptor-{gamma} (PPAR{gamma}) Modulator Blocks Adipocyte Differentiation but Stimulates Glucose Uptake in 3T3-L1 Adipocytes

Ranjan Mukherjee1, Patricia A. Hoener, Lily Jow, James Bilakovics, Kay Klausing, Dale E. Mais, Amy Faulkner, Glenn E. Croston2 and James R. Paterniti Jr.

Departments of Pharmacology (R.M., P.A.H., L.J., J.B., J.R.P.), Endocrine Research (D.E.M.), Medicinal Chemistry (A.F.), Retinoid Research (K.K.), and New Leads Discovery (G.E.C.) Ligand Pharmaceuticals, Inc. San Diego, California 92121


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) agonists such as the thiazolidinediones are insulin sensitizers used in the treatment of type 2 diabetes. These compounds induce adipogenesis in cell culture models and increase weight gain in rodents and humans. We have identified a novel PPAR{gamma} ligand, LG100641, that does not activate PPAR{gamma} but selectively and competitively blocks thiazolidinedione-induced PPAR{gamma} activation and adipocyte conversion. It also antagonizes target gene activation as well as repression in agonist-treated 3T3-L1 adipocytes. This novel PPAR{gamma} antagonist does not block adipocyte differentiation induced by a ligand for the retinoid X receptor (RXR), the heterodimeric partner for PPAR{gamma}, or by a differentiation cocktail containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Surprisingly, LG100641, like the PPAR{gamma} agonist rosiglitazone, increases glucose uptake in 3T3-L1 adipocytes. Such selective PPAR{gamma} antagonists may help determine whether insulin sensitization by thiazolidinediones is mediated solely through PPAR{gamma} activation, and whether there are PPAR{gamma}-ligand-independent pathways for adipocyte differentiation. If selective PPAR{gamma} modulators block adipogenesis in vivo, they may prevent obesity, lower insulin resistance, and delay the onset of type 2 diabetes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) is a member of the intracellular receptor family of transcription factors (1). PPAR{gamma} is expressed at high levels in fat, spleen, and colon but is also detectable in skeletal muscle, liver, and in other tissues (1, 2). Interest in this receptor increased when it was clear that insulin sensitizers of the thiazolidinedione class [troglitazone, rosiglitazone (BRL 49653), Pioglitazone], are high-affinity ligands for PPAR{gamma} (3). A correlation was reported between the affinity of thiazolidinediones for PPAR{gamma} and the minimum effective dose required to lower glucose levels in diabetic rodent models (4). It was therefore proposed that the antidiabetic activity of these compounds reflects their ability to bind and activate PPAR{gamma}. However, antidiabetic actions of thiazolidinediones may also be partly independent of PPAR{gamma}.

Preadipocyte cell lines such as 3T3-L1 differentiate into adipocytes after treatment with dexamethasone, insulin, and 1-methyl-3-isobutylxanthine (IBMX) (5, 6). Ectopic expression and ligand activation of PPAR{gamma} also induces differentiation of preadipocytes to adipocytes (7). PPAR{gamma} may therefore play a key role in adipogenesis.

To understand better the role of PPAR{gamma} in adipogenesis, we identified a compound, LG100641, that binds to PPAR{gamma} but does not activate gene expression. This ligand displaces thiazolidinediones from the receptor, thereby blocking its transcriptional activity.

We report here that LG100641 is a novel, PPAR{gamma}-specific antagonist that blocks thiazolidinedioneinduced adipocyte differentiation but stimulates insulin-mediated glucose uptake in adipocytes. It also antagonizes target gene activation as well as repression in agonist-treated 3T3-L1 adipocytes. Surprisingly, it does not block differentiation induced by retinoid X receptor (RXR)-selective agonists or by the combination of dexamethasone, insulin, and IBMX. Such a selective antagonist will be a valuable tool to determine whether PPAR{gamma} activation is necessary for inducing adipogenesis and for elucidating the mechanism of insulin sensitization by thiazolidinediones. Compounds of this class could determine whether blocking PPAR{gamma} activity in vivo gives rise to animals with less adipose tissue and increased insulin sensitivity. If that is the case, then PPAR{gamma} ligands with activities very different from the thiazolidinediones may be useful in the treatment of obesity, insulin resistance, and type 2 diabetes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have identified a novel PPAR{gamma} ligand LG100641 (Fig. 1AGo) that displaces [3H]rosiglitazone from PPAR{gamma} with an inhibition constant (Ki ) of 435 nM (Fig. 1BGo). While the affinity is less than that of rosiglitazone (3), it is higher than that of troglitazone (8). Structurally, LG100641 is very different from thiazolidinediones but similar to RXR selective ligands or rexinoids (9, 10). However, LG100641 shows very weak binding to RXRs (Ki of 25 µM for RXR{alpha}, Fig. 1CGo) and no detectable binding to RARs (Ki > 30 µM, data not shown).



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Figure 1. LG100641 Is a PPAR{gamma} Ligand

Structure of LG100641 (A). Competition assays were performed with [3H]rosiglitazone and recombinant baculovirus-expressed hPPAR{gamma}1 (B) or [3H]9-cis-retinoic acid and recombinant baculovirus-expressed RXR{alpha} (C).

 
In a cotransfection assay, LG100641 does not activate PPAR{gamma} (Fig. 2AGo) whereas the PPAR{gamma} ligand and agonist, rosiglitazone, shows significant activity. Since LG100641 binds to PPAR{gamma} but does not transactivate, we next determined whether it antagonizes ligand-induced PPAR{gamma} activation. Indeed, dose-dependent antagonism of rosiglitazone-induced activation of PPAR{gamma} was observed (Fig. 2AGo). LG100641 is a PPAR{gamma}-specific antagonist since it does not antagonize activation of PPAR{alpha} by GW2331, a PPAR{alpha} ligand and agonist (11) (Fig. 2BGo) or that of RXR{alpha} by LG100268 (9, 10) (Fig. 2CGo). It did not activate the glucocorticoid receptor or PPARß ({delta}) (data not shown). Hence, LG100641 is a PPAR{gamma} ligand that is transcriptionally neutral and competitively antagonizes activation of PPAR{gamma} by thiazolidinediones.



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Figure 2. LG100641 Is a PPAR{gamma} Antagonist

Cotransfection assays were performed in CV-1 cells with expression vectors for PPAR{gamma} (A), PPAR{alpha} (B), and RXR{alpha} (C) and PPRE-tk-LUC reporter. Each data point represents the mean ± SD.

 
Ligand activation of nuclear receptors depends upon recruitment of coactivators (12). One such coactivator, the steroid receptor coactivator-1 (SRC-1), is recruited to rosiglitazone-bound PPAR{gamma} (13). Unlike rosiglitazone, LG100641-bound PPAR{gamma} does not recruit SRC-1 (Fig. 3Go). When present in molar excess, LG100641 prevents rosiglitazone from binding to PPAR{gamma} and recruiting SRC-1. Hence, a possible mechanism of LG100641 antagonism is by preventing SRC-1 recruitment by a PPAR{gamma} agonist.



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Figure 3. LG100641 Does Not Recruit SRC-1 and Blocks Thiazolidinedione-Induced SRC-1 Recruitment to PPAR{gamma}

Immunopulldown assays were performed with 1 µl labeled in vitro translated SRC-1, 5 µg recombinant baculovirus-expressed PPAR{gamma} protein extract and DMSO (vehicle), 1 µM rosiglitazone (BRL), 10 µM LG100641 (641) or the combination of rosiglitazone and LG1000641 (1 µM and 10 µM, respectively). Lane 9 shows the band intensity obtained with cell extracts without PPAR{gamma} and denotes labeled SRC-1 nonspecifically pulled down by the antibody.

 
PPAR{gamma} is a key transcriptional factor controlling adipocyte differentiation. Overexpression of PPAR{gamma} in nonadipogenic cell lines (e.g. fibroblasts) and transcriptional activation by a PPAR{gamma} agonist is sufficient to induce the adipocyte phenotype (7). Indeed, induction of adipogenesis is a well documented response of PPAR{gamma} agonists both in vitro (3) and in vivo (14). Since LG100641 antagonizes the effect of rosiglitazone in cotransfection assays, we next determined whether it blocks thiazolidinedione-induced differentiation of 3T3-L1 cells. Figure 4AGo demonstrates induction of adipocyte differentiation by rosiglitazone as shown by an increase in triglyceride content. LG100641 does not increase triglyceride levels, which is consistent with the cotransfection data. It results in a dose-dependent decrease in triglyceride accumulation when added together with rosiglitazone. A similar result was obtained by Oil Red O staining (see illustration on cover and data not shown). We conclude LG100641 antagonizes adipocyte differentiation induced by thiazolidinedione. LG100641 does not, however, block adipocyte differentiation induced by dexamethasone, insulin, and IBMX (Fig. 4BGo).



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Figure 4. LG100641 Blocks Thiazolidinedione-Induced 3T3-L1 Cell Differentiation (A) but not Differentiation Induced with Dexamethasone, Insulin, and IBMX (B) or the RXR Ligand LG100268 (C)

3T3-L1 cells were induced to differentiate with rosiglitazone (BRL 49653), LG100641, LG100268, dexamethasone, insulin, and IBMX or combinations of inducer and antagonist at the concentrations shown. Triglyceride quantification was performed after 7 days of differentiation. Data are the mean ± SEM. Statistically significant differences in the rosiglitazone plus LG100641- treated cells compared with cells treated with rosiglitazone alone were determined by Student’s t test.

 
RXR agonists also induce adipogenesis (15). However, LG100641 does not block LG100268-induced adipogenesis (Fig. 4CGo). LG100641 does not displace LG100268 from RXR and is therefore unable to antagonize LG100268-induced differentiation. We have previously shown that an RXR antagonist (LG100754) does not antagonize PPAR{gamma} activation by rosiglitazone (16). Here we demonstrate the converse: that a PPAR{gamma} antagonist does not antagonize a biological pathway driven by RXR ligands via PPAR{gamma}/RXR. This result also demonstrates the selectivity of LG100641 action for PPAR{gamma}. This is surprising and indicates that adipocyte differentiation may occur via pathways that do not require PPAR{gamma} activation.

LG100641 also blocks PPAR{gamma}-induced 3T3-L1 adipocyte differentiation as measured by expression of aP2, an adipocyte-specific marker gene. The aP2 mRNA is highly expressed in 3T3-L1 cells treated with rosiglitazone, but the induction is severely blocked when LG100641 is added together with rosiglitazone (Fig. 5AGo). LG100641 alone does not induce aP2 gene expression.



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Figure 5. LG100641 Blocks Thiazolidinedione-Induced Gene Expression

A, LG100641 blocks rosiglitazone-mediated aP2 mRNA induction during adipocyte differentiation. Northern blot analysis was performed with RNA from 3T3-L1 cells induced to differentiate with DMSO (vehicle), 1 µM rosiglitazone (BRL), 30 µM LG100641 (LG641), or the combination of rosiglitazone and LG100641 (1 µM and 30 µM), respectively. RNA was harvested from cells after 7 days of differentiation. B, LG100641 antagonizes rosiglitazone-induced gene expression in 3T3-L1 adipocytes. 3T3-L1 cells were fully differentiated into adipocytes with dexamethasone, insulin, and IBMX. After 7 days of differentiation, ligands were added for 3 more days. RNA was harvested and analyzed by Northern blot analysis. Ligand concentrations were the same as in panel A.

 
To determine whether LG100641 antagonizes induction of PPAR{gamma} target genes in fully differentiated adipocytes, 3T3-L1 adipocytes were treated for 3 days with rosiglitazone in the presence or absence of LG100641 and RNA levels of aP2 and adipsin, two known target genes for PPAR{gamma} measured by Northern blot analysis (Fig. 5BGo). Induction of aP2 RNA was observed in the presence of rosiglitazone. This induction was abolished in the presence of the antagonist. Surprisingly, rosiglitazone decreased adipsin RNA levels, and this repression was attenuated in the presence of the antagonist. LG100641 by itself did not have an appreciable effect on the expression of these two genes. However, in adipocytes, it antagonized modulation of these two target genes by the PPAR{gamma} agonist rosiglitazone. To the best of our knowledge, this is the first demonstration of antagonism of thiazolidinedione-induced target gene modulation in mature adipocytes.

These results together demonstrate that LG100641 1) prevents rosiglitazone-induced coactivator recruitment and transactivation of PPAR{gamma}, 2) blocks conversion of preadipocytes to adipocytes by rosiglitazone, and 3) antagonizes thiazolidinedione-induced target gene expression in adipocytes. Therefore, LG100641 is an antagonist for PPAR{gamma}.

PPAR{gamma} agonists (thiazolidenediones) are insulin sensitizers (17); they decrease hyperglycemia and hyperinsulinemia and improve insulin sensitivity in peripheral tissues in vivo (18). The mechanism by which thiazolidinediones improve insulin sensitivity is not known. Insulin-stimulated glucose uptake in 3T3-L1 adipocytes is a well established assay in cell culture and a relevant measure of insulin action in peripheral tissues. PPAR{gamma} agonists increase glucose uptake in 3T3-L1 adipocytes (19). Since LG100641 is a PPAR{gamma} antagonist, we tested its effect on the magnitude of glucose uptake in 3T3-L1 adipocytes.

In this assay, rosiglitazone increases 2-deoxyglucose uptake (Fig. 6AGo) in agreement with results reported for other thiazolidinediones (19). Tumor necrosis factor-{alpha} (TNF{alpha}), a cytokine that induces insulin resistance in 3T3-L1 adipocytes (20) and in vivo (21), decreases insulin-stimulated glucose uptake in these cells. This result demonstrates that the assay identifies compounds that stimulate insulin sensitivity, e.g. rosiglitazone, as well as those that induce insulin resistance, e.g. TNF{alpha}. However, LG100641, even at the highest dose of 30 µM, does not decrease basal or insulin-stimulated glucose uptake. In fact, there is a small but significant increase in glucose uptake at the higher concentrations. Therefore, at a concentration that clearly blocks adipocyte differentiation (Fig. 4AGo), LG100641 does not decrease glucose uptake in 3T3-L1 adipocytes. Hence, LG100641 is a selective modulator of PPAR{gamma} activity. When both ligands were added together, LG100641 attenuates the high response induced by rosiglitazone consistent with its ability to displace rosiglitazone from PPAR{gamma} (Fig. 6BGo).



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Figure 6. LG100641 Increases 2-Deoxyglucose Uptake in 3T3-L1 Adipocytes

A. 3T3-L1 adipocytes were treated with vehicle (control), rosiglitazone (BRL 49653), LG100641, and TNF{alpha} at the concentrations indicated for 3 days, and [3H]2-deoxyglucose uptake assay was performed as described in Materials and Methods. Data are the mean ± SEM. Statistically significant differences compared with the vehicle-treated cells were determined by Student’s t test and are indicated. LG100641 antagonizes rosiglitazone-induced stimulation of 2-deoxyglucose uptake in 3T3-L1 adipocytes (B). The assay was performed as above with the indicated concentrations of ligands. Statistically significant decreases in LG100641-treated samples compared with BRL 49653 treatment alone are indicated.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We report the identification of a PPAR{gamma} antagonist, LG100641, a novel PPAR{gamma} ligand structurally different from the thiazolidinediones but related to a certain rexinoid selective structure, LG100268. Surprisingly, it binds very poorly to RXRs but has a greater affinity for PPAR{gamma} than troglitazone. In binding assays carried out against all three RXR subtypes up to a concentration of 30 µM, LG100641 failed to achieve a 50% inhibition of [3H]-9-cis-RA, (Fig. 1CGo and data not shown). However, extrapolating to obtain an IC50 value allowed approximate Ki values to be obtained in the 15–25 µM range for the three RXR subtypes. Unlike the thiazolidinediones, it does not transactivate PPAR{gamma} but, instead, acts as an antagonist. LG100641 may induce a conformational change in PPAR{gamma} different from that of an agonist, resulting in failure to recruit a coactivator (e.g. SRC-1, Fig. 3Go). Hence, LG100641-bound PPAR{gamma} is transcriptionally silent.

LG100641 blocks rosiglitazone-induced 3T3-L1 cell differentiation (Fig. 4AGo) but not adipocyte differentiation induced by dexamethasone, insulin, and IBMX. This suggests that there may be at least two pathways for 3T3-L1 adipocyte differentiation. One is regulated by PPAR{gamma} and its ligand and is blocked by a PPAR{gamma} antagonist. The second pathway is independent of PPAR{gamma} ligand activation and therefore cannot be blocked by the PPAR{gamma} antagonist. Differentiation by insulin, dexamethasone, and IBMX acting through the second pathway may involve activation of genes like C/EBP{alpha} (22). Hence, a PPAR{gamma} antagonist is a useful tool to dissect the process of adipogenesis.

LG100641 does not block RXR agonist-induced adipogenesis. In transactivation, the transcriptional response of an agonist to one partner of the PPAR/RXR heterodimer is enhanced by an agonist bound to the other partner (9, 16). However, a competitive antagonist bound to one partner does not antagonize the transcriptional response from an agonist bound to the other. This is true for both RXR antagonists (e.g. LG100754) (16) and PPAR{gamma} antagonists (e.g. LG100641). It is also possible that a different class of antagonists, when bound to PPAR, induces a conformational change in the PPAR/RXR heterodimer such that it disrupts binding of the RXR ligand and vice versa. Such ligands would antagonize transactivation from either side of the heterodimer.

LG100641 inhibits thiazolidinedione-induced conversion of preadipocytes to adipocytes. In adipocytes it antagonized the ability of rosiglitazone to induce aP2 expression. We show that although rosiglitazone induced adipsin expression during adipocyte differentiation (23), it unexpectedly repressed adipsin gene expression in mature adipocytes. LG100641 blocked agonist-mediated repression of adipsin. Thus, in mature adipocytes it also acts as a PPAR{gamma} antagonist effectively attenuating agonist-mediated transactivation and transrepression of target genes.

Such an antagonist might be expected to decrease glucose uptake. However, it did not inhibit basal and insulin-mediated glucose disposal in adipocytes. In fact, LG100641 at high concentrations acts as a weak agonist in glucose uptake assays. Identification of such selective PPAR{gamma} modulators that block adipocyte differentiation but increase glucose uptake offer the potential for improved therapy for metabolic diseases. A partial PPAR{gamma} agonist (GW0072) that inhibits adipocyte differentiation has been recently described (23). LG100641 is structurally different from GW0072, and in contrast to GW0072, LG100641 in the cotransfection assays described shows no agonism of PPAR{gamma}.

Heterozygous PPAR{gamma} knockout mice show increased insulin sensitivity in response to a high-fat diet compared with wild-type animals (24). This suggests that in some cases (e.g. obesity) down-regulating PPAR{gamma} activity may increase insulin sensitivity.

Since PPAR{gamma} activation is crucial for adipogenesis, PPAR{gamma} antagonists may be useful in the treatment of obesity. Obese individuals have an increased body fat content above a normal standard range. They often have elevated levels of FFA, which are recognized PPAR ligands. 15-Deoxy-{Delta} 12,14-PGJ2, is a naturally occurring ligand and agonist for PPAR{gamma} (25, 26). Treatment with current PPAR{gamma} agonists, the thiazolidinediones, leads to increased adiposity and body weight gain in rodents (14) and weight gain in human patients (27, 28). Our data suggest that compounds such as LG100641 could prevent adipogenesis, insulin resistance, and obesity by blocking endogenous ligand-induced activation of PPAR{gamma} and the consequent differentiation of preadipocytes into adipocytes. In addition, these compounds may increase glucose disposal in peripheral tissues.

Controlling obesity in rhesus monkeys prevents the onset of diabetes mellitus (29). In addition, an abundant literature documents that weight loss in type 2 diabetic humans improves insulin sensitivity. An effective antiobesity agent may lower insulin resistance and prevent or significantly delay the onset of type 2 diabetes. Our data suggest that such selective PPAR{gamma} modulators might prevent obesity without inducing insulin resistance in peripheral tissues. In vivo studies are necessary to further explore this hypothesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
2-[(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydro-2-naphthyl) carbonyl]benzoic acid (LG100641), GW2331(3), rosiglitazone (BRL 49653), and LG100268 were synthesized at Ligand Pharmaceuticals, Inc. (San Diego, CA). Stock solutions were made by dissolving the compounds in dimethylsulfoxide (DMSO). TNF{alpha} and insulin were purchased from Genzyme Corp. and Sigma (St. Louis, MO), respectively.

Ligand Binding Assay
Binding affinities of LG100641 to recombinant baculovirus-expressed hPPAR{gamma}1 and hRXR were determined using [3H] rosiglitazone and [3H] 9-cis-retinoic acid as the radioligand, respectively (30). The Ki values were calculated by measuring IC50 values and the application of Cheng-Prusoff equation (31).

Transfection Assays
Cotransfection assays were performed in CV-1 cells (9) using expression vectors for hPPAR{gamma}1 (16), hPPAR{alpha} (32), and hRXR{alpha} (33) and the PPRE-tk-LUC reporter (34).

PPAR-SRC-1 Immunopulldown Assays
In vitro translated, labeled SRC-1 (12), and 5 µg recombinant baculovirus-expressed hPPAR{gamma}1 protein extract were incubated with ligand in IP buffer (10 mM HEPES, pH 7.8, 50 mM KCl, 1 mM DTT, 2.5 mM MgCl2, 20% glycerol, and 0.1% NP40) for 1 h at 4 C. Twenty microliters of protein A Sepharose (Pharmacia Biotech, Piscataway, NJ) stock made by adding 1.5 g powder in 16 ml IP buffer, and 2 µl of PPAR{gamma} polyclonal antibody were added in 100 µl of IP buffer, rotated for 1 h at 4 C and centrifuged for 6 min at 14,000 rpm in an Eppendorf 5415C microcentrifuge. The beads were washed three times with cold IP buffer and boiled in Laemmli buffer, and the supernatant was electrophoresed on 10% TrisGlycine gels (Novex, San Diego, CA). The bands were visualized by autoradiography using the Amplify detection system (Pharmacia Biotech, Piscataway, NJ).

3T3-L1 Cell Culture and Differentiation Assays
3T3-L1 cells (American Type Culture Collection, Manassas, VA) were grown in DMEM supplemented with 10% heat-inactivated calf serum. Two days after reaching confluence, differentiation was induced in DMEM supplemented with 10% FCS (HyClone Laboratories, Inc., Logan, UT), insulin (10 µg/ml, Sigma, St. Louis, MO) and PPAR{gamma} or RXR ligands. After 3 days the medium was replaced by DMEM supplemented with 10% FCS, insulin, and PPAR{gamma} or RXR ligands. Two days later the cells were refed with DMEM plus FCS only. After 7 days of differentiation, cells were harvested for RNA and triglyceride analysis, or stained by Oil Red O, an indicator of cell lipid content.

Alternatively, cells were induced to differentiate with the combination of dexamethasone, insulin and IBMX (35) with or without PPAR{gamma} ligand present during the first 5 days of differentiation. Cells were harvested for triglyceride analysis after 7 days of differentiation.

Triglyceride Assay
Treated 3T3-L1 adipocytes were washed gently with PBS and lysed with PBS supplemented with 0.1% NP40. Triglyceride content was measured using the Triglyceride GPO-Trinder kit (Sigma). The absorbance was measured at 540 nm, and triglyceride was normalized to protein content determined by the Bradford assay.

[3H]-2-deoxy-D-Glucose Uptake Assay in Differentiated 3T3-L1 Adipocytes
3T3-L1 cells were grown in 12-well tissue culture plates (Corning, Inc. Costar, Corning, NY) and differentiated into adipocytes with dexamethasone, insulin, and IBMX (35). Seven days after induction of differentiation, test compounds were added for an additional 3 days. After two rinses with serum-free DMEM, cells were incubated for 3 h in serum-free DMEM and rinsed at room temperature four times with freshly prepared KRPH buffer (5 mM phosphate, pH 7.4 (NaH2PO4-H2O + Na2HPO4-7H2O), 20 mM HEPES pH 7.4, 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM KCl). The buffer was removed and the cells were incubated with or without 100 nM insulin in KRPH buffer at 37 C for 20 min. The buffer was replaced with 1 µCi/well of [3H]-2-deoxy-D-glucose (NEN Life Science Products; Boston, MA) in KRPH buffer supplemented with 100 µM 2-deoxy-D-glucose (Aldrich, Milwaukee, WI) with incubation for 10 min at room temperature. The supernatant was removed, and plates were rinsed carefully four times with cold PBS. Plates were drained briefly and cells lysed overnight in 0.5 ml/well 0.1 N NaOH. Four hundred microliters of lysate were added to a scintillation vial, neutralized with 40 µl of 1 N HCl, and 4 ml of Ecoscint A scintillation fluid (National Diagnostics; Atlanta, GA) were added. The vials were mixed and counted.

Northern Blot Analysis
3T3-L1 adipocyte RNA (10 µg per lane) was loaded onto a 1% agarose-formaldehyde gel. Northern blotting was done by standard procedures with cDNA fragments labeled by random priming. The bands were visualized by autoradiography. To control for loading, the blots were stripped and hybridized with pTRI RNA 28S riboprobe (Ambion, Inc. Austin, TX).


    ACKNOWLEDGMENTS
 
We gratefully acknowledge the help of Kathy Ogilvie, Marcus Boehm, Luc Farmer, and members of New Leads Discovery. We also thank Drs. Bruce Spiegelman for aP2 and adipsin and Ming-Jer-Tsai for the SRC-1 cDNA.


    FOOTNOTES
 
Address requests for reprints to: James R. Paterniti, Jr. Ligand Pharmaceuticals, Inc., 10275 Science Center Drive, San Diego, California 92121 or Ranjan Mukherjee, DuPont Pharmaceuticals Co., Experimental Station, E400, Wilmington, DE 19880.

Ranjan Mukherjee and James R. Paterniti are joint corresponding authors.

1 Present address: Dupont Pharmaceuticals Company, Experimental Station, E/400, Wilmington, DE 19880-0400. Back

2 Present address: Acadia Pharmaceuticals, 3911 Sorrento Valley Boulevard, San Diego, California 92121-1402. Back

{hd2}Note Added in Proof

{smtexf}While this manuscript was in preparation, Wright et al., identified a synthetic antagonist for PPAR{gamma}, BADGE with a Kd(app) of 100 µM (H. M. Wright, C. B. Clish, T. Mikami, S. Hauser, K. Yanagi, R. Hiramatsu, C. N. Serhan, B. M. Spiegelman, J Biol Chem 275:1873–1877, 2000). This compound inhibited adipogenesis, but its effect on glucose uptake was not shown.

Received for publication March 1, 2000. Revision received June 1, 2000. Accepted for publication June 8, 2000.


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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