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Thiazolidinediones and Rexinoids Induce Peroxisome Proliferator-Activated Receptor-Coactivator (PGC)-1 Gene Transcription: An Autoregulatory Loop Controls PGC-1 Expression in Adipocytes via Peroxisome Proliferator-Activated Receptor- Coactivation

Department of Biochemistry and Molecular Biology, University of Barcelona, 08028 Barcelona, Spain

Address all correspondence and requests for reprints to: Francesc Villarroya, Ph.D., Department of Biochemistry and Molecular Biology, University of Barcelona, Avda Diagonal 645, 08028 Barcelona, Spain. E-mail: fvillarroya{at}ub.edu.

	Abstract

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Thiazolidinediones (TZDs) are insulin-sensitizing drugs currently used to treat type 2 diabetes. They are activators of peroxisome proliferator-activated receptor (PPAR)-, and adipose tissue constitutes a major site for their biological effects. PPAR coactivator (PGC)-1 is a transcriptional coactivator of PPAR and other transcription factors. It is involved in the control of mitochondrial biogenesis, and its activity has been linked to insulin sensitization. Here we report that PGC-1 gene expression in brown and white adipocytes is a direct target of TZDs via PPAR activation. Activators of the retinoid X receptor also induce PGC-1 gene expression. This is due to the presence of a PPAR-responsive element in the distal region of the PGC-1 gene promoter that binds PPAR/retinoid X receptor heterodimers. Moreover, there is a positive autoregulatory loop of control of the PGC-1 gene through coactivation of PPAR responsiveness to TZDs by PGC-1 itself. These data indicate that some of the effects of TZDs, especially promotion of mitochondrial biogenesis and oxidative pathways in adipose depots, entail PGC-1 up-regulation via enhanced transcription of the PGC-1 gene.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THIAZOLIDINEDIONES (TZDS) ARE insulin-sensitizing drugs of current use in type 2 diabetes. They are strong agonists of peroxisome proliferator-activated receptor (PPAR)-, and most of their effects are believed to involve the activation of this receptor (1). The mechanisms of insulin sensitization elicited by TZDs are not fully understood. Although TZDs can act on skeletal muscle, adipose tissue is their major target. PPAR is highly expressed in adipose tissue and it acts as a master transcription factor of adipogenesis. Thus, it has been proposed that the main mechanisms of action of TZDs include stimulation of adipogenesis and subsequent fatty acid storage in adipose tissue, sparing other tissues such as liver, muscle, or ß-cells from excessive exposure to free fatty acids (2). However, the effects of TZDs are complex, and several other mechanisms have been implicated in insulin sensitization, including the promotion of mitochondrial oxidative activity in adipose tissue (3).

PPAR-coactivator (PGC)-1 is a transcriptional coactivator involved in the control of energy metabolism. It coactivates members of the type II class of nuclear hormone receptors, including PPAR as well as nuclear respiratory factors-1 and -2, responsible for concerted activation of genes determining mitochondrial respiratory capacity (4). PGC-1 is implicated in the control of multiple biological pathways related to energy metabolism in liver, muscle, and adipose tissues. It is required for efficient activation of gluconeogenesis in the liver (5), and it promotes mitochondrial biogenesis in skeletal muscle (6). PGC-1 is strongly expressed in brown adipose tissue from rodents and, to a lesser extent, white adipose tissue of humans and rodents (7, 8). When overexpressed in either rodent or human adipocytes, PGC-1 drives differentiation toward a brown adipocyte phenotype, characterized by the expression of the mitochondrial uncoupling protein (UCP)-1 and enhancement of overall mitochondrial oxidative activity (7, 9). It has been proposed that PGC-1 mediates the conversion of white adipocytes from storage to fat-oxidizing cells in response to such signals as leptin (10).

A deficient mitochondrial oxidative capacity associated with reduced expression of PGC-1 target genes has recently been identified as a primary event associated with insulin resistance in skeletal muscle (11, 12), and recent studies indicate that reduced expression of PGC-1 accounts for a reduction in the oxidative capacity in subjects prone to insulin resistance (13). Whereas this impairment of oxidative capacity has been reported mainly in skeletal muscle, recent data indicate that it may also be relevant in adipose tissue. Corvera and collaborators (14, 15) identified mitochondrial biogenesis as a key process in the differentiation of white adipose tissue, similarly to brown fat differentiation. They recently reported that TZDs favor this process, including the induction of PGC-1, in white adipose tissue depots in rodents. An overall reduction in marker genes characteristic of brown adipose phenotype, including PGC-1, in sc adipose tissue of human adults has been associated with insulin resistance (16). This has led to the proposal that up-regulation of PGC-1 could constitute a strategy for preventing and reversing insulin resistance and obesity (17). PGC-1 gene transcription is induced by cAMP-dependent pathways. In response to adrenergic stimulus in brown adipose tissue, transcriptional activation is mediated by phosphorylation of activating transcription factor (ATF)-2 via p38 MAPK, which is activated by protein kinase A (PKA) in response to cAMP (18). A similar mechanism involving direct phosphorylation of cAMP response element-binding protein (CREB) by PKA takes place in liver in response to glucagon (19). ATF-2 and CREB action on the PGC-1 gene occurs through a cAMP-responsive site in the proximal promoter region. Myocyte enhancer factor-2, a specific myogenic transcription factor, is another transcription factor implicated in PGC-1 gene expression in skeletal muscle and its responsiveness to calcium (20).

Considering that TZDs exert most of their effects on adipose tissue and recent data indicating that they may up-regulate oxidative activity in adipose tissue of diabetic patients (3), we examined their effect on the transcription of the PGC-1 gene in brown and white adipose cells. We identified PGC-1 as a direct target of TZDs acting via a PPAR-responsive element in the PGC-1 gene promoter as well as a positive autoregulatory loop of control of the PGC-1 gene through coactivation of PPAR responsiveness by PGC-1 itself.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Rosiglitazone and ciglitazone were from Cayman Chemicals (Ann Arbor, MI). Troglitazone was provided by Glaxo Wellcome (Greenford, UK). Norepinephrine, 9-cis retinoic acid (9-cis RA), T₃, insulin, propanolol, prazosin, and cycloheximide were from Sigma (St. Louis, MO). AGN194204 was a gift from Dr. R. A. Chandraratna (Allergan Pharmaceuticals, Irvine, CA). TTNPB, GW9662, and H89 were from EMD Biosciences (La Jolla, CA).

Cell culture
The care and use of mice were in accordance with European Community Council Directive 86/609/EEC and were approved by the Comitè Ètic d’Experimentació Animal of University of Barcelona. Primary cultures of brown adipocytes from mice were performed as described previously (21), and experiments were done on d 9 of culture when 80–90% of the cells were differentiated. HIB-1B cells were cultured and differentiated as described elsewhere (22). 3T3-L1 cells were maintained and differentiated following standard procedures (23) and were studied 1 wk after induction of differentiation when more than 90% of the cells were differentiated on the basis of lipid accumulation. Human preplated adipocytes, corresponding to a mix of cells from six healthy individuals with normal body mass index, were obtained from Zen-Bio (Research Triangle Park, NC), cultured according to supplier’s instructions, and studied 2 wk after induction of differentiation. Cells were exposed to 10 µM rosiglitazone, 10 µM troglitazone, 30 µM ciglitazone, or 1 µM 9-cis RA for 24 h, unless otherwise indicated. Treatment with norepinephrine was at 0.5 µM for 6 h. The effects of PPAR inhibition were determined by using 30 µM GW9662 and those of retinoid X receptor (RXR) activation with 1 µM AGN194204. Propanolol plus prazosin were used at 10 µM for 24 h. H89 was used at 10 µM and cycloheximide was used at 5 µg/ml, as reported elsewhere (21).

RNA isolation, Northern blot, and quantitative real-time PCR analyses
RNA was extracted using the RNeasy minikit (QIAGEN, Hilden, Germany). Northern blot analysis and hybridization were carried out as described (24), using as probes the cDNA for mouse PGC-1 (7), rat UCP1 (25), adipocyte P2 (aP2)/fatty acid binding protein (FABP) (26), mouse cytochrome oxidase subunit II (COII) (27) and subunit IV (COIV) (American Type Culture Collection, Manassas, VA), and mouse ß-actin (28). Hybridization signals were quantified using Molecular Image System GS-525 (Bio-Rad Laboratories, Foster City, CA). After individual densitometric analysis of the 6.5- and 5.0-kb transcript signals for PGC-1, no differential changes in their expression were detected in any of the experimental treatments studied, except for the positive control norepinephrine (see Fig. 1), and both transcripts were modified in parallel in response to the TZDs or rexinoids. Therefore, data are expressed by integrating densitometry signals for both transcripts. For quantitative real-time PCR analysis of mRNA expression, TaqMan RT-PCR was performed on the ABI PRISM 7700HT sequence detection system and reagents (Applied Biosystems, Foster City, CA). One microgram of RNA was transcribed into cDNA using MultiScribe reverse transcriptase and random-hexamer primers. The reaction was performed in a final volume of 25 µl using TaqMan universal PCR master mix, No-AmpErase UNG reagent, and the specific gene expression primer pair probes. The Assay-on-Demand probes used were Mm00447183 for mouse PGC-1, Hs00173304 for human PGC-1, Hs00222453 for human UCP1, Mm00437762 for mouse ß₂-microglobulin, and Hs99999904 for human cyclophilin A (housekeeping reference controls). Human COII mRNA was analyzed (Assay-by-Design, Applied Biosystems) using as primers CAAACCACTTTCACCGCTACAC (forward) and GGACGATGGGCATGAAACTGT (reverse), and the FAM-labeled probe was AAATCTGTGGAGCAAACC. The relative amount of PGC-1 mRNA in each sample was normalized to that of the reference control using the comparative (2^–CT) method according to the manufacturer’s instructions.

View larger version (30K): [in this window] [in a new window]   FIG. 1. Effects of rosiglitazone, 9-cis-RA, and norepinephrine on PGC-1 mRNA in brown adipocytes in primary culture. Murine brown adipocytes differentiated in primary culture were used for all figure panels. Cells were treated 24 h with 10 µM or the other indicated concentrations of rosiglitazone, 1 µM 9-cis RA, 1 µM AGN 194204, 30 µM GW9662, 10 µM propanolol plus prazosin or for 6 h with 0.5 µM norepinephrine. A, Example of Northern blot analysis PGC-1 mRNA expression. B, Dose response of the effects of rosiglitazone on PGC-1 mRNA levels. C, Effects of 10 µM rosiglitazone on UCP1 mRNA, COII mRNA, and COIV mRNA expression. D, Effects of the PPAR inhibitor GW 9662, the adrenergic inhibitors propanolol plus prazosin, and the PKA inhibitor H89 on the induction of PGC-1 mRNA by rosiglitazone, 9-cis RA, or norepinephrine. Results are the mean ± SEM of four to five independent experiments. *, Significant differences (P < 0.05) with respect to controls; #, Significant differences due to GW9662, propanolol plus prazosin, or H89 for each experimental situation.

Plasmids and transfection assays
The plasmid –2553-PGC-1-Luc, in which the region –2553 to +78 of the mouse PGC-1 gene drives the promoterless luciferase gene from firefly, was a gift from Dr. B. Spiegelman (Dana-Farber Cancer Institute and Department of Cell Biology, Harvard Medical School, Boston, MA). The plasmid –2553-PPREmut-PGC-1-Luc, a version containing point mutations at the putative PPAR-responsive element (PPRE) (GT instead of AG at sites –2043 and –2044, and GCT instead of AGG at sites –2050 to –2052) was generated using the Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and checked by direct DNA sequencing. The plasmid in which the proximal region containing the cAMP-responsive element at –146/–129 (19) had been removed (–2553-CREmut-PGC-1-Luc) was obtained by digestion of the parental plasmid –2553-PGC-1-Luc with PvuII and ZraI and further ligation. Plasmid expression vectors pSG5-PPAR (29), pRSV-RXR (30), pSV-PGC-1 (7), and SR-PKA (31) are described elsewhere. HIB-1B cells were transiently transfected using FuGENE-6 (Roche Molecular Biochemicals, Indianapolis, IN) for 16 h. Unless otherwise indicated, each transfection contained 1.5 µg of –2553-PGC-1-Luc, 0.3 µg of plasmid expression vectors, and 3 ng of pRL-CMV (Promega Corp., Madison, WI), an expression vector of Renilla reniformis luciferase. Cells were incubated for 48 h after transfection and, when indicated, were treated for 24 h before harvest with or without 10 µM rosiglitazone or 1 µM 9-cis RA. Luciferase activities were measured in a Turner Designs luminometer TD20/20 using the dual luciferase reporter assay system (Promega). Luciferase activity elicited by wild-type and mutant –2553-PGC-1-Luc constructs was normalized for variation in transfection efficiency using Renilla luciferase as internal standard.

EMSA experiments
Nuclear proteins were isolated from rat brown adipose tissue or differentiated primary brown adipocytes as described elsewhere (32). cDNAs for mouse PPAR and human RXR were transcribed and translated in vitro using the TNT quick-coupled transcription/translation systems (Promega). The PGC-1-PPRE double-stranded oligonucleotide used in EMSAs corresponds to positions –2036 to –2062 of the mouse PGC-1 gene, and its sequence is 5'-GTAAAATTCAGGACAAAGGTCATGGGCTC-3' (wild type). The mutated version of PGC-1-PPRE designed to have impaired PPAR/RXR binding had GT instead of AG at sites –2043 and –2044 and GCT instead of AGG at sites –2050 to –2052. The ³²P-labeled PGC-1-PPRE wild-type or mutated oligonucleotides (10,000–20,000 cpm) were incubated for 30 min at 25 C with 5 µg of nuclear protein extract or 5 µl of in vitro-transcribed/translated proteins. Reactions were carried out in a volume of 20 µl containing 10 mM Tris-HCl (pH 8.0), 0.05% Nonidet P-40, 1 mM dithiothreitol, 40 mM KCl, 6% glycerol, and 2 µg of poly(dI)/(dC). Samples were analyzed by electrophoresis at 4 C in nondenaturing 5% polyacrylamide gels in 0.5 x 44.5 mM Tris, 44.5 mM borate, and 1 mM EDTA. In the competition experiments, 100-fold molar excess of unlabeled oligonucleotide was included. When indicated, 1 µl of antiserum against PPAR (sc-7196; Santa Cruz Biotechnology) or CCAAT/enhancer-binding protein (C/EBP) (sc-151; Santa Cruz Biotechnology) was used.

Chromatin immunoprecipitation (ChIP) assay
HIB-1B cells were transfected with the PPAR expression vector and exposed to 10 µM rosiglitazone as described above. When indicated, cells were transfected with –2553-PGC-1-Luc or –2553-PPREmut-PGC-1-Luc. Cells were chilled at 4 C for 10 min and treated with 5 mM HEPES-KOH (pH 8.0), 0.1 mM EDTA, 0.05 mM EGTA, 10 mM NaCl, and 1.1% formaldehyde. After centrifugation (600 g, 10 min at 4 C), cell pellets were lysed in ice-cold lysis buffer [50 mM HEPES-KOH (pH 7.4), 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 0.25% Triton X-100] supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 µg/ml each of aprotinin, leupeptin, and pepstatin A). After incubation at 4 C for 10 min, crude nuclei were collected by centrifugation (600 x g, for 5 min at 4 C). Nuclei were resuspended in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, and 200 mM NaCl plus protease inhibitors, collected as above, and resuspended in radioimmunoprecipitation assay buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 5% glycerol, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100, supplemented with protease inhibitors]. Samples were sonicated with Branson Sonifier 250 and lysates were cleared by centrifugation (16,000 x g, 15 min at 4 C) and used for immunoprecipitation with 2 µg of anti-PPAR antibody (sc-7196, Santa Cruz Biotechnology) or an equal amount of an unrelated immunoglobulin (sc-9314, Santa Cruz Biotechnology).

Immunoprecipitations were performed for 16 h at 4 C, and immune complexes were collected by nutating the lysates for 1 h at 4 C with 30 µl/sample of 50% slurry protein G agarose beads (Santa Cruz Biotechnology) preincubated with 100 µg/ml salmon sperm DNA (Life Technologies). The beads were washed with radioimmunoprecipitation assay buffer supplemented with 1 mM phenylmethylsulfonyl fluoride and 100 µg/ml yeast tRNA (Life Technologies). The beads were then incubated in 100 µl of Tris/EDTA buffer, 0.5% sodium dodecyl sulfate, and 200 µg/ml proteinase K (Sigma) for 3 h at 55 C, and cross-links were reversed for 6 h at 65 C. After phenol-chloroform extraction, DNA was used for PCR analysis. Primers for amplifying a 378-bp fragment encompassing the putative PPRE in the PGC-1 gene were: forward, 5'GTATCAGTTACCATCAGG 3' and reverse, 5'AACAAGATGGCCAACAGC3'; those amplifying a 237-bp fragment of the murine cyclophilin A gene, used as control, were: forward, 5' AAGACTGAATGGCTGGATGG 3' and reverse, 5' TTACAGGACATTGCGAGCAG 3'. After 35 cycles of amplification, PCR products were electrophoresed in a 1.5% agarose gel and visualized by ethidium bromide staining.

Transduction of brown adipocytes with an adenoviral vector driving PGC-1
Differentiated brown adipocytes were infected with an adenoviral vector driving murine PGC-1 (AdCMV-PGC-1, provided by B. Spiegelman) or AdCMV-GFP (control) at a multiplicity of infection of 400 for 4 h in DMEM/F12 medium. Further culture of cells with regular culture medium for brown adipocytes (21) was performed for 48 h. This treatment led to an efficiency of transduction of about 80%, on the basis of assessment of green fluorescent protein (GFP) fluorescence. In these conditions, transduction increased the 120-kDa immunoblot signal for PGC-1 protein at least 10-fold with respect to controls.

Statistical analysis were performed using the Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TZDs and rexinoids induce PGC-1 gene expression in brown adipocytes
Rosiglitazone increased PGC-1 mRNA expression in mouse primary brown adipocytes differentiated in culture (Fig. 1A). This effect was dose and time dependent (Fig. 1B). PGC-1 mRNA expression was significantly higher than the controls 4 h after exposure to 10 µM rosiglitazone (4.8 ± 0.5-fold induction), peaked 24 h after exposure (see Fig. 1B), and remained high for 48 h (not shown). Other TZDs, such as troglitazone or ciglitazone, had similar effects. Thus, 10 µM troglitazone caused a 5.3 ± 0.4-fold induction in PGC-1 mRNA levels (P < 0.05); and 30 µM ciglitazone caused a 3.9 ± 0.3-fold induction in PGC-1 mRNA (P < 0.05). Treatment of cells with 9-cis RA also induced PGC-1 mRNA expression (Fig. 1A). Similar activation by AGN194204, a specific RXR agonist (Fig. 1A), but not TTNPB, a retinoic acid receptor activator (data not shown), indicated that RXR activation was responsible for the effects of 9-cis RA. Parallel experiments in which brown adipocytes were exposed to norepinephrine indicated a robust induction, consistent with the known adrenergic, cAMP-dependent, activation of PGC-1 gene transcription (18). However, whereas the induction elicited by norepinephrine occurred mainly for the 5.0-kb PGC-1 mRNA transcript, changes elicited by TZDs and rexinoids occurred equally for the 5.0- and 6.5-kb transcripts. A concomitant increase in the mRNAs for the genes encoding the mitochondrial proteins UCP1, COII, and COIV, known targets of PGC-1 effects, was observed in response to rosiglitazone (Fig. 1C). Rosiglitazone also increased significantly PGC-1 mRNA in HIB-1B cells, a cell line representative of brown adipocytes (7.3 ± 0.9-fold induction, P < 0.05) as did 9-cis RA, albeit to a lesser extent (3.4 ± 0.5-fold induction, P < 0.05).

The induction of PGC-1 by TZDs and rexinoids is mediated by a PPAR-dependent pathway
The effects of both rosiglitazone and 9-cis RA on PGC-1 gene expression were blunted in the presence of GW9662, a specific PPAR antagonist, thus indicating that rosiglitazone acted through PPAR and that 9-cis RA effects via RXR also required an active PPAR (Fig. 1D). This indicates that PPAR/RXR heterodimers mediate TZD and 9-cis RA effects on PGC-1 gene expression. The PPAR antagonist GW9662 did not alter the induction of PGC-1 mRNA levels by norepinephrine, thus indicating that TZDs and adrenergic, cAMP-mediated activation of PGC-1 gene expression take place through distinct mechanisms. This was further established by the use of a mixture of ß- and -adrenergic antagonists, propanolol plus prazosin, which reduced the norepinephrine effects on PGC-1 mRNA expression, whereas it did not alter the action of rosiglitazone or 9-cis RA (Fig. 1D). The PKA inhibitor H89 also reduced the action of norepinephrine on PGC-1 mRNA expression, whereas it did not have any effect on rosiglitazone- and 9-cis RA-dependent induction (Fig. 1D). Cycloheximide did not affect the induction of PGC-1 by rosiglitazone (5.8 ± 0.7-fold induction by rosiglitazone in control cells, 6.5 ± 0.9-fold induction by rosiglitazone in cycloheximide-treated cells), thus indicating that rosiglitazone acts on PGC-1 gene expression through protein synthesis-independent mechanisms, compatible with a ligand-dependent activation of PPAR.

TZDs induce the expression of the PGC-1 protein in brown adipocytes
We determined whether the observed induction of PGC-1 mRNA levels by TZDs result in an increase in the PGC-1 protein in brown adipocytes. A robust time-dependent induction of PGC-1 protein abundance in brown adipocyte protein extracts was observed as a consequence of treatment with rosiglitazone (Fig. 2).

View larger version (24K): [in this window] [in a new window]   FIG. 2. Effects of rosiglitazone on PGC-1 protein levels in brown adipocytes in primary culture. Murine brown adipocytes differentiated in primary culture were treated with rosiglitazone at the indicated times and concentration. Data are means ± SEM of at least three independent experiments and are shown as the ratio of the densitometric intensity of the immunoreactive signal of PGC-1 protein normalized by the immunoreactive signal for ß-actin (top). *, Significant differences (P < 0.05) with respect to controls. Representative immunoblot analysis of PGC-1 protein levels (bottom). Control corresponds to the immunoreactive signal using a protein extract of HEK-293 cells stably transduced with a cytomegalovirus-driven adenoviral construct for murine PGC-1 expression.

TZDs and rexinoids activate PGC-1 gene expression in white adipocytes

View larger version (20K): [in this window] [in a new window]   FIG. 3. Effects of rosiglitazone and 9-cis RA on PGC-1 mRNA in white adipocytes. Murine 3T3-L1 adipocytes and human white adipocytes differentiated in primary culture were treated for 24 h with 10 µM rosiglitazone or 1 µM 9-cis RA. Relative abundance of PGC-1 mRNA was determined by quantitative real-time PCR (see Materials and Methods). Results are the mean ± SEM of three to four independent experiments. *, Significant differences (P < 0.05) with respect to controls.

The PGC-1 gene is a target of PPAR-mediated transcriptional activation via a PPAR-responsive element in the promoter region

View larger version (15K): [in this window] [in a new window]   FIG. 4. Effects of rosiglitazone, 9-cis RA, and PPAR on PGC-1 gene promoter activity. Transient transfection experiments were performed in HIB-1B cells with –2553 PGC-1-Luc. A, Dose-response effects of rosiglitazone on –2553 PGC-1-Luc activity in the absence or presence of cotransfected PPAR. B, When indicated, the expression vectors for PPAR, RXR, or for constitutively active PKA were cotransfected and 24 h previous to harvest 10 µM rosiglitazone or 1 µM 9-cis RA were added to the cells. B, Results show the mean ± SEM of at least three independent experiments done in triplicate. *, Statistically significant differences with respect to untreated controls (P < 0.05).

View larger version (20K): [in this window] [in a new window]   FIG. 5. Identification of a PPAR-responsive element in the PGC-1 gene promoter. A, Sequence of the PPAR-response element in the mouse PGC-1 gene promoter and comparison with similar sequences in PGC-1 promoters from other species. Arrows indicate one-base spaced direct repeat alignment. B, Effects of points mutations in the PPAR-response element (shown as asterisks in A) or deletion mutation of the proximal cAMP response element (see Materials and Methods) on the responsiveness of the PGC-1 gene promoter activity to rosiglitazone, PPAR, and PKA. The wild-type (2553 PGC-1-Luc) and mutated (2553-PPREmutPGC-1-Luc, 2553-CREmutPGC-1-Luc) plasmid constructs were transfected and treated as in Fig. 4. Results show the mean ± SEM of three to five independent experiments done in triplicate. *, Statistically significant differences (P < 0.05) due to rosiglitazone treatment or PKA cotransfection; #, Statistically significant differences (P < 0.05) observed after comparing either constructs at equal conditions of cotransfection or treatment.

View larger version (55K): [in this window] [in a new window]   FIG. 6. EMSA of PPAR binding to the PPAR-response element in the PGC-1 gene promoter. Double-stranded oligonucleotides corresponding to –2036/–2062 region of the mouse PGC-1 gene promoter (–2036/–2062 wt) or a mutated version designed to impair PPAR/RXR binding (–2036/–2062 mut) (see Materials and Methods) were used as a labeled probes. Five microliters of in vitro-transcribed/translated PPAR plus RXR protein extract or unprogrammed lysate (left), 5 µg of nuclear protein extracts from mouse brown adipose tissue (center), or 5 µg of nuclear protein extracts from brown adipocytes in primary culture (right) were incubated as described in Materials and Methods and when indicated, antibodies (Ab) against PPAR and C/EBP (negative control) were added. x 100 wt means addition of 100-fold excess unlabeled nonmutated probe (Comp). Arrows indicate super-shifted (up) or weakened (down) bands.

View larger version (29K): [in this window] [in a new window]   FIG. 7. ChIP analysis of PPAR binding to the PGC-1 gene promoter. PPAR antibody (+) or an unrelated Ig (–) was used to immunoprecipitate the protein-DNA complexes (see Materials and Methods). A, ChIP analysis of the PPAR binding to the endogenous PGC-1 gene promoter in HIB-1B cells transfected with the PPAR expression vector and treated with 10 µM rosiglitazone. Arrows indicate the 378-bp PGC-1 and 237-bp cyclophylin A (Cyp) PCR products. No DNA and control represent negative and positive PCR controls. B, HIB-1B cells were transfected with the plasmids –2553-PGC-1-Luc and –2553-PPREmut-PGC-1-Luc always in the presence of PPAR expression vector and 10 µM rosiglitazone. Immunoprecipitation and PCR amplification were performed as in A.

PGC-1 coactivate the PPAR-dependent activation of its own gene in response to TZDs

View larger version (37K): [in this window] [in a new window]   FIG. 8. PGC-1 coactivate the PPAR-mediated effects of rosiglitazone on its own gene expression. A, Representative Northern blot analysis of RNA from murine brown adipocytes in primary culture transduced with an adenoviral vector driving recombinant PGC-1 expression (Ad-PGC-1) or a control vector (Ad-GFP), treated or not with 10 µM rosiglitazone. Blot was probed with PGC-1 cDNA as well as with the UCP1 cDNA and aP2/FABP cDNA probes to detect recombinant, adenoviral mediated PGC-1 transcript and the endogenous PGC-1 mRNA. B, Effects of cotransfection with a PGC-1 expression vector on PGC-1 promoter activity in response to rosiglitazone and cotransfected PPAR. Experimental details were as in Fig. 5. Results show the mean ± SEM of at least three independent experiments done in triplicate. *, Statistically significant differences (P < 0.05) with respect to controls; #, Statistically significant differences (P < 0.05) due to PGC-1 cotransfection.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The presence of a PPAR-responsive element in the PGC-1 gene explains the induction of PGC-1 gene expression in white adipose tissue depots after chronic treatment of rodents with TZDs (14) and even with non-TZD PPAR activators (35). Moreover, the presence of a PPAR-responsive element in the PGC-1 gene that binds PPAR/RXR heterodimers is consistent with the observed induction of the PGC-1 gene by rexinoids, selective RXR activators, considering the known ligand-dependent sensitivity of the RXR moiety in the PPAR/RXR heterodimers (36). Treatment of experimental animals with rexinoids mimics some of the effects of PPAR activation by TZDs, including insulin sensitization. However, the more pleiotropic effects of rexinoids caused by the capacity of RXR to dimerize with members of the nuclear hormone receptor superfamily other than PPAR have precluded to date their clinical use in type 2 diabetes (37). Finally, the presence of a PPAR-responsive element in the PGC-1 gene raises the possibility that this element could also mediate regulation by other PPAR subtypes. In some target genes of PPAR, such as lipoprotein lipase gene, the PPAR-responsive element mediates response to TZDs in adipose tissue but also response to fibrates in liver, via PPAR-dependent activation (38). It has been reported that PPAR activators induce PGC-1 gene expression in differentiated muscle cells, whereas activators of PPAR do not (39). Further research will be needed to assess the action of PPAR subtypes on the PGC-1 gene and their relation to the tissue-specific effects of drugs capable of activating these receptors.

The effects of PGC-1 induction by TZDs in adipocytes may entail enhancement of oxidative capacity in adipose tissue, which could constitute a major therapeutic mechanism of TZD action. PGC-1 overexpression in rodent or human white adipose cells in culture promotes mitochondriogenesis (6, 9). Moreover, the present findings may explain recent reports indicating that TZDs induce a coordinate up-regulation of genes encoding the mitochondrial oxidation machinery in human adipose tissue from diabetic patients (3, 40). Accordingly, experimental treatments in rodents had already evidenced mitochondrial remodeling and enhanced energy expenditure in white fat in response to TZDs (15), including increased PGC-1 expression (14). Impaired mitochondrial oxidative capacity in skeletal muscle, particularly of genes that are targets of PGC-1, has been identified as an early event associated with insulin resistance and type 2 diabetes (11, 12). Although this has not been unequivocally established in adipose tissue, experimental interventions promoting white adipose tissue oxidative capacity, i.e. through ectopic expression of the brown fat UCP1, improve overall energy expenditure and protect against diet-induced obesity (41). In fact, some of the biological effects of TZDs acting via the induction of PGC-1 gene expression would consist in promoting a brown fat-like phenotype in adipose depots. It has recently been demonstrated that reduced expression of marker genes characteristic of the brown adipocyte phenotype is associated with insulin resistance in patients with type 2 diabetes (16). Enhancement of brown fat-like features in adipose depots, i.e. high PGC-1 expression and promotion of mitochondrial oxidative activity and energy expenditure in response to TZDs, could be a major element in the insulin sensitizing action of these drugs because it would enhance fatty acid oxidation inside the adipose depots, thus contributing to the lowering in free fatty acids and subsequent prevention of lipotoxicity.

	Acknowledgments

 
We thank Drs. B. Spiegelman and P. Puigserver for PGC-1 expression vectors and promoter construct and for HIB-1B cells and Drs. R. Evans and H. H. Samuels for expression vectors.

	Footnotes

 
This work was supported by Grants SAF2002-03648 and SAF2005-01722 from Ministerio de Ciencia y Tecnologia and Grant C03/08 from Instituto Carlos III. Ministerio de Sanidad y Consumo, Spain. O.M. was supported by Generalitat de Catalunya (2004PIV210) and Programa de Apoyo para la Superación del Personal Académico, Universidad Nacional Autónoma de México.

Present address for O.M.: Laboratorio de Rumiología y Metabolismo Nutricional. Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México.

Disclosure statement: E.H., O.M., P.Y., M.R.d.l.C., R.I., M.G., and F.V. have nothing to declare.

First Published Online March 2, 2006

Abbreviations: aP2, Adipocyte P2; C/EBP, CCAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation; 9-cis RA, 9-cis retinoic acid; COII, cytochrome oxidase subunit II; COIV, cytochrome oxidase subunit IV; CREB, cAMP response element-binding protein; FABP, fatty acid binding protein; GFP, green fluorescent protein; PGC, PPAR coactivator; PKA, protein kinase A; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR-responsive element; RXR, retinoid X receptor; TZD, thiazolidinedione; UCP, uncoupling protein.

Received January 18, 2006.

Accepted for publication February 23, 2006.

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