Peroxisome Proliferator-activated Receptor α (PPARα) Induces PPARγ Coactivator 1α (PGC-1α) Gene Expression and Contributes to Thermogenic Activation of Brown Fat

Background: PPARα is a distinctive marker of the brown-versus-white fat phenotype. Results: PPARα induces PGC-1α gene transcription in brown adipocytes through mechanisms involving PRDM16. Conclusion: PPARα regulates brown fat thermogenesis via induction of PGC-1α and PRDM16 gene expression. Significance: Activation of PGC-1α by PPARα provides a molecular mechanism for concerted induction of thermogenic genes (UCP1, mitochondrial genes, and lipid oxidation genes) in brown fat. Peroxisome proliferator activated receptor α (PPARα) is a distinctive marker of the brown fat phenotype that has been proposed to coordinate the transcriptional activation of genes for lipid oxidation and for thermogenic uncoupling protein 1 in brown adipose tissue. Here, we investigated the involvement of PPARα in the transcriptional control of the PPARγ coactivator (PGC)-1α gene. Treatment with PPARα agonists induced PGC-1α mRNA expression in brown fat in vivo and in primary brown adipocytes. This enhancement of PGC-1α transcription was mediated by PPARα binding to a PPAR-responsive element in the distal PGC-1α gene promoter. PGC-1α gene expression was decreased in PPARα-null brown fat, both under basal conditions and in response to thermogenic activation. Moreover, PPARα- and cAMP-mediated pathways interacted to control PGC-1α transcription. PRDM16 (PRD1-BF1-RIZ1 homologous domain-containing 16) promoted PPARα induction of PGC-1α gene transcription, especially under conditions in which protein kinase A pathways were activated. This enhancement was associated with the interaction of PRDM16 with the PGC-1α promoter at the PPARα-binding site. In addition, PPARα promoted the expression of the PRDM16 gene in brown adipocytes, and activation of PPARα in human white adipocytes led to the appearance of a brown adipocyte pattern of gene expression, including induction of PGC-1α and PRDM16. Collectively, these results suggest that PPARα acts as a key component of brown fat thermogenesis by coordinately regulating lipid catabolism and thermogenic gene expression via induction of PGC-1α and PRDM16.

Mammals possess two specialized types of fat cells that serve opposite functions. White adipocytes store excess energy as triacylglycerols in large lipid droplets. When needed, this stored energy can be mobilized by activating lipolysis with the consequent release of free fatty acids into the circulation. In contrast, brown adipocytes oxidize endogenous triacylglycerols to generate heat (thermogenesis), a process made possible by brown fat-specific uncoupling protein (UCP) 3 1 present in their abundant mitochondria (1). Thermogenesis in brown adipose tissue (BAT) is mainly controlled by norepinephrine, which is released from sympathetic terminals innervating the tissue in response to cold or dietary stimuli. Norepinephrine, through ␤-adrenergic receptors, leads to cAMP/protein kinase A (PKA)-mediated activation of lipolysis and thermogenic activity. The clear role of BAT in the defense against hypothermia and obesity described in rodent model studies is now being reevaluated in humans in light of the recent demonstration that considerable amounts of metabolically active BAT are present in adult humans (reviewed in Ref. 2).
Despite the differences in the functional and developmental characteristics of brown and white adipocytes, their terminal differentiation processes are mainly regulated by transcriptional factors in common: peroxisome proliferator-activated receptor ␥ (PPAR␥/NR1C3) and CCAAT/enhancer binding proteins (C/EBPs) (3). In fact, both PPAR␥ and C/EBP␣ induce UCP1 gene transcription (4,5), but UCP1 can still be induced in their absence (6,7). Recently, comprehensible advances have been achieved on the identification of several transcriptional factors and coregulators that specifically promote embryonic development and acquisition of the BAT-specific gene expression profile, including UCP1, among them PRDM16 (PRD1-BF1-RIZ1 homologous domain-containing 16) and PGC-1␣ (PPAR␥ coactivator 1␣) (reviewed in Ref. 8).
PGC-1␣ is a transcriptional coactivator involved in the control of energy metabolism that is highly expressed in BAT (9). PGC-1␣ is critical for the cAMP-dependent activation of BAT thermogenesis, playing a role in inducing UCP1 gene expression and enhancing overall mitochondrial oxidative activity (9). PGC-1␣ is not essential for BAT differentiation because it can be replaced by PGC-1␤ (10). However, ectopic expression of PGC-1␣ in white adipocytes induces acquisition of BAT features, including expression of mitochondrial and fatty acid-oxidation and thermogenic genes (9,11). Therefore, increasing PGC-1␣ may be a plausible strategy for the treatment of obesity. In response to an adrenergic stimulus, PGC-1␣ gene expression is up-regulated by cAMP-mediated pathways in BAT. The underlying mechanism involves phosphorylation and binding of activating transcription factor 2 to a cAMPresponsive element (CRE) in the proximal PGC-1␣ promoter region (12). Furthermore, we previously reported that PPAR␥ is also a powerful inducer of PGC-1␣ gene transcription, acting through a PPAR-responsive element (PPRE) in the distal promoter region (13).
PRDM16, which is also highly expressed in BAT, has been identified as a transcriptional coactivator responsible for determining the BAT lineage (6,14). When ectopically expressed in white adipocyte or myogenic precursor cells, PRDM16 induces the BAT-specific gene expression program (6,14). Conversely, PRDM16 knockdown in brown adipocytes ablates brown-specific gene expression in association with induction of skeletal muscle-specific gene expression (14). PRDM16 is thus recognized as a critical determinant of the differentiation of the BAT lineage from myogenic progenitors during embryonic development, although it is unclear whether it plays a role in fully differentiated brown adipocytes. Recently, PRDM16 was reported to promote the induction of the thermogenic program in subcutaneous white adipose tissue (WAT) of rodents (15). PPAR␣ (NR1C1) plays an important role in the overall regulation of lipid metabolism. Many PPAR␣ target genes are involved in cellular fatty acid uptake (e.g. lipoprotein lipase) and in mitochondrial and peroxisomal ␤-oxidation of fatty acids (16). Consistent with this function, PPAR␣ is highly expressed in BAT (17), and actually, it is considered a distinctive marker of the BAT with respect to WAT phenotype (18). Moreover, PPAR␣ regulates the expression of UCP1, the specific protein that enables brown adipocytes to perform thermogenesis (19). Here, we identify PPAR␣ as a direct activator of PGC-1␣ and further demonstrate that the interaction of PPAR␣ with PRDM16 and the cAMP-mediated pathways is necessary for full thermogenic activation of PGC-1␣ gene transcription in BAT.
Animals-Mice were cared for and used in accordance with European Community Council Directive 86/609/EEC, and animal protocols were approved by the Comitè Ètic d'Experimentació Animal of the University of Barcelona. For studies in Swiss neonates, pups were placed in a humidified, thermostatically controlled chamber at 28°C and injected intraperitoneally 2 h after birth with Wy14,643 (50 g/g body weight) or equivalent volumes of a 20% (v/v) dimethyl sulfoxide/saline solution. Pups were studied 15 h after treatment. Two-month-old female, 15-day lactating mice were also treated with a single intraperitoneal injection of Wy14,643 (50 g/g body weight) or bezafibrate (100 g/g body weight) in 50% dimethyl sulfoxide/saline. Controls were given equivalent volumes of the vehicle, and mice were studied 6 h after injections. Studies in PPAR␣-null (PPAR␣ Ϫ/Ϫ ) mice (The Jackson Laboratory, Bar Harbor, ME) and strain-matched wild-type (PPAR␣ ϩ/ϩ ) mice were performed to determine the effects of acute cold exposure. Two-month-old mice were acclimated to a thermoneutral environmental temperature (28°C) for 1 week and then exposed to 4°C for 4 or 24 h. In all experiments, animals were killed by decapitation, and interscapular BAT was dissected and frozen in liquid nitrogen. At least three different litters per experiment were analyzed.
Cell Culture-Primary cultures of brown adipocytes from mice were established and maintained as described previously (20). After culturing for 9 days, a time when 80 -90% of cells had differentiated, cells were exposed to 10 M rosiglitazone, 10 M Wy14,643, 1 M GW7647, 1 M GW501516, 0.5 M norepinephrine, or 1 mM dibutyryl-cAMP for 24 h (except where indicated otherwise) and then harvested. The effects of PPAR␣ inhibition were determined using 10 M GW6471. Mouse embryonic fibroblasts (MEFs) from PGC-1␣ Ϫ/Ϫ and PGC-1␣ ϩ/ϩ mice were isolated and cultured as described previously (21). The SGBS human adipocyte cell line was cultured and differentiated as described previously (22).
The relative amount of mRNA in each sample was normalized to that of the reference control 18 S rRNA using the comparative (2 Ϫ⌬CT ) method according to the manufacturer's instructions.
ChIP Assays-ChIP assays in HIB-1B cells and brown adipocytes were performed as described previously (13). HIB-1B cells were transfected with the PPAR␣ expression vector and exposed to 10 M Wy 14,643 or 1 M GW7647 as described above. Where indicated, cells were transfected with Ϫ2553-PGC-1␣-Luc or Ϫ2553-PPREmut-PGC-1␣-Luc and cotransfected with PRDM16 and PKA expression vectors or treated with 0.5 mM IBMX. Brown adipocytes were treated for 24 h with 1 M GW7647or 1 mM dibutyryl-cAMP as indicated. The ChIP assay in BAT was performed as described (28). Chromatin samples were immunoprecipitated with 8 g of anti-PPAR␣ antibody (H98, Santa Cruz Biotechnology, Santa Cruz, CA), 3 g of anti-PRDM16 antibody (EB 05579, Everest Biotech, UK), or an equal amount of an unrelated immunoglobulin (sc-9314, Santa Cruz Biotechnology). After phenol-chloroform extraction, immunoprecipitated chromatin DNA was used for PCR analysis. The primers used to amplify a 378-bp fragment encompassing the putative PPRE in the PGC-1␣ gene were 5Ј-GTA TCA GTT ACC ATC AGG-3Ј (forward) and 5Ј-AAC AAG ATG GCC AAC AGC-3Ј (reverse).
Adenovirus Transduction-Differentiated SGBS (day 12) adipocytes in DMEM/F12 medium were infected with adenoviral vectors driving human PPAR␣ (AdCMV-hPPAR␣) (29) or AdCMV-GFP (control) at a multiplicity of infection of 400 for 4 h. Experiments were performed following an additional 48-hour incubation in fresh differentiation media. This treatment led to an efficiency of transduction of about 80% on the basis of an assessment of GFP fluorescence. Cells were treated for 24 h with the PPAR␣-selective ligand GW7647 (1 M) or vehicle (dimethyl sulfoxide).
Statistics-Student's t test was used for statistical analyses.

RESULTS
PPAR␣ Activation Induces PGC-1␣ Gene Transcription in Brown Adipocytes-Treatment with the PPAR␣ agonists GW7647 and Wy14,643 increased PGC-1␣ mRNA expression in mouse primary brown adipocytes differentiated in vitro (Fig.  1A). The specific PPAR␥ agonist rosiglitazone, which was previously reported to activate PGC-1␣ gene expression (13), had similar effects. We next established primary cultures of brown adipocytes from PPAR␣-null mice. Morphological differentiation was unchanged in PPAR␣-null brown adipocytes, as described elsewhere (30 and data not shown), as were basal levels of PGC-1␣ mRNA expression. However, PPAR␣ agonistinduced increases in PGC-1␣ mRNA were totally suppressed in PPAR␣-null brown adipocytes, whereas the effect of rosiglitazone was essentially unchanged.
The effects of acute treatment of mice with the PPAR␣-specific agonist Wy14,643 were studied both in adult and neonatal mice. For adult studies, lactating dams were used, as previous studies had indicated that sensitivity to PPAR␣ is enhanced in physiological states involving low levels of circulating free fatty acids (19). Wy14,643 significantly increased PGC-1␣ gene expression in BAT in vivo in both physiological contexts (Fig.  1B). The PPAR-panagonist bezafibrate induced PGC-1␣ expression to a similar extent. These results demonstrate that PPAR␣ agonists acutely regulate the PGC-1␣ gene both in vivo and in differentiated primary brown adipocytes.
We next performed transient transfection experiments in the brown adipocyte-derived HIB-1B cell line using a plasmid containing 2 kb of the mouse PGC-1␣ gene promoter region fused to the luciferase reporter gene. Cotransfection with PPAR␣, and particularly further addition of the PPAR␣ agonists Wy14,643 or GW7647, increased PGC-1␣ promoter activity (Fig. 1C). Conversely, responsiveness to PPAR␣ was abolished in a mutated construct (mutPPRE-PGC-1␣-luc) in which the PPRE, which had previously been shown to mediate sensitivity to PPAR␥ (13), was mutated ( Fig. 1D, asterisks). Notably, this PPRE did not behave as a PPAR␤/␦-responsive element in this brown adipocyte context (108 Ϯ 18% activity of the mutPPRE-PGC-1␣-luc with respect to the wild-type PGC-1␣-luc construct in the presence of cotransfected PPAR␤/␦ expression vector plus the PPAR␤/␦-specific ligand GW501516).
To assess whether PPAR␣ binds specifically to the endogenous PGC-1␣ gene, we performed ChIP assays in brown fat and primary brown adipocytes (Fig. 1D, left panel). Incubation with an anti-PPAR␣ antibody significantly enriched the specific PGC-1␣-promoter PCR product, thus indicating that endogenous PPAR␣ protein does indeed bind to the endogenous PGC-1␣ gene in brown fat. When analyzed in primary brown adipocytes, we found that activation of the endogenous PPAR␣ led to a significant enhancement of its recruitment to the endogenous PGC-1␣ gene (Fig. 1D, center panel). Moreover, ChIP was also performed in HIB-1B cells transfected with the 2kb-PGC-1␣ or mutPPRE-PGC-1␣ promoter constructs. Results showed that enrichment was impaired when the construct in which the PPRE had been mutated was transfected (Fig. 1D, right panel). These results confirm that PPAR␣ binds and activates the PPRE element in the distal region of the PGC-1␣ gene promoter in brown adipocytes.
Interaction between PPAR␣-and cAMP-mediated Pathways in the Control of PGC-1␣ Gene Transcription-To further study the role of PPAR␣ in the control of the PGC-1␣ gene, we analyzed the response of the PGC-1␣ gene to acute cold induction in PPAR␣-null mice. Basal expression of PGC-1␣ mRNA was significantly decreased in BAT from PPAR␣-null mice reared at a thermoneutral temperature compared with that in wild-type animals ( Fig. 2A). Exposure of mice to cold (4°C for 24 h) caused a significant increase in PGC-1␣ mRNA levels in wild-type BAT, as reported previously (9). This increase was significantly blunted in the PPAR␣-null mice, suggesting that PPAR␣ is involved in the noradrenergic regulatory pathway in BAT in response to thermogenic activation.
We next used the specific PPAR␣ antagonist GW6471 to analyze whether an active PPAR␣-dependent regulatory pathway is required for effective noradrenergic induction of the PGC-1␣ gene in primary cultures of brown adipocytes (Fig. 2B). This drug not only suppressed the action of the PPAR␣ agonist GW7647, it also significantly reduced the action of norepinephrine, indicating the existence of cross-talk between the PPAR␣dependent and the noradrenergic pathways that regulate PGC-1␣ gene expression.
This PPAR␣/noradrenergic cross-talk was further analyzed at the transcriptional level (Fig. 2C). The increase in cAMP levels caused by the addition of IBMX led to a significant induction of PGC-1␣ promoter activity, an increase that was compa-  DECEMBER 16, 2011 • VOLUME 286 • NUMBER 50 rable with that achieved via PPAR␣ activation. The degree of induction was even greater following cotransfection with an expression vector driving the catalytic subunit of PKA, and when cotransfected together with PPAR␣ plus its ligand, a robust interaction was detected. Mutation of a previously reported CRE in the proximal region of the PGC-1␣ gene promoter (31) significantly impaired cAMP-and PKA-dependent induction but had no effect on sensitivity to activation by PPAR␣. In contrast, the PPRE-mutant form of the PGC-1␣ promoter (13) showed a significantly diminished capacity to respond to both PPAR␣and cAMP/PKA-dependent activation. Taken together, these results indicate that PPAR␣ and cAMP-mediated pathways interact to regulate PGC-1␣ gene transcription, a regulatory mechanism that requires the PPRE.

PPAR␣ and PRDM16 Induce PGC-1␣ Gene Transcription
Involvement of PRDM16 in PPAR␣-mediated Regulation of PGC-1␣ Gene Transcription-The requirement of an intact PPAR␣ signaling pathway for cAMP responsiveness of PGC-1␣ gene expression led us to analyze whether other transcriptional regulators could also be involved. We first analyzed PGC-1␣ itself. In contrast to what has been reported for PPAR␥-dependent activation of PGC-1␣ (13), where PGC-1␣ serves as a coactivator of its own gene, there was no evidence for a PPAR␣dependent autoinduction mechanism (2.6 Ϯ 0.4-fold induction by cotransfection of PPAR␣ versus 2.0 Ϯ 0.3-fold induction by cotransfection of PPAR␣ and PGC-1␣). We next analyzed PRDM16, which has been reported as another PPAR␥-coactivating protein involved in determining BAT fate (14). Cotransfection of a PRDM16 expression vector did not stimulate basal PGC-1␣ promoter activity and tended to activate PPAR␣ induction of PGC-1␣ gene transcription, although this last effect did not reach statistical significance (p Ͻ 0.061). Furthermore, this effect was significantly potentiated when the PKA pathway was also activated (Fig. 3A).
To assess whether PRDM16 binds to the PGC-1␣ gene promoter, we performed ChIP experiments. Results indicated a significant recruitment of endogenous PRDM16 protein to the endogenous PGC-1␣ gene in brown adipose tissue (Fig. 3B, left  panel). Treatment with either GW7647 or dibutyryl-cAMP significantly increased the recruitment of endogenous PRDM16 to the endogenous PGC-1␣ gene in primary brown adipocytes. This recruitment was maximal when both compounds were added (Fig. 3B, right panel). We next performed ChIP assays in HIB-1B cells cotransfected with either 2kb-PGC-1␣ or mutP-PRE-PGC-1␣ promoter constructs together with expression vectors for PPAR␣ and PRDM16 in the presence of either IBMX or a PKA stimulus. Results indicated that PRDM16 binding to the PGC-1␣ gene promoter occurs at the PPRE site (Fig.  3C). Given the recent demonstration of a direct physical inter-

FIGURE 2. PPAR␣-and cAMP-mediated pathways interact to control PGC-1␣ gene expression in brown adipocytes.
A, wild-type and PPAR␣-null mice were acclimated to a thermoneutral temperature (TN, 28°C) and then exposed to 4°C ambient temperature for 24 h (Cold) or maintained at 28°C. Data are presented as fold induction of PGC-1␣ mRNA levels relative to values in BAT from wild-type mice at 28°C. **, p Ͻ 0.01 for differences because of cold exposure for each genetic background; #, p Ͻ 0.05 for differences between wild-type and PPAR␣-null mice under each temperature condition. B, differentiated brown adipocytes in culture were treated with 1 M GW7647 for 24 h or with 0.5 M norepinephrine (NE) for 3 h in the presence or absence of the PPAR␣ antagonist GW7647 (10 M). Data are presented as mean Ϯ S.E. of three independent experiments done in triplicate. *, p Ͻ 0.05 versus controls; #, p Ͻ 0.05 for differences because of the PPAR␣ antagonist. C, HIB-1B cells were transfected with the 2kbPGC-1␣-luciferase construct (WT), the PPRE-mutated construct (PPREmut), or the CRE-mutated plasmid (CREmut). Where indicated, cells were cotransfected with an expression vector for the constitutively active form of PKA or were exposed to 0.5 mM IBMX in the presence or absence of the PPAR␣ expression vector plus 1 M GW7647. Results are presented as mean Ϯ S.E. of the fold change in promoter activity relative to the untreated, non-cotransfected WT. *, p Ͻ 0.05 versus controls; #, p Ͻ 0.05 for comparisons of mutated constructs with the WT construct under equivalent cotransfection or treatment conditions. action between PRDM16 and PPAR␣ (14), it is likely that the recruitment of PRDM16 to the PGC-1␣ promoter involves PRDM16 binding to PPAR␣. Although it has been reported that PRDM16 and PPAR␣ interact in a non-ligand-dependent manner in coimmunoprecipitation assays (14), the binding of PRDM16 to the endogenous PGC-1␣ gene is induced by addition of the PPAR␣-ligand in brown adipocytes (Fig. 3B).
PRDM16 Coactivates PPAR␣-and PKA-dependent Induction of UCP1 Gene Transcription-We next studied whether UCP1, another key thermogenesis gene that shares with PGC-1␣ the property of dual regulation by PPAR␣ and PKA, was also a direct target of PRDM16 coactivation. As shown in Fig. 4A, the transcriptional activity of a luciferase reporter construct driven by a 4.5-kb upstream region of the rat Ucp1 gene was induced by cotransfected PPAR␣ or PKA, as reported previously (19). An additional increase in luciferase activity was observed upon cotransfection of PPAR␣ and PKA together. Cotransfection of PRDM16 did not affect basal UCP1 promoter activity but significantly enhanced its response to PKA (Fig.  4A). Moreover, PRDM16 tended to increase PPAR␣ respon-siveness of the UCP1 promoter (p Ͻ 0.057) and, when the PKA pathway was also activated, maximal effects of PRDM16 were observed.

PRDM16 Coactivates a Consensus PPAR␣-driven Promoter, an Effect That Is Favored by PKA but Does Not Require PGC-1␣-To
further analyze the involvement of PRDM16 in the regulation of gene transcription by PPAR␣, we transiently transfected HIB-1B brown adipocytes with a promoter-reporter construct in which expression of the luciferase reporter gene is driven by a consensus PPAR␣-responsive promoter. PRDM16 alone had no effect, but it did significantly coactivate PPAR␣-mediated induction of PPRE-dependent promoter activity (Fig. 4B). Cotransfection of an expression vector for PKA alone did not affect PPRE promoter activity but potently enhanced PRDM16/ PPAR␣ coactivation. These results indicate that PRDM16 coactivation of PPAR␣ in brown adipocytes is just dependent on the presence of a PPAR␣-responsive element and enhanced when PKA pathways are activated.
PRDM16 has been reported to coactivate and bind PGC-1␣ (6). Because PGC-1␣ is highly expressed in HIB-1B brown adi- pocytes, involvement of PGC-1␣ in the molecular mechanism responsible for PRDM16 coactivation of PPAR␣ could not be ruled out. Therefore, we analyzed whether PRDM16 coactivation of PPAR␣ occurs in cells lacking PGC-1␣. For this purpose, we performed transient transfection experiments of the consensus PPRE promoter construct using CV-1 cells. As shown in Fig. 4C, the extent of induction of PPAR␣-dependent transcriptional activity by PRDM16 was similar with and without transfection of a PGC-1␣ expression vector. The same results were obtained using COS-7 cells (not shown). Transient transfection experiments were also performed using MEFs obtained from wild-type and PGC-1␣-null mice. PPAR␣-mediated induction and its coactivation by PRDM16 were unaltered in PGC-1␣null MEFs (Fig. 4D), indicating that PGC-1␣ is not required for PRDM16/PPAR␣ regulation of consensus PPRE promoter activity.

PRDM16 Gene Expression Is Controlled by PPAR␣ and by Noradrenergic, cAMP-mediated Mechanisms in Brown
Adipocytes-We next analyzed whether PRDM16 expression is affected by PPAR␣ and/or noradrenergic regulation. Acute cold exposure of mice (4°C for 24 h) significantly induced PRDM16 mRNA expression in BAT (Fig. 5A), although this effect was not observed at short times of cold exposure (0.82 Ϯ 0.20 fold-change after 4 h at 4°C versus controls), in agreement with previous reports (6). In PPAR␣-null mice, PRDM16 mRNA levels were lower under basal conditions, and their response to cold was greatly diminished (Fig. 5A).
Exposure of differentiated brown adipocytes to the PPAR␣specific activator GW7647 resulted in a significant increase in PRDM16 mRNA levels (Fig. 5B). Norepinephrine and cAMP also significantly increased PRDM16 mRNA expression. Basal levels of PRDM16 mRNA were unaltered in PPAR␣-null brown adipocytes, whereas the effects of GW7647 were blocked. Induction by norepinephrine and cAMP, although not completely impaired, was significantly reduced in brown adipocytes lacking PPAR␣, providing further support for cross-talk between PPAR␣-dependent and noradrenergic regulation of PRDM16 gene expression.
Activation of PPAR␣ in Human White Adipocytes Leads to Activation of the Brown Adipocyte-specific Pattern of Gene Expression, including Induction of PGC-1␣ and PRDM16-To further analyze the role of PPAR␣ in the control of PGC-1␣ and PRDM16 gene expression, we investigated whether increasing PPAR␣ expression in white adipocytes could confer on these cells the capacity to activate the brown-fat specific gene expression pattern. Adenoviral transduction of human SGBS white adipocytes with PPAR␣ increased PPAR␣ mRNA levels by approximately 30-fold compared with those in basal conditions. These high levels are comparable with those of endogenous PPAR␣ mRNA levels in mouse BAT. In the presence of GW7647, PPAR␣ expression led to a significant induction of PGC-1␣ and PRDM16 mRNA expression in human SGBS white adipocytes (Fig. 6). Furthermore, expression of other key BAT-selective genes, such as UCP1 and type 2 deiodinase (DIO2), were also induced, as was expression of the ␤3-adrenergic receptor (ADR␤3), the main ␤-adrenergic receptor involved in the regulation of thermogenesis. PPAR␣ activation in SGBS white adipocytes also increased the mRNA levels for several mitochondrial proteins that are known to be enriched in brown relative to white adipocytes. These include UCP3; ubiquinol-cytochrome c reductase core protein 1 (UQCRC1), a component of mitochondrial respiratory complex III; cytochrome c oxidase-2 (COII) and Ϫ4 (COIV), subunits of mitochondrial respiratory complex IV; and medium-chain acyl-coenzyme A dehydrogenase (MCAD), a metabolic protein involved in lipid catabolism. PPAR␣ activation also induced gene expression of lipoprotein lipase (LDL) and glucose transporter type 4 (GLUT4), which are involved in lipid and glucose uptake, respectively. In contrast, expression of the adipogenic genes PPAR␥ and fatty acid binding protein 4 (FABP4) remained unchanged. This confirmed that the action of PPAR␣ on adipocyte gene expression favors the acquisition of BAT-like features through induction of PGC-1␣ and PRDM16, master regulators of the brown adipocyte-specific phenotype.

DISCUSSION
PPAR␣ plays an important role in the overall regulation of energy metabolism, mainly by controlling genes involved in lipid metabolism. Furthermore, PPAR␣ constitutes a specific marker of mature brown adipocytes, where together with PGC-1␣, it regulates key components of the thermogenic function, such as UCP1 (18,19). In this study, we identified PGC-1␣ as a direct target of PPAR␣ transcriptional regulation in BAT. PPAR␣ activators regulated the expression of the PGC-1␣ gene in brown adipocytes through a PPAR␣-responsive element in the distal promoter region. This PPRE binds both PPAR␣ (these results) and PPAR␥ (13) and behaves as a promiscuous responsive site to activation by either PPAR␣ or PPAR␥, but not PPAR␦. Transcriptional control by PPAR subtypes through a unique PPRE has been reported previously for mammalian genes (16). This mechanism provides tissue-specific regulation and/or specific responsiveness to metabolic challenges in a par- ticular cell-type and probably relies on the interaction of PPARs with tissue-specific coregulators, such as PRDM16 in brown adipocytes. Dual PPAR␣ and PPAR␥ (but not PPAR␦) regulation has been reported for the UCP1 gene (4,19) and probably reflects the regulatory dynamics of thermogenic gene expression in BAT, namely PPAR␥-dependent induction of thermogenic genes in association with brown adipocyte differentiation, and coordination between PPAR␣-induced fatty acid oxidation and thermogenesis in fully differentiated brown adipocytes (18). Both PGC-1␣ and UCP1 genes also share a common transcriptional regulatory mechanism involving PPARand cAMP-responsive elements located in distal and proximal promoter regions, respectively (4,12,13,19,32). Moreover, the present results provide evidence for a robust interaction between these two pathways in the regulation of UCP1 and PGC-1␣ genes that is mediated at the molecular level by PPAR␣ and PRDM16, as discussed below and schematized in Fig. 7.
Although PPAR␥ is essential for fat cell differentiation and lipid storage in both WAT and BAT, PPAR␣ is specifically induced in the terminal steps of brown adipocyte differentiation (17) as well as in brown-like adipocytes ("brite" adipocytes) appearing in WAT after cold exposure of mice (33). Cold exposure induces the expression and nuclear translocation of PPAR␣ in rodent BAT (34), and genetic analyses have revealed that PPAR␣ is involved in PGC-1␣ and UCP1 gene induction in mice (35). The role of PPAR␣ in brown adipocytes may be related to its capacity to respond to lipid ligands originating from lipolysis after noradrenergic activation of brown adi-pocytes. The activation of PGC-1␣ gene expression by PPAR␣ may provide a mechanism for concerted induction of thermogenic genes (UCP1, fatty acid oxidation genes, mitochondrial genes) in response to the enhanced intracellular availability of fatty acids sensed by PPAR␣. PPAR␣-null mice, despite being resistant to cold and having overtly normal BAT morphology (35,36,30), show thermogenesis-related disturbances in FIGURE 6. Effects of PPAR␣ on gene expression in human white adipocytes. Human SGBS white adipocytes differentiated in culture were transduced with adenoviral vectors driving PPAR␣ or GFP as a control and were then exposed to 1 M GW7647. Shown is the analysis of the transcript levels of the indicated genes by real-time RT-PCR. Bars represent the mean Ϯ S.E. from three independent experiments done in duplicate. Results are expressed as fold induction with respect to values in control GFP adipocytes. *, p Ͻ 0.05 versus control. response to cold stress, such as a marked suppression of BAT growth concurrent with a prominent decrease in fatty acid oxidative and thermogenic activities (37,38,18). Consistent with our current findings of a role for PPAR␣ in the control of PGC-1␣ gene transcription, we observed that PGC-1␣ gene expression is decreased in PPAR␣-null BAT both under basal conditions and in response to acute thermogenic activation.
We found that PPAR␣-and cAMP-mediated pathways strongly interacted in the control of PGC-1␣ gene transcription. This dynamic was greatly potentiated by PRDM16, which we found binds to the PGC-1␣ gene promoter at the PPAR␣binding site. Because the action of PRDM16 regulating brownfat-specific gene expression occurs independently from binding to DNA (6) and PRDM16 can bind PPAR␣ (14), we propose that PRDM16 coactivation activity upon the PGC-1␣ gene relies on direct PRDM16 interaction with PPAR␣ bound to the PPRE. PRDM16 has been identified as the key molecular switch that determines the development of brown adipocytes during embryonic development (6) and the appearance of brown adipocytes in WAT depots in adult mice (15). It has been suggested that the ability of PRDM16 to stimulate a BAT phenotype is due, in part, to its association with PGC-1 coactivators and PPAR␥ (6,14). These results further indicate that the interaction of PPAR␣ with PRDM16 is required for the full thermogenic activation of PGC-1␣ gene transcription in BAT. Hence, we propose a novel role for PRDM16 in brown adipocytes. By interacting with PPAR␣, PRDM16 coordinately regulates the expression of genes required for active thermogenesis through induction of the PGC-1␣ gene.
We also demonstrate for the first time that PRDM16 gene expression is induced by both norepinephrine and PPAR␣ activation in brown adipocytes. Furthermore, an active PPAR␣-dependent pathway is required for maximal adrenergic stimulation of PRDM16 expression, both in BAT in vivo and in cultured brown adipocytes. This PPAR␣-and cAMP-mediated regulation of PRDM16 gene expression is expected to contribute to the cross-talk between PPAR␣and cAMP-dependent signaling pathways and further suggests that PRDM16 is linked to activation of BAT thermogenesis. In addition, it has been reported that overexpression of PRDM16 induces PPAR␣ gene expression (6). Thus, a feed-forward loop of PPAR␣ and PRDM16 gene regulation may provide a way to maintain high levels of both PPAR␣ and PRDM16 in thermogenically active BAT. Accordingly, PRDM16 may play a pivotal role not only in early differentiation processes of the brown adipose lineage but also in specific metabolic regulation in relation to thermogenesis in the already differentiated and functional brown adipocyte.
Whether PPAR␣-dependent pathways affect energy expenditure in adult humans remains to be determined. Recently, several laboratories have reported the presence of discrete, metabolically active BAT depots in healthy adult humans that can be activated by cold exposure and blocked using ␤-adrenergic antagonists (39 -41). Fibrates are synthetic ligands of PPAR␣ that are currently used as hypolipidemic drugs. It is possible that increased fatty acid uptake and subsequent oxidation by BAT via activation of PPAR␣ contribute to the hypolipidemic effect of fibrates. On the other hand, the expression of PPAR␣ in human WAT is low but has been reported to be negatively correlated with body mass index (42). According to our current findings in human SGBS white adipocytes, one aspect of the biological effects of PPAR␣ activation would be to promote a BAT-like phenotype in WAT depots, acting through the induction of PGC-1␣ and PRDM16 gene expression. This is consistent with previous observations that PPAR␣ agonists induce the expression of genes involved in lipid oxidation and mitochondrial biogenesis in human white adipocytes in vitro (43,44). Those parameters are also induced by pharmacological activation of the cAMP/PKA pathway, which also increases PPAR␣ expression in human white adipocytes (43). In rodents, chronic cold exposure and ␤-adrenergic stimulation induce the appearance of brown-like adipocytes, now referred to as brite adipocytes (33), in WAT depots (45)(46)(47). PPAR␣, which is up-regulated by ␤3-adrenergic stimulation, may be involved in this process because PPAR␣-null mice exhibit impaired induction of lipid oxidation and mitochondrial biogenesis in WAT in response to ␤3-adrenergic agonist treatment (48).
In mice, BAT has been described as having antiobesity and antidiabetic effects (49,50). In adult humans, the possibility of activating BAT metabolism by stimulating specific BAT depots and/or by facilitating conversion of WAT into BAT could be a promising approach for treating obesity and related metabolic disorders. Unfortunately, many candidate agonists of the ␤3-adrenergic receptor have failed in human clinical trials, although these drugs have been efficacious in rodent models of obesity (51,52). Because this failure has been related to the low number of ␤3-adrenergic receptors in human adipose tissue, the observation that PPAR␣ induces ␤3-adrenergic receptor expression in human white adipocytes, reported here, suggests that combined treatment with ␤-adrenergic and PPAR␣-specific agonists might have the potential to promote energy expenditure in adipose depots.
In summary, we have established that PPAR␣ is an important actor in the control of BAT thermogenic activity via induction of PGC-1␣ and PRDM16, key players in the acquisition of the thermogenic competence of brown adipocytes.