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J. Biol. Chem., Vol. 278, Issue 33, 30614-30623, August 15, 2003

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Peroxisome Proliferator-activated Receptor- Represses GLUT4 Promoter Activity in Primary Adipocytes, and Rosiglitazone Alleviates This Effect^*

Michal Armoni  , Natalia Kritz , Chava Harel , Fabiana Bar-Yoseph , Hui Chen ¶, Michael J. Quon ¶ and Eddy Karnieli 

From the Institute of Endocrinology, Diabetes, and Metabolism, Rambam Medical Center and Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel and the ^¶NCCAM, National Institutes of Health, Bethesda, Maryland 20892-1632

Received for publication, May 5, 2003 , and in revised form, May 29, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The synthetic thiazolidinedione ligands of peroxisome proliferator-activated receptor- (PPAR) improve insulin sensitivity in type II diabetes and induce GLUT4 mRNA expression in fat and muscle. However, the molecular mechanisms involved are still unclear. We studied the regulatory effects of PPAR and its ligands on GLUT4 gene expression in primary rat adipocytes and CHO-K1 cells cotransfected with PPAR and the GLUT4 promoter reporter. PPAR1 and PPAR2 repressed the activity of the GLUT4 promoter in a dose-dependent manner. Whereas this repression was augmented by the natural ligand 15-prostaglandin J₂, it was completely alleviated by rosiglitazone (Rg). Ligand binding-defective mutants PPAR1-L468A/E471A and PPAR2-L496A/E499A retained the repression effect, which was unaffected by Rg, whereas the PPAR2-S112A mutant exhibited a 50% reduced capacity to repress GLUT4 promoter activity. The –66/+163 bp GLUT4 promoter region was sufficient to mediate PPAR inhibitory effects. The PPAR/retinoid X receptor- heterodimer directly bound to this region, whereas binding was abolished in the presence of Rg. Thus, we show that PPAR represses transcriptional activity of the GLUT4 promoter via direct and specific binding of PPAR/retinoid X receptor- to the GLUT4 promoter. This effect requires an intact Ser¹¹² phosphorylation site on PPAR and is completely alleviated by Rg, acting via its ligand-binding domain. These data suggest a novel mechanism by which Rg exerts its antidiabetic effects via detaching PPAR from the GLUT4 gene promoter, thus leading to increased GLUT4 expression and enhanced insulin sensitivity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Glucose uptake in eukaryotic cells is mediated by the GLUT family of glucose transport proteins. Six isoforms of GLUT proteins have been identified and cloned, and their tissue specificity has been extensively studied (1). The GLUT4 isoform is expressed in insulin target tissues, i.e. adipose and muscle, where it mediates an increase in glucose uptake in response to acute insulin stimulation. Since glucose transport is a rate-limiting step in glucose metabolism, GLUT4 expression is tightly regulated at both the mRNA and protein levels (2).

The critical importance of tissue-specific regulation of GLUT4 expression for maintaining normal glucose homeostasis is amplified in altered metabolic states. Insulin resistance in type II diabetes, obesity, and aging has been associated with marked reduction in the intracellular pool of GLUT4 proteins; this, in turn, impairs insulin stimulation of glucose transport (3–5). Similarly, in insulin-deficient states such as fasting and streptozotocin-induced diabetes, the intracellular pools of GLUT4 protein were dramatically depleted (6, 7), leading to post-receptor insulin resistance. GLUT4 knockout mice exhibit mild hyperglycemia (8), whereas glycemic control is markedly improved when GLUT4 is overexpressed (9).

Identification of the peroxisome proliferator-activated receptor (PPAR)¹ family of nuclear transcription factors provided new insight into the molecular mechanisms involved in adipocyte differentiation. Three PPAR isoforms (, , and ) have been identified and cloned, and these differ in their tissue distribution and ligand specificity (10). Whereas PPAR (also called NUC1 in humans) is ubiquitously expressed in many tissues, PPAR is found predominantly in hepatocytes, cardiomyocytes, and entrocytes (11). PPAR is a transcription factor that plays a pivotal role in adipocyte differentiation and in expression of adipocyte-specific genes (12). Recently, two human PPAR isoforms have been identified (PPAR1 and PPAR2) that arise from different promoter usage and alternative splicing (13, 14). Both isoforms have a ligand-dependent binding domain and a ligand-independent activation domain (referred to as A/B). Transcriptional activity of PPAR is induced by binding to a diverse group of ligands, including natural fatty acid derivatives, antidiabetic thiazolidinediones, and nonsteroidal anti-inflammatory drugs (15). Ligand binding by PPAR, as well as by the entire nuclear receptor superfamily, is independently mediated by the carboxyl-terminal ligand-binding domain of the receptor. The two isoforms differ only in their N termini, with PPAR2 having an additional 28 amino acids at its amino terminus. These N termini contain distinct ligand-independent activation domains, which confer distinct activation capacities to each isoform and different responsiveness to insulin (16).

The identification of subtype-selective ligands for PPAR has led to the discovery that these ligands play a role in the regulation of lipid metabolism and glucose homeostasis. The antidiabetic thiazolidinedione (TZD) agents have been shown to act as potent agonists of PPAR (17). Both troglitazone (Tg) and rosiglitazone (Rg), novel hypoglycemic agents of the TZD family, were found to increase insulin sensitivity and responsiveness; to correct hyperglycemia, hyperinsulinemia, and hyper-triglyceridemia in type II diabetic patients; and to enhance differentiation, basal glucose uptake, and GLUT1 protein levels in adipose cells (for reviews, see Refs. 12 and 18). On the other hand, studies regarding GLUT4 gene regulation have thus far provided unclear and even conflicting results. Whereas treatment of obese Zucker fa/fa rats with TZD drugs enhances adipogenic differentiation and increases GLUT4 mRNA levels (19), incubation of 3T3-L1 cells with other PPAR ligands results in down-regulation of GLUT4 mRNA levels (20). Whereas insulin stimulates the ligand-independent activation of PPAR1 and PPAR2 (21), obesity and nutritional factors influence the expression of only PPAR2 in human adipocytes. Furthermore, whereas the TZD ligands are considered beneficial for insulin sensitivity, heterozygous PPAR knockout mice exhibit improved insulin sensitivity and are protected from the development of insulin resistance due to adipocyte hypertrophy on a high fat diet (22, 23). Thus, PPAR appears to be a transcription factor that has highly complex and versatile modes of action, including intramolecular communication between its amino-terminal A/B domain and its carboxyl-terminal ligand-binding domain. The effects of PPAR depend on the specific ligands, growth factors, cofactors, and kinases present in the tissue(s) where it is expressed. Whereas PPAR is expressed mainly in adipose tissue, natural PPAR ligands discovered to date have been rarely evaluated in adipocyte cellular models. Therefore, this study was undertaken to identify the molecular mechanisms by which PPAR and its ligands regulate the expression of the GLUT4 gene at the level of transcription in bona fide insulin target cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Materials—The thiazolidinedione PPAR ligand troglitazone was obtained from Parke-Davis, and rosiglitazone was from GlaxoSmithKline (West Sussex, UK). Two commercially available 15-deoxy-12,14-prostaglandin J₂ (15-PGJ₂) preparations were used, one from Cayman Chemical Co., Inc. (Ann Arbor, MI) and one from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). Compounds were handled exactly as instructed by the manufacturer and used within <1 year. Experiments with either compound yielded similar results. The luciferase reporter assay kit, calf intestine alkaline phosphatase, and restriction endonucleases were obtained from Promega (Madison, WI). T4 polynucleotide kinase was obtained from Roche Applied Science (Ottweiler, Germany). [-³²P]ATP (6000 Ci/mmol) and cell labeling-grade L-[³⁵S]methionine were obtained from Amersham Biosciences (Buckinghamshire, UK). Cell culture reagents were from Invitrogen (Paisley, UK).

PPAR Expression Vectors—pSVSPORT1 expression vectors for wild-type mouse PPAR2 (mPPAR2), mutant mPPAR2-S112A, and wild-type human retinoid X receptor- (RXR) were kindly provide by Dr. Bruce Spiegelman (Dana-Farber Cancer Institute, Boston, MA) and have been described previously (24). mPPAR2-S112A contains a serine-to-alanine substitution at position 112, which abolishes the MAPK phosphorylation site. The PPAR2-L496A/E499A double mutant was derived from wild-type mPPAR2 using mutagenic oligonucleotides 5'-GAC-ATG-AGC-CTT-CAC-CCC-GCG-CTC-CAG-GCG-ATC-TAC-AAG-GAC-TTG-3' and 5'-CAA-GTC-CTT-GTA-GAT-CGC-CTG-GAG-CGC-GGG-GTG-AAG-GCT-CAT-GTC-3' and the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and its sequence was confirmed by direct sequencing. This mutant carries alanine at both positions 496 and 499 to replace the highly conserved residues Leu⁴⁹⁶ and Glu⁴⁹⁹ in helix 12 of the ligand-binding domain. Constructs of human PPAR1 (hPPAR1), which is 98% homologous to mouse PPAR1, and its mutant PPAR1-L468A/E471A, corresponding to the same sites in mPPAR2-L496A/E499A, were obtained from Dr. V. K. Chatterjee (University of Cambridge, Cambridge, UK) and have been described (25, 26). Both hPPAR1 constructs were subcloned into the pcDNA3 vector.

GLUT4- and PPRE-Luciferase Promoter Reporters—The pGEM7-Luc construct was generated as described by Quon et al. (27) by subcloning the firefly luciferase gene (as a HindIII/SmaI fragment from pRSV-Luc) into the pGEM7zf(+) plasmid (Promega). This vector is referred to as p0-Luc, as it is devoid of any eukaryotic promoter, and was used as a negative control throughout these experiments. The GLUT4 promoter reporter (pGLUT4-P-Luc) was generated by subcloning the upstream 5'-region of the rat GLUT4 gene (from –2213 to +163 bp relative to the transcription start site) into p0-Luc upstream of the luciferase gene. GLUT4 promoter deletion mutants were generated from pGLUT4-P-Luc by excising specific fragments with the appropriate restriction endonuclease enzymes, followed by religation. The (AOX)₃-Luc reporter, containing an acyl-coenzyme A oxidase triple PPAR-response element (PPRE), was obtained from Dr. Christopher Glass (University of California at San Diego, La Jolla, CA) and has been described previously (28).

Transient Transfection and GLUT4 Promoter Activity Assays— CHO-K1 fibroblasts were plated in 100-mm dishes (750,000 cell/dish) and transfected 24 h later with a total of 15 µg of affinity-purified plasmid DNA (EndoFree plasmid purification kit, QIAGEN Inc., Hilden, Germany) using the calcium phosphate DNA precipitation method (29). Cells were transfected with 7.5 µg of pGLUT4-P-Luc reporter cDNA, 0–6 µg of PPAR cDNA (wild-type 1 or 2 isoform or a related mutant), and 1.5 µg of pCMV--galactosidase expression vector. Five hours later, the DNA-containing medium was washed and replaced with incubation medium (supplemented with stripped fetal bovine serum), and the cells were incubated for additional 48 h at 37 °C.

For experiments in insulin target cells, isolated adipocytes were prepared from rat epididymal fat pads according to procedures detailed by us previously (3). Cells were grown in primary cultures and transfected according to the technique originally developed by Quon (30). Briefly, adipocytes were cotransfected by electroporation (three pulses at 960 V, 50 microfarads; Bio-Rad GenePulser) with 2.0 µg of pGLUT4-P-Luc reporter DNA, 0–6 µg of expression vector for PPAR1 or PPAR2 (wild-type or mutant), and 0.5 µg of pCMV--galactosidase. One hour later, an equal volume of incubation medium (supplemented with 7% bovine serum albumin) was added to the DNA-containing medium, and the cells were incubated for an additional 18 h at 37 °C.

One set of dishes cotransfected with the (AOX)₃-Luc promoter reporter was used as a positive control for PPAR transcriptional activation. To determine nonspecific activity, one set of dishes was cotransfected with the control vector p0-Luc and either 0 or 6 µg of PPAR expression vector. In each set of experiments, this promoterless p0-Luc reporter exhibited only background levels of activity that were unaffected by PPAR itself (data not shown), thus ruling out the possibility of global, nonspecific activity of the expression vectors tested. In each experiment, the total amount of DNA transfected was held constant by adding the relevant insertless expression vector to account for squelching by the promoter itself. Luciferase activity was assayed at room temperature using the luciferase reporter assay kit and a Lumat LB9501 luminometer (Berthold Systems Inc., Nashua, NH). Luciferase activity was normalized to -galactosidase activity as an internal control (29). Within each experiment, values are expressed as a percentage of the induced basal GLUT4 promoter activity, i.e. the activity obtained in cells transfected with the pGLUT4-P-Luc reporter alone. Cell viability was assesses by trypan blue exclusion, and transfection efficiency was monitored by fluorescence microscopy of pCIS2-EGFP-transfected cells. Each experiment was repeated three to six times, with each sample analyzed in triplicates (CHO-K1 cells) or in quadruplicates (adipocytes).

In Vitro Translation and Electrophoretic Mobility Shift Assays—The TNT SP6/T7-coupled reticulocyte lysate system (Promega) was used to generate in vitro translated RXR and PPAR proteins from the corresponding cDNA. The resulting protein lysate was used in electrophoretic mobility shift assays (EMSAs). Protein expression was confirmed by SDS-PAGE, followed by PhosphorImager analysis of proteins translated in the presence of [³⁵S]methionine. In vitro translation reactions generated sufficient protein to use in EMSA studies. EMSA studies were performed as detailed by us before (31). Briefly, a GLUT4 promoter-derived –66/+163 bp DNA probe was prepared by NcoI/Hin- dIII digestion of pGLUT4-P-Luc and end-labeled with [-³²P]ATP in the presence of polynucleotide kinase. EMSA was then performed using the gel shift assay system kit from Pharmacia, with minor modifications. Protein/DNA binding reactions were assembled in a total volume of 20 µl, which included 2–4 µl of in vitro translated PPAR2 and/or RXR protein lysate, 50,000 cpm radiolabeled probe, and 4 µg of poly(dI-dC) in buffer containing 10 mM HEPES (pH 7.9), 1 mM dithiothreitol, 1 mM EDTA, 4% Ficoll, and 50 mM KCl. Competition experiments were performed in the presence of a 50–100-fold molar excess of unlabeled DNA probe, which was added 10 min prior to addition of the radiolabeled probe. An OCT1-derived oligonucleotide (supplied with the kit and containing no known PPRE motifs) was included as a nonspecific inhibitor. For supershift assays, 2 µl of anti-PPAR antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) were preincubated with the protein lysate for 10 min prior to addition of the labeled probe. After incubating the samples for 30 min at 25 °C, protein·DNA complexes were resolved by electrophoresis on 5% nondenaturing polyacrylamide gels at 150 V at 4 °C in 0.5x buffer containing 45 mM Tris (pH 8.3), 45 mM borate, and 1.0 mM EDTA. Gels were fixed in 10% acetic acid for 15 min, dried, and analyzed by phosphorimaging.

Statistical Analysis—Wherever stated, the data were analyzed statistically using two-tailed Student's t test for unpaired samples and were considered significant at p < 0.05. Group results are expressed as means ± S.E. of individual data from three to six assays, with each sample analyzed in triplicates (CHO-K1 cells) or in quadruplicates (adipocytes).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Wild-type PPAR1 and PPAR2 Repress Transcriptional Activity of the GLUT4 Promoter
The effects of PPAR on transcriptional activity of the GLUT4 promoter were studied in primary cultures of rat adipocytes as well as in CHO-K1 cells, and the data are shown in Fig. 1. Surprisingly, we found that coexpression of wild-type PPAR2 repressed transcriptional activity of the GLUT4 promoter reporter in a dose-dependent manner to as much as 40–50% below basal levels (i.e. levels obtained in cells transfected with the reporter alone) in both CHO-K1 fibroblasts and rat adipocytes (Fig. 1, A and C, respectively). The wild-type PPAR1 isoform also repressed GLUT4 promoter activity, but to a much greater extent compared with PPAR2, reaching a maximum effect of 75% trans-repression in both cell types (Fig. 1, B and D). Under similar conditions, both PPAR isoforms dose-dependently activated the PPRE from the (AOX)₃-Luc reporter, which was used as a positive control (Fig. 1, E and F), thus excluding the possibility of either cytotoxic or squelching effects in the expression system. The two isoforms differ only in their N termini, with PPAR2 having an additional 28 amino acids at its amino terminus. Werman et al. (16) have found that the N termini of PPAR1 and PPAR2 have distinct ligand-independent activation capacities. Indeed, the present data show that PPAR1 is a more potent transcriptional repressor than PPAR2, at least with respect to the GLUT4 gene. Thus, our data indicate that, in the absence of any ligand, both the PPAR1 and PPAR2 isoforms exhibit an inherent ligand-independent capacity to trans-repress transcriptional activity of the GLUT4 promoter. These findings appear to contradict the current dogma of PPAR biology, at least with respect to glucose homeostasis. However, Miles et al. (23) found that insulin sensitivity is enhanced in knockout mice heterozygous for PPAR deficiency, although the molecular basis for this effect is unknown. These investigators suggested that, whereas exogenous pharmacological activation of PPAR improves insulin sensitivity, endogenous activation of the receptor by its natural ligands might serve to dampen insulin action, thereby promoting insulin resistance. Our present data support this hypothesis and, for the first time, provide evidence for a molecular mechanism by which PPAR can inhibit insulin action.

View larger version (27K): [in this window] [in a new window]   FIG. 1. Ligand-independent effects of PPAR on GLUT4 promoter activity. A and B, effects in CHO-K1 cells. CHO-K1 cells were cultured at described under "Experimental Procedures." Twenty-four hours after plating, the cells were cotransfected with 7.5 µg of full-length pGLUT4-P-Luc promoter reporter together with 0–6 µg of pSVSPORT1 expression vector for wild-type mPPAR2 (), mPPAR2-S112A (), or mPPAR2-L496A/E499A () (A) or of pcDNA3 expression vector for either wild-type hPPAR1 () or hPPAR1-L468A/E471A () (B). C and D, effects in primary rat adipocytes. Isolated rat adipocytes were prepared and cultured as described under "Experimental Procedures." Isolated adipocytes were cotransfected with 2 µg of full-length pGLUT4-P-Luc promoter reporter together with 0–6 µg of pSVS-PORT1 expression vector for wild-type mPPAR2 (), mPPAR2-S112A (), or mPPAR2-L496A/E499A () (C) or of pcDNA3 expression vector for either wild-type hPPAR1 or hPPAR1-L468A/E471A ()(D). E and F, effects on (AOX)3-Luc. CHO-K1 cells () or isolated rat adipocytes () were cotransfected with 2 µg of (AOX)3-Luc promoter reporter together with 0–5 µg of DNA from either wild-type mPPAR2 (E) or wild-type hPPAR1 (F). The relevant empty vector was added to keep the total amount of DNA transfected constant. GLUT4 promoter (GLUT4-P) activity was determined by measuring luciferase and -galactosidase activities as described under "Experimental Procedures." Within each experiment, the results are expressed as a percentage of the basal GLUT4 promoter activity, i.e. the activity obtained from the expressed pGLUT4-P-Luc reporter alone. The data are expressed as means ± S.E. of three to six experiments, with each sample analyzed in triplicate. A schematic structure of the PPAR protein is depicted above the graph, with the site of mutation denoted with a thick bar. LBD, ligand-binding domain.

Various PPAR Domains Make Differential Contributions to GLUT4 Promoter Repression
Like other members of the nuclear receptor/transcription factor family, PPAR can exert its transcriptional regulatory effects via either ligand-independent or ligand-dependent pathways. Distinct domains of the PPAR protein mediate these activities; AF-1 mediates ligand-independent activity and AF-2 mediates ligand-dependent activity, respectively. To further characterize the specific contribution of the various PPAR functional domains to GLUT4 repression, we used PPAR mutants that are defective in either AF-1 (ligand-independent) or AF-2 (ligand-dependent) activity.

Contribution of the AF-1 Domain of PPAR—The contribution of the AF-1 ligand-independent component(s) of PPAR trans-repression was studied in cells cotransfected with mPPAR2-S112A. In the absence of ligands, PPAR demonstrates a basal transcriptional activity that is exerted in a ligand-independent pathway via MAPK phosphorylation of serine at position 112 (32). In mPPAR2-S112A, Ser¹¹² (which corresponds to Ser¹¹⁴ in the human ortholog) was substituted with alanine, thereby rendering the protein unable to be phosphorylated by MAPK and hence defective in its AF-1 function. Our data show that this non-phosphorylatable mutant exhibits significantly reduced capacity to trans-repress transcription from the GLUT4 promoter in both CHO-K1 cells and primary adipocytes (Fig. 1, A and C, respectively).

Contribution of the AF-2 Domain of PPAR—The contribution of the AF-2 ligand-dependent component(s) of PPAR trans-repression was studied in cells cotransfected with either hPPAR1-L468A/E471A or the corresponding mutant mPPAR2-L496A/E499A. These mutants were constructed following heterozygous mutations in the ligand-binding domain of PPAR that were found in three subjects with severe insulin resistance, type II diabetes mellitus, and hypertension (25). In the PPAR crystal structure, these mutations destabilized helix 12, which mediates transactivation, consistent with marked impairment in transcriptional activation and dominant-negative inhibition of coexpressed wild-type PPAR. Our data show that both the PPAR1 and PPAR2 double mutants, although lacking ligand binding capacity, retain the capacity to repress transcription from the GLUT4 promoter, similar to wild-type PPAR, in both CHO-K1 cells (Fig. 1, A and B) and primary adipocytes (Fig. 1, C and D). These results indicate that impairment of the AF-1 ligand-independent (but not AF-2 ligand-dependent) function of PPAR1 and PPAR2 significantly impairs the basal capacity of this transcription factor to trans-repress transcriptional activity of the GLUT4 promoter compared with the intact protein.

Effects of TZD on GLUT4 Promoter Regulation by PPAR1 and PPAR2
Because TZDs enhance insulin sensitivity, it has been assumed that PPAR (and its ligands) enhances insulin sensitivity by ensuring proper expression levels of key glucoregulatory genes. However, our findings show that PPAR repressed transcription of the insulin-responsive GLUT4 gene. Therefore, we studied the molecular basis of TZD action affecting PPAR-induced repression of the GLUT4 promoter. We examined the effects of two hypoglycemic TZD drugs that are well known synthetic ligands of PPAR, troglitazone and rosiglitazone. Isolated primary adipocytes as well as CHO-K1 fibroblasts that had been cotransfected with pGLUT4-P-Luc and with either wild-type PPAR2 or the expression vector alone were incubated in the presence of the indicated levels of Rg or Tg (and in otherwise ligand-free medium). These levels were chosen based on the range of TZD levels measured in the sera of patients treated with a single oral dose of TZD (33, 34). The effects of Tg and Rg on GLUT4 trans-repression by wild-type PPAR2 in both cell types are presented in Fig. 2. Studying the effects of Rg in rat adipocytes, we found that addition of up to 1.0 µM Rg abrogated the ability of PPAR2 to repress the GLUT4 promoter and that the GLUT4 promoter was even transactivated. These induced Rg effects were significantly higher than the unrepressed activity over the whole range of Rg concentrations examined, i.e. the differences observed between the basal unrepressed and the PPAR-regulated GLUT4 promoter activities at 0–1.0 µM Rg are statistically significant (p < 0.01) (Fig. 2D), and the differences observed between the repressed GLUT4 promoter activities in the absence of Rg and in the presence of 0.01–1.0 µM Rg are statistically significant (p < 0.001). Interestingly, this alleviation effect was unique to isolated adipocytes that are bona fide insulin target cells and was not observed in CHO-K1 fibroblasts (i.e. the effect of PPAR to repress GLUT4 promoter activity in the absence of Rg is only insignificantly different from the extent of the repression shown at Rg concentrations of 0.01–1.0 µM; p = 0.11–0.83) (Fig. 2B). Although exhibiting similar trends, Tg was less potent in alleviating PPAR effects compared with Rg. Furthermore, under no circumstances could Tg induce GLUT4 activation beyond basal levels in both cell types examined (Fig. 2, A and C). To exclude the possibility of cytotoxic effects of the drugs, another set of experiments was included in which cells were transfected with the (AOX)₃-Luc reporter (used as a positive control) under similar conditions. Indeed, Rg activated the PPRE from the (AOX)₃-Luc reporter by as much as 250% in CHO-K1 cells and to a smaller extent in primary adipocytes (Fig. 2, E and F, respectively), and Tg had similar yet smaller (160–170%) transactivation effects (data not shown). These data show that, contrary to the concept that TZD compounds are pure activators of PPAR, at least some may act as partial agonists, making them, in effect, also partial antagonists, able to displace putative endogenous full agonists. To gain deeper insight into these data, we compared the effects of Rg on GLUT4 trans-repression induced by wild-type PPAR2 versus the PPAR2-L496A/E499A double mutant (Fig. 2, B and D). We found that, although addition of Rg (0.01–1.0 µM) completely abolished GLUT4 promoter repression by wild-type PPAR2 in primary adipocytes, Rg only insignificantly affected the repression capacity of PPAR2-L496A/E499A. Similar results were observed for the corresponding mutant PPAR1-L468A/E471A (data not shown). These results further support our hypothesis that the inherent PPAR activity to repress the GLUT4 promoter is ligand-independent, whereas GLUT4 gene activation represents an alleviation of this effect by rosiglitazone, acting via the AF-2 domain/capacity of PPAR. In accordance with our data, Gurnell et al. (26) found that, in 3T3-L1 adipocytes, the AF-2-defective double mutant exhibited reduced transactivation due to impaired recruitment of coactivator CBP (cAMP-response element-binding protein-binding protein) and SRC-1 (steroid receptor coactivator-1), as well as delayed ligand-dependent corepressor release.

View larger version (37K): [in this window] [in a new window]   FIG. 2. TZD-dependent effects of PPAR on GLUT4 promoter activity. A and B, TZD-dependent effects of PPAR2 in CHO-K1 cells. Cells were cotransfected with 7.5 µg of full-length pGLUT4-P-Luc promoter reporter together with 5 µg of pSVSPORT1 expression vector for either wild-type mPPAR2 (black bars) or mPPAR2-L496A/E499A (gray bars) or of pSVSPORT1 alone (white bars). Cells were then incubated with the indicated doses of the TZD compounds Tg (A) and Rg (B) for an additional 48 h at 37 °C. C and D, TZD-dependent effects of PPAR2 in primary rat adipocytes. Cells were cotransfected with 2 µg of full-length pGLUT4-P-Luc promoter reporter together with 5 µg of pSVSPORT1 expression vector for either wild-type mPPAR2 (black bars) or mPPAR2-L496A/E499A (gray bars) or of pSVSPORT1 alone (white bars). Cells were then incubated with the indicated doses of Tg (C) or Rg (D) for an additional 20 h at 37 °C. GLUT4 promoter (GLUT4-P) activity was determined by measuring luciferase and -galactosidase activities as described under "Experimental Procedures." E and F, effects on (AOX)3-Luc. CHO-K1 cells (E) or primary rat adipocytes (F) were cotransfected with 2 µg of (AOX)3-Luc promoter reporter together with 5 µg of DNA for either pSVSPORT1 expression vector for wild-type mPPAR2 (black bars) or pSVSPORT1 alone (white bars). Cells were then incubated with the indicated doses of rosiglitazone as described above. For clarity, within each experiment, the TZD-mediated effects of PPAR2 are expressed as a percentage of the basal promoter activity at the same TZD dose. The data are expressed as means ± S.E. of three to six experiments, with each sample analyzed in quadruplicates.

Effects of 15-PGJ₂ on GLUT4 Promoter Regulation by PPAR
Based on these findings, one explanation for the insulin-sensitizing effects of Rg may be due to its ability to partially inhibit the dampening effect of insulin action with a natural ligand (or ligands) acting through PPAR. The prostaglandin J₂ metabolite 15-PGJ₂ has been shown to be the most potent natural ligand of PPAR and regulates adipocyte differentiation (35, 36). However, natural PPAR ligands discovered to date have been rarely evaluated in adipocyte cellular models, and few studies have been conducted on adipocytes using 15-PGJ₂, although this ligand was identified as a promoter of adipocyte differentiation. Therefore, we examined the effects of 15-PGJ₂ on GLUT4 gene repression in rat adipocytes compared with preadipose-like CHO-K1 cells (Fig. 3). We found that, in CHO-K1 cells, 15-PGJ₂ had no significant effect on either basal activity of the GLUT4 promoter reporter or its repression by PPAR2 (Fig. 3A). In contrast, 15-PGJ₂ dose-dependently repressed basal activity of the GLUT4 promoter in primary rat adipocytes by a maximum of 50% (Fig. 3B). This tissue-specific effect of 15-PGJ₂ that was observed in adipocytes (but not in CHO-K1 cells) may be due to the fact that adipocytes (but not CHO-K1 cells) endogenously express PPAR2 receptors. Importantly, 15-PGJ₂ enhanced the basal capacity of PPAR2 to repress GLUT4 promoter activity from 50% in the absence of ligand to as much as 70% in the presence of 5 µM 15-PGJ₂ (Fig. 3B). Under similar conditions, 15-PGJ₂ dose-dependently enhanced PPAR transcriptional activation of the PPRE from the (AOX)₃-Luc reporter in both CHO-K1 and primary adipose cells (Fig. 3, C and D), thus excluding any cytotoxic effects of the drug. Furthermore, addition of 15-PGJ₂ had no effect on the non-PPAR-regulated cytomegalovirus promoter since -galactosidase activity from the coexpressed pCMV--galactosidase expression vector included in all transfections was unaffected. These data support the notion that the endogenous receptor, along with its natural ligands, serves to dampen insulin action, thereby promoting insulin resistance. Teleologically, this could serve to offset exaggerated effects on glucose metabolism caused by unrestrained activation of the PPAR-induced differentiation program. Most importantly, our work provides the first example of differential regulation of a gene by the natural 15-PGJ₂ and the synthetic TZD ligands of PPAR in insulin target cells.

View larger version (28K): [in this window] [in a new window]   FIG. 3. 15-PGJ2-dependent effects of PPAR2 on GLUT4 promoter activity. A and C, effects in CHO-K1 cells. CHO-K1 cells were cotransfected with 7.5 µg of either full-length pGLUT4-P-Luc (A) or (AOX)3-Luc promoter reporter together with 5 µg of either pSVSPORT1 expression vector for wild-type mPPAR2() or pSVSPORT1 alone (). Cells were incubated with the indicated doses of 15-PGJ2 for48hat37 °C, and then GLUT4 promoter (GLUT4-P) activity was determined by measuring luciferase and -galactosidase activities as described under "Experimental Procedures." B and D, effects in primary rat adipocytes. Isolated rat adipocytes were prepared and cultured as described under "Experimental Procedures." Isolated adipocytes were cotransfected with 2 µg of full-length pGLUT4-P-Luc (B) or (AOX)3-Luc (D) promoter reporter together with 5 µg of either pSVSPORT1 expression vector for wild-type mPPAR2 () or pSVSPORT1 alone (). Cells were then incubated with the indicated doses of 15-PGJ2 for an additional 20 h at 37 °C, and then GLUT4 promoter (GLUT4-P) activity was determined by measuring luciferase and -galactosidase activities as described under "Experimental Procedures." Within each experiment, the results are expressed as a percentage of the basal GLUT4 promoter activity, i.e. the activity obtained from the expressed pGLUT4-P-Luc reporter alone. The data are expressed as means ± S.E. of three experiments, with each sample analyzed in triplicates.

Effects of RXR Coexpression on GLUT4 Promoter Regulation by PPAR
As PPAR receptors need to heterodimerize with RXR to exert their transcriptional regulation, we examined the effects of RXR coexpression on regulation of the GLUT4 promoter by PPAR2. The data obtained in CHO-K1 cells show that RXR coexpression repressed GLUT4 promoter activity to a similar level compared with PPAR2 coexpression (Fig. 4A). In contrast, coexpression of RXR in primary adipocytes had no additional effect on GLUT4 promoter activity beyond that exerted by PPAR itself (Fig. 4B). It has been suggested that the ability of nuclear receptors to inhibit gene expression in the absence of ligand may be due to passive inhibition occurring as a result of competition with other nuclear receptors for RXR heterodimerization or formation of homodimer pairs that are transcriptionally inactive (37). However, these data indicate that this may not be the case for GLUT4 trans-repression in adipocytes; RXR was not a rate-limiting component for PPAR effects, and its overexpression alongside with PPAR did not abolish GLUT4 trans-repression. The specific effect of RXR in CHO-K1 cells (but not in adipocytes) may also reflect the tissue-specific expression pattern of this receptor.

View larger version (24K): [in this window] [in a new window]   FIG. 4. RXR-dependent effects of PPAR2on GLUT4 promoter activity. A, effects in CHO-K1 cells. CHO-K1 cells were cotransfected with 7.5 µg of full-length pGLUT4-P-Luc promoter reporter together with 4.0 µg of pSVSPORT1 expression vector for wild-type mPPAR2, wild-type human RXR, or both. Appropriate amounts of empty pSVSPORT1 vector were added to the cells to keep the total amount of DNA transfected constant. After 48 h at 37 °C, GLUT4 promoter (GLUT4-P) activity was determined by measuring luciferase and -galactosidase activities as described under "Experimental Procedures." B, effects in primary rat adipocytes. Cells were cotransfected with 2.0 µg of full-length pGLUT4-P-Luc promoter reporter together with 4.0 µg of pSVSPORT1 expression vector for wild-type mPPAR2, wild-type human RXR, or both. Empty pSVSPORT1 vector was added to the cells to keep the total amount of DNA transfected constant. After 20 h at 37 °C, GLUT4 promoter activity was determined by measuring luciferase and -galactosidase activities as described under "Experimental Procedures."

Detection of cis-Elements in the GLUT4 Promoter That Mediate Its Repression by PPAR
Previous studies regarding GLUT4 regulation by PPAR and its ligands have yielded inconclusive data (see the Introduction). Hence, we aimed at identifying PPAR-response elements in the GLUT4 promoter that may serve as potential PPAR-binding sites. We examined the 5'-flanking and promoter regions (–2213/+163 bp relative to the transcription start site) of the rat GLUT4 gene for the presence of known PPREs of the DR1 type. As sequence analysis of this region revealed only incomplete PPREs, we performed progressive 5'-deletion analysis of the GLUT4 promoter in an attempt to identify cis-elements in the GLUT4 promoter that could potentially mediate its trans-repression by PPAR (Fig. 5). The 5'-deletion promoter reporter constructs used are shown in Fig. 5A. For clarity, the effect of PPAR is expressed as a percentage of the basal promoter activity in each construct. This analysis clearly revealed a tissue-specific expression pattern. In CHO-K1 cells, we found that a minimum promoter region from –66 to +163 bp was sufficient to retain the full repression effect (Fig. 5B). In contrast, two promoter regions that regulate GLUT4 promoter activity were identified in rat adipocytes (Fig. 5C). When the region spanning from –1887 to –1110 bp was removed, repression of the GLUT4 promoter was lost. A second region spanning from –66 to +163 bp was sufficient to retain the full repression effect, similar to what was observed in CHO-K1 cells. This tissue-specific pattern may reflect a different set of transcription factors that are present in each cell type, as well as the presence of various coactivators and corepressors that modulate PPAR activity. These findings underscore the importance of studying the regulation of GLUT4 gene expression in the context of genuine insulin target cells, as in this study.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Progressive 5'-deletion analysis of the GLUT4 promoter. A, pGLUT4-P-Luc promoter reporters. The full-length GLUT4 promoter (GLUT4-P) reporter pGLUT4-P-Luc and a series of progressive 5'-deletion mutants were used as described under "Experimental Procedures." The restriction sites used to generate each construct are indicated and numbered according to the first nucleotide in the site. B, GLUT4 promoter activity in CHO-K1 cells. Cells were transiently cotransfected with 7.5 µg of the various promoter reporter constructs, as indicated, along with 5 µg of either pSVSPORT1 expression vector alone (gray bars) or pSVSPORT1 expression vector for mPPAR2 (black bars). C, GLUT4 promoter activity in primary rat adipocytes. Cells were transiently cotransfected with 2 µg of the various promoter reporter constructs, as indicated, along with 5 µg of either pSVSPORT1 expression vector alone (gray bars) or pSVSPORT1 expression vector for mPPAR2 (black bars). GLUT4 promoter (GLUT4-P) activity was determined by measuring luciferase and -galactosidase activities as described under "Experimental Procedures." For clarity, the effect of PPAR is expressed as a percentage of the basal promoter activity in each construct. The data are expressed as means ± S.E. of six experiments, performed in triplicates. TSS, transcription start site.

PPAR/RXR Heterodimer Binds to the –66/+163 bp GLUT4 Promoter Region
To further investigate whether GLUT4 trans-repression by PPAR involves a direct protein/DNA interaction, PPAR and RXR proteins were translated in vitro and tested for their ability to bind the –66/+163 bp GLUT4 promoter region. Data from one representative EMSA are shown in Fig. 6. Full-length PPAR and RXR proteins were expressed at the expected sizes, as determined by SDS-PAGE analysis (data not shown). Our data show that neither PPAR nor RXR alone bound to the GLUT4 probe. However, addition of both PPAR and RXR proteins led to clear complex formation (indicated by the black arrow in Fig. 6). This binding was specific, as it could be competed by a 100-fold molar excess of unlabeled probe, but not by nonspecific DNA. The specificity of this interaction was further observed by the supershift assays, showing a further retardation in the electrophoretic mobility of the PPAR/RXR·DNA complex in the presence of specific anti-PPAR antibodies compared with the protein·DNA complex alone. These data indicate that, in the absence of ligands, PPAR repression of transcriptional activity of the GLUT4 promoter occurs via direct and specific binding of the PPAR/RXR heterodimer to the –66/+163 bp GLUT4 promoter region. Most importantly, complex formation became increasingly undetectable in the presence of increased µM doses of Rg. Thus, we show, for the first time, that the presence of rosiglitazone dose-dependently interferes with this binding, in accordance with its ability to alleviate GLUT4 trans-repression in transfection assays.

View larger version (59K): [in this window] [in a new window]   FIG. 6. EMSAs of PPAR/RXR binding to the –66/+163 bp GLUT4 promoter region. Binding reactions for EMSAs included the 32P-labeled –66/+163 bp GLUT4 promoter restriction fragment and in vitro translated mPPAR2 and human RXR protein lysates as indicated above each lane. An unlabeled –66/+163 bp GLUT4 promoter fragment was used as a specific DNA competitor, whereas an OCT1-derived oligonucleotide was used as a nonspecific competitor. The -fold molar excess of specific competitor DNA or addition of nonspecific DNA (NS) is also indicated above the relevant lanes. Where indicated, rosiglitazone was added to the protein/DNA mixture and incubated for 30 min at 25 °C. Complex supershift was induced by addition of 2 µl of anti-PPAR antibody (Ab) 10 min prior to addition of the probe. The black and white arrows indicate motilities of bound and free probes, respectively.

On the whole, our data are in line with the current concept of the mechanisms of PPAR action as presented by Glass and Rosenfeld (37). These investigators have shown that, in the absence of ligand ("unliganded" state), the PPAR/RXR heterodimer is bound to the PPRE within the promoter domains of target genes in association with a multiprotein corepressor complex that contains histone deacetylase activity, and it is the deacetylated state of histone that keeps the nucleosome in a state in which basal transcription is inhibited. After PPAR ligand binding, the corepressor complex is dismissed, and a coactivator complex is recruited to the heterodimer. The coactivator complex contains histone acetylase activity, leading to chromatin remodeling and facilitating active transcription. Similarly, it is possible that Rg binding to PPAR/RXR induces a conformational change that leads to the complex dissociation from the GLUT4 promoter. Consequently, Rg may exert its beneficial insulin-sensitizing effect at the level of gene regulation by directly interfering with PPAR/RXR binding and subsequent transcriptional repression of the GLUT4 promoter, thus restoring insulin responsiveness.

Conclusion
In conclusion, we suggest the following model for PPAR-induced regulation of GLUT4 gene transcription, as summarized in Fig. 7. (a) PPAR represses transcriptional activity of the GLUT4 promoter via direct and specific binding of the PPAR/RXR heterodimer to a –66/+163 bp GLUT4 promoter region; (b) this effect requires an intact Ser¹¹² phosphorylation site on PPAR; and (c) PPAR-induced repression is alleviated by Rg, which acts via its ligand-binding domain. The general paradigm for PPAR action suggests that this transcription factor enhances gene expression by inducing promoter activity rather than by repressing it. However, at least for the GLUT4 gene, our data demonstrate that native PPAR represses the activity of the GLUT4 promoter. As shown in Fig. 1, this phenomenon occurs in both non-insulin-responsive cells and insulin-responsive adipocytes. Furthermore, we have recently also observed similar repression of the GLUT4 promoter by PPAR2 in human adipocytes.² Although these findings appear to oppose the present dogma about PPAR, they corroborate findings from knockout mice heterozygous for PPAR deficiency. These mice exhibit improved insulin sensitivity and protection from the development of insulin resistance due to adipocyte hypertrophy on a high fat diet (22, 23). Our work provides the first example of differential regulation of a gene by natural and synthetic PPAR ligands in authentic insulin target cells. Whereas PPAR is mainly expressed in adipose tissue, natural PPAR ligands discovered to date have been rarely evaluated in adipocyte cellular models. Our data support the notion that endogenous PPAR ligands such as prostaglandin J₂ further augment the natural effect of PPAR, as demonstrated by the enhancement of PPAR-induced repression of the GLUT4 promoter. This repression is exerted via direct and specific binding of the PPAR/RXR heterodimer to the –66/+163 bp GLUT4 promoter region, although a perfect PPRE of the DR1 type was not detected in this region. Finally, in accordance with the notion that Rg enhances insulin sensitivity in diabetic patients, we have shown that, in adipocytes, PPAR-induced repression is alleviated by Rg and that this occurs by directly interfering with PPAR binding to and transcriptional repression of the GLUT4 gene. These data represent a novel mechanism by which Rg exerts its beneficial antidiabetic effects to enhance insulin sensitivity and GLUT4 mRNA expression in insulin target cells. Taken together, these findings may have important clinical and therapeutic implications, as the identification of the GLUT4 promoter as a down-stream molecular target for PPAR and the mechanism of PPAR and rosiglitazone regulation of GLUT4 gene expression will endow the design of better therapeutic and pharmacological interventions for the treatment and prevention of diabetes mellitus.

View larger version (24K): [in this window] [in a new window]   FIG. 7. Schematic model of PPAR and rosiglitazone regulation of GLUT4 gene expression. Details of the proposed model for PPAR- and rosiglitazone-induced regulation of GLUT4 gene transcription are discussed under "Conclusion." A schematic model of the PPAR protein is shown at the top. S112 denotes the MAPK phosphorylation site (Ser112) in mPPAR2 (corresponding to Ser82 in hPPAR1); this serine was mutated to alanine in PPAR2-S112A, thereby rendering it unable to be phosphorylated by MAPK and resulting in impaired ligand-independent activity. LBD, ligand-binding domain of PPAR. The highly conserved hydrophobic and charged residues Leu496 and Glu499 in helix 12 of this domain were substituted with alanine to generate PPAR2-L496A/E499A cDNA, thereby rendering the protein defective in ligand binding. A schematic description of the GLUT4 promoter (GLUT4-P) region is shown at the bottom, with the numbers of the first nucleotide in each restriction site used to generate each of the 5'-deleted constructs indicated below.

	FOOTNOTES

 
^* This work was supported in part by Grant 358/99-2 from the Israel Science Foundation of the Israel Academy of Science and Humanities and by grants from the General Apotropus Fund-Israel Ministry of Health and the L. R. Diamond Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Unit of Molecular Endocrinology, Inst. of Endocrinology, Diabetes, and Metabolism, Rambam Medical Center, Haifa 31096, Israel. Tel.: 972-4-854-3514; Fax: 972-4-854-2746; E-mail: amichal{at}tx.technion.ac.il.

¹ The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; mPPAR2, mouse peroxisome proliferator-activated receptor-2; hPPAR1, human peroxisome proliferator-activated receptor-1; TZD, thiazolidinedione; Tg, troglitazone; Rg, rosiglitazone; 15-PGJ₂, 15-deoxy-12,14-prostaglandin J₂; RXR, retinoid X receptor-; MAPK, mitogen-activated protein kinase; Luc, luciferase; AOX, acylcoenzyme A oxidase; PPRE, PPAR-response element; EMSA, electrophoretic mobility shift assay.

² M. Armoni, N. Kritz, C. Harel, F. Bar-Yoseph, H. Chen, M. J. Quon, and E. Karnieli, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Dr. Dana Beitner-Johnson (Biomed-Editors) for providing scientific editing and valuable critical comments on this manuscript.

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