Peroxisome Proliferator-activated Receptor alpha  Activates Transcription of the Brown Fat Uncoupling Protein-1 Gene

A LINK BETWEEN REGULATION OF THE THERMOGENIC AND LIPID OXIDATION PATHWAYS IN THE BROWN FAT CELL*

M. José Barberá, Agatha Schlüter, Neus Pedraza, Roser Iglesias, Francesc Villarroya, and Marta GiraltDagger

From the Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Avda Diagonal 645, 08028 Barcelona, Spain

Received for publication, July 14, 2000, and in revised form, October 3, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

High expression of the peroxisome proliferator-activated receptor alpha  (PPARalpha ) differentiates brown fat from white, and is related to its high capacity of lipid oxidation. We analyzed the effects of PPARalpha activation on expression of the brown fat-specific uncoupling protein-1 (ucp-1) gene. Activators of PPARalpha increased UCP-1 mRNA levels severalfold both in primary brown adipocytes and in brown fat in vivo. Transient transfection assays indicated that the (-4551)UCP1-CAT construct, containing the 5'-regulatory region of the rat ucp-1 gene, was activated by PPARalpha co-transfection in a dose-dependent manner and this activation was potentiated by Wy 14,643 and retinoid X receptor alpha . The coactivators CBP and PPARgamma -coactivator-1 (PGC-1), which is highly expressed in brown fat, also enhanced the PPARalpha -dependent regulation of the ucp-1 gene. Deletion and point-mutation mapping analysis indicated that the PPARalpha -responsive element was located in the upstream enhancer region of the ucp-1 gene. This -2485/-2458 element bound PPARalpha and PPARgamma from brown fat nuclei. Moreover, this element behaved as a promiscuous responsive site to either PPARalpha or PPARgamma activation, and we propose that it mediates ucp-1 gene up-regulation associated with adipogenic differentiation (via PPARgamma ) or in coordination with gene expression for the fatty acid oxidation machinery required for active thermogenesis (via PPARalpha ).



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The peroxisome proliferator-activated receptor alpha  (PPARalpha )1 is a fatty acid-activated transcription factor that plays a key role in the transcriptional regulation of genes involved in cellular lipid metabolism (1). PPARalpha together with PPARgamma and PPARdelta /beta belong to a subgroup of the nuclear hormone receptor superfamily that heterodimerizes with the 9-cis-retinoic acid receptors (RXRs) (2-5). The PPAR-RXR heterodimer binds to specific response elements (PPREs), which consist of a direct repeat of the consensus half-site motif spaced by one nucleotide (DR-1) (6). Fatty acids, peroxisome proliferators, and fibrate hypolipidemic drugs can activate PPARalpha (1, 4), and natural (leukotrine B4) or synthetic (fibrate Wy 14,643) specific ligands for PPARalpha have been identified (7). In contrast, 15-deoxy-Delta 12,14-prostaglandin J2 and thiazolidinedione antidiabetic agents are selective ligands for PPARgamma (8-10). In addition to ligand selectivity, PPAR subtypes have been involved in different biological functions. PPARalpha is mostly expressed in tissues with high rates of fatty acid oxidation and peroxisomal metabolism, such as brown fat, liver, or heart (1, 11). Recent studies of PPARalpha -null mice have confirmed that PPARalpha is necessary in vivo for hepatic fatty acid oxidation and ketone body synthesis during starvation (12). PPARdelta , which is ubiquitously expressed, seems to be involved in basic lipid metabolism (11). High expression of PPARgamma is mainly restricted to white (WAT) and brown (BAT) adipose tissue (13). Hence, in contrast to the role of PPARalpha in cellular lipid catabolism, PPARgamma regulates adipogenesis (i.e. lipid deposition) (13, 14).

BAT is a major site for nonshivering thermogenesis in mammals. Its thermogenic capacity relies on the presence of an inner mitochondrial protein uniquely expressed in brown adipocytes, the uncoupling protein (UCP) (15), now referred to as UCP-1 since the discovery of the more widely expressed UCP-2 and UCP-3 (for review, see Ref. 16). Brown fat thermogenesis is mainly controlled by norepinephrine released from sympathetic terminals innervating the tissue, although nuclear receptor-mediated pathways have also been described. Thus, activation of PPARgamma promotes HIB-1B brown adipocyte differentiation (17), and up-regulates ucp-1 gene expression (18). Furthermore, we demonstrated that retinoic acid is a powerful inducer of ucp-1 gene transcription, acting through retinoic acid receptors and RXRs (19, 20). The 5'-flanking region of the rat ucp-1 gene contains the proximal regulatory promoter, including C/EBP-regulated sites (21) and the main cAMP-regulatory element (22), and an upstream enhancer involved in complex regulation by retinoic acid receptors, RXR, and thyroid hormone nuclear receptors (19, 20, 23). A site responsive to PPARgamma activators has also been located in the upstream enhancer of the murine ucp-1 gene (18).

BAT highly coexpresses not only PPARgamma and PPARdelta subtypes but also PPARalpha (24). BAT stores triglycerides but, in contrast to WAT, it uses lipids as oxidative substrates to generate heat. Since PPARalpha induces the expression of fatty acid oxidation enzymes in tissues other than BAT (6), it may do so in BAT in association with thermogenic requirements. Here we report that PPARalpha activators induce ucp-1 gene expression in brown adipocytes and in BAT in vivo, acting through a PPRE located in the upstream enhancer of the ucp-1 gene that is also responsible for PPARgamma -dependent regulation. PPARalpha is proposed to coordinate the activation of lipid oxidation and thermogenic activity in brown fat.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Wy 14,643 (pirixinic acid) and 15-deoxy-Delta 12,14-prostaglandin J2 were obtained from Cayman Chemicals. Troglitazone and BRL 49653 were kind gifts from Dr. T. Leff (Parke-Davis Research) and Dr. L. Casteilla (Toulouse, France), respectively. Clofibrate, fenofibrate, bezafibrate, Ly171883, 3,5,3'-triiodothyronine, insulin, norepinephrine, and 8-bromo-cAMP were obtained from Sigma. Methoprene was from Promochem.

Cell Culture-- Primary culture of differentiated brown adipocytes was performed as described previously (19), and grown in 5 ml of Dulbecco's modified Eagle's medium-Ham's F-12 medium (1:1) supplemented with 10% fetal calf serum, 20 nM insulin, 2 nM 3,5,3'-triiodothyronine, and 100 µM ascorbate. Experiments were performed on day 9 of culture when 80-90% of the cells were considered to be differentiated on the basis of lipid accumulation and acquisition of brown adipocyte morphology. Brown adipocytes were exposed to 10 µM Wy 14,643 for 24 h, or at the concentrations and times indicated in the experiments. Cells were also exposed to various PPAR agonists for 24 h, except for 15-deoxy-Delta 12,14-prostaglandin J2 which was added at a final concentration of 10 µM for 6 h. As indicated, cycloheximide (Sigma) was used at a dose of 5 µg/ml as reported (19).

HepG2 human hepatoma cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The HIB-1B brown adipocyte cell line, kindly provided by Dr. B. Spiegelman, was cultured in Dulbecco's modified Eagle's medium/F-12 (1:1) supplemented with 10% heat-inactivated fetal calf serum and 4 mg/liter biotin.

RNA Isolation and Northern Blot Analysis-- Total RNA was extracted using the RNeasy Mini Kit (Quiagen). Northern blot analysis and hybridization were carried out as described (24). Blots were hybridized using as probes the full-length cDNA for rat UCP-1 (25) and 0.5 kb of the cDNA for mouse mitochondrial-genome-encoded cytochrome oxidase subunit II (COII) (26), which was used as a control. Hybridization signals were quantified using Molecular Image System GS-525 (Bio-Rad). Statistical analysis was performed by Student's t test.

Oligonucleotides and Plasmids-- Oligonucleotides were chemically synthesized by Roche Diagnostics. The UCP1-PPRE double-stranded oligonucleotide corresponds to positions -2485 to -2458 of the rat ucp-1 gene and its sequence is 5'-GTGGGTCAGTCACCCTTGATCACACTGC-3', flanked by HindIII/XbaI ends. The mutated version mutUCP1-PPRE corresponds to the sequence 5'-AGTCACAATTGATCACACTGC-3' also flanked by HindIII/XbaI ends.

The plasmid (-4551)UCP1-CAT, in which the region -4551 to +110 of the rat ucp-1 gene drives the promoterless chloramphenicol acetyltransferase (CAT) gene, was a kind gift from Dr. D. Ricquier (27). The derived plasmids containing 5'-deletion mutations (-3628)UCP1-CAT and (-141)UCP1-CAT, an internal deletion mutation (Delta -2469/-2283)UCP1-CAT, or the plasmid (-3628/-2283)UCP1-CAT corresponding to the fragment -3628/-2283 linked to (-141)UCP1-CAT, have been described previously (19). The plasmid (-2767/-2283)UCP1-CAT was obtained by digesting the plasmid (-3628/-2283)UCP1-CAT with SpeI. The plasmid (-2534/-2283) was constructed by polymerase chain reaction using an oligonucleotide corresponding to the -2534/-2522 fragment (5'-ACATGGGCGGCGAG-3') and (-3628/-2283)UCP1-CAT as template. The plasmids (-172)UCP1-CAT and (-2494/-2318)UCP1-CAT, in which the fragment -2494/-2318 was placed upstream in (-172)UCP1-CAT, are described elsewhere (20). The mutated versions of (-3628/-2283)UCP1-CAT and (-2494/-2318)UCP1-CAT, containing AA instead of CC at sites -2473 and -2472, were generated using the Quikchange site-directed mutagenesis kit (Stratagene).

Mammalian expression vectors that contain the murine cDNAs of the respective PPAR isoforms are driven by the simian virus-40 promoter (1, 4, 5). pRSV-RXRalpha was an expression vector for the alpha -subtype of human RXR (28). Expression plasmids driving murine RXR isoforms were kindly provided by Dr. P. Chambon. The expression vectors for human CBP, pCMX-CBP (29), and murine PGC-1, pSV-PGC1 (30), are described elsewhere.

Transfection Assays-- Murine primary brown adipocytes differentiated in culture were transiently transfected by the calcium phosphate precipitation method on day 9 of culture (22). Each transfection contained 12 µg of (-4551)UCP1-CAT and included or not 3 µg of the expression vector pSG5-PPARalpha . When indicated 10 µM Wy 14,643, 10 µM BRL 49653, or 30 µM Ly171883 was added after transfection. 1 µg of cytomegalovirus-beta -galactosidase was also included to assess the efficiency of separate transfections. The cells were incubated for 24 h and, for each condition, at least three plates were pooled.

HepG2 and HIB-1B cells were transfected using the FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals) for 16 h and cells were harvested 24 h later. Unless otherwise indicated, each transfection contained between 0.5 and 1 µg of UCP1-CAT vector, 0.1 µg of cytomegalovirus-beta -galactosidase, and included or not 0.3 µg of pSG5-PPARalpha or pSG5-PPARgamma expression vector, and/or 0.1 µg of pRSV-RXRalpha . When indicated, 0.1 µg of the expression vector pCMX-CBP or pSV-PGC1 was added.

Analysis of CAT activity was determined by thin layer chromatography (22) and quantified by radioactivity counting (AMBIS). The amount of cell extract used was adjusted to maintain a percentage conversion of chloramphenicol between 1 and 20%. The CAT activity was normalized for variation in transfection efficiency using the beta -galactosidase activity measured for each sample as a standard.

DNA Binding Experiments-- Nuclear proteins were isolated from rat BAT or differentiated primary brown adipocytes as described elsewhere (21, 22). cDNAs for mPPARalpha , mPPARgamma , and hRXRalpha were transcribed and translated in vitro by using the TNT Quick Coupled Transcription/translation Systems (Promega) according to the manufacturer's instructions.

For the gel retardation assays, the UCP1-PPRE oligonucleotide (10,000-20,000 cpm) was incubated for 30 min at 25 °C with 5 µg of nuclear protein extract from BAT or differentiated brown adipocytes, or 5 µl of in vitro transcribed/translated proteins. Reactions were carried out in a final 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 for 60-80 min in nondenaturing 5% polyacrylamide gels in 0.5 × TBE (44.5 mM Tris, 44.5 mM borate, 1 mM EDTA). In the competition experiments, 100-fold molar excess of unlabeled oligonucleotide was included in each respective binding reaction. When indicated, 1 µl of antiserum against PPARalpha (N-19), PPARgamma (N-20), RXRalpha (D-20), or ETS (C-275) from Santa Cruz was used.

Tissue Samples-- BAT was extracted from two-month-old female, 15-day lactating, or newborn Swiss mice. Adult mice were treated with a single intraperitoneal injection of Wy 14,643 (50 µg/g body weight) or troglitazone (100 µg/g body weight) in 50% dimethyl sulfoxide/saline. Controls were given equivalent volumes of the vehicle and mice were studied 6 h after injections. Neonates were placed in a humidified thermostated chamber at 28 °C, and injected intraperitoneally 2 h after birth with Wy 14,643 (50 µg/g body weight), BRL 49653 (50 µg/g body weight), or equivalent volumes of the 20% dimethyl sulfoxide/saline vehicle solution. Pups were studied 15 h after treatment.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activators of PPARalpha Induce the Expression of the ucp-1 Gene in Differentiated Brown Adipocytes-- To analyze whether PPARalpha agonists modulate the expression of the ucp-1 gene, primary cultures of murine brown adipocytes were used since they express all three PPAR subtypes (24). As shown in Fig. 1, exposure of brown adipocytes differentiated in culture (day 9) to PPARgamma activators resulted in a 2-fold (15-deoxy-Delta 12,14-prostaglandin J2) to 8-fold (10 µM BRL 49653) increase in UCP-1 mRNA levels. When PPARalpha activators, such as several fibrates and the PPARalpha -specific ligand Wy 14,643, were tested an even higher (3-12-fold) increase in UCP-1 mRNA expression was detected. In contrast, COII mRNA expression did not respond to PPAR activators, thus indicating that the effect of PPAR activators is specific for UCP-1 mRNA.



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Fig. 1.   Effects of PPARalpha and PPARgamma agonists on ucp-1 gene expression in primary brown adipocytes. Brown adipocytes differentiated in culture from stromal vascular cells (day 9) were exposed for 24 h (except 15-deoxy-Delta 12,14-prostaglandin J2 for 6 h) to the following concentrations of PPARgamma -specific (10 µM 15-deoxy-Delta 12,14-prostaglandin J2, 10 µM BRL 49653) or PPARalpha -specific (30 µM Ly171883, 500 µM clofibrate, 500 µM fenofibrate, 100 µM bezafibrate, 10 µM Wy 14,643) agonists. Northern blot analyses were performed with 10 µg of total RNA extracted from three pooled plates. Bars are means from two to four independent experiments on different cultures and S.E. is indicated when n >=  3. An example of the Northern blot analysis is depicted in the bottom of the figure. Arrows indicate the position of the two UCP-1 mRNA species in mice (1.6 and 1.9 kb) and the mitochondrial genome-encoded COII mRNA (0.8 kb), which was used as a control.

Exposure to Wy 14,643 led to a dose-dependent increase in UCP-1 mRNA expression (Fig. 2A) and maximum induction was attained at 10 µM, a concentration at which it selectively activates PPARalpha (4), whereas at 100 µM it activates all three PPAR subtypes (10). The effects of 10 µM Wy 14,643 were maximal after 12 h of exposure to the PPARalpha ligand, and maintained after 24 h (Fig. 2B). The maximal effect of 10 µM Wy 14,643 on UCP-1 mRNA levels resulted in an induction that was around 40% that of 0.5 µM norepinephrine (20-fold ± 2.7) and 180% that of 0.5 mM 8-bromo-cAMP, a nonmetabolizable cAMP derivative (4.3-fold ± 0.6).



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Fig. 2.   Dose-response and time course curves for the effects of the PPARalpha ligand Wy 14,643 on ucp-1 gene expression. Brown adipocytes differentiated in culture (day 9) were exposed to the indicated concentrations of Wy 14,643 for 24 h (A) or exposed to 10 µM Wy 14,643 for the indicated times (B). Total RNA (10 µg) was analyzed by Northern blot. Points are means from three independent experiments with triplicate plates, in which the variation within the experimental groups is less than 15%.

The Stimulation of ucp-1 Gene Expression by the PPARalpha Ligand Wy 14,643 Is Independent of Protein Synthesis and Synergizes with the Effects of an RXR-specific Agonist-- Brown adipocytes were exposed for 12 h to 10 µM Wy 14,643 in the absence or presence of 5 µg/ml cycloheximide, an inhibitor of protein synthesis (Fig. 3A). Cycloheximide treatment led to lower basal expression of UCP-1 mRNA, as already described (19), but it did not affect the ability of Wy 14,643 to increase UCP-1 mRNA.



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Fig. 3.   Effects of 10 µM Wy 14,643 in the presence of cycloheximide or of an RXR-agonist on UCP-1 mRNA expression. Brown adipocytes differentiated in culture (day 9) were exposed to 10 µM Wy 14,643 for 12 h in the presence or not of 5 µg/ml cycloheximide (CHX) (A) or exposed to 10 µM Wy 14,643 and/or 100 µM methoprene for 24 h (B). Total RNA (10 µg) was analyzed by Northern blot. Treatments are indicated as + whereas untreated cells are shown as -. Bars are means from two to three independent experiments on different cultures and are expressed relative to untreated cells, which were set as 1.

When the effects of the RXR-specific agonist methoprene were analyzed (Fig. 3B), results showed that besides its reported direct action upon UCP-1 mRNA expression (20), there was a synergistic effect when both the PPARalpha and the RXR ligands were added, suggesting a PPARalpha -RXR heterodimer-mediated effect on ucp-1 gene expression.

PPARalpha Induces the Rat ucp-1 Gene Promoter Activity-- Primary brown adipocytes were transiently transfected with a plasmid containing the upstream 4.5 kb of the rat ucp-1 gene fused to a CAT reporter gene. As shown in Fig. 4, PPARalpha activators increased the (-4551)UCP1-CAT activity at least 2-fold, in the same range of the effect caused by BRL 49653. Responsiveness of (-4551)UCP1-CAT to PPAR activators was enhanced 6-fold by co-transfection of the expression vector for PPARalpha . Thus, expression of both endogenous ucp-1 gene and transfected ucp-1 gene promoter are up-regulated by PPARalpha activators in primary brown adipocytes.



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Fig. 4.   Effects of PPARalpha and PPARgamma agonists on (-4551)UCP1-CAT expression in transiently transfected brown adipocytes. Brown adipocytes differentiated in culture (day 9) were transfected with 12 µg of (-4551)UCP1-CAT. When indicated, 3 µg of the expression vector pSG5-PPARalpha was co-transfected. After transfection, cells were exposed or not to 10 µM Wy 14,643, 10 µM BRL 49653, or 30 µM Ly171883. Results are expressed as CAT activity relative to control, which is set to 1, and are means of two independent experiments, each one performed in triplicate.

RXRalpha Enhances the PPARalpha -dependent Induction of the ucp-1 Gene Promoter-- To further investigate the transcriptional regulation by PPARalpha of the ucp-1 gene promoter, we used the brown adipocyte-derived HIB-1B cell line. These cells express PPARgamma and PPARdelta but not PPARalpha (24). Thus, HIB-1B cells provide a useful model of brown fat-derived cell in which PPARalpha -dependent regulation rely on transfected receptor. In agreement, Wy 14,643 did not modify (-4551)UCP1-CAT activity (Fig. 5A). However, co-transfection of pSG5-PPARalpha induced (-4551)UCP1-CAT activity 3-fold in the absence and nearly 7-fold in the presence of 10 µM Wy 14,643. Co-transfection of pRSV-RXRalpha caused a synergistic increase in the PPARalpha -dependent effect upon (-4551)UCP1-CAT activity. We next performed co-transfection experiments using HepG2 cells to avoid any interference of PPARgamma in the observed effects. The HepG2 cell line was chosen because, in contrast to HIB-1B cells, does not express PPARgamma nor PPARalpha (31), and has been widely used to analyze PPARalpha regulation of gene transcription (31-33). As shown in Fig. 5B, co-transfection of pSG5-PPARalpha enhanced (-4551)UCP1-CAT activity and its responsiveness to Wy 14,643 in a dose-dependent manner, and maximal effects were observed at 0.3 µg of pSG5-PPARalpha . This amount of vector was the same at which the maximum synergistic enhancement by co-transfection of pRSV-RXRalpha was found (Fig. 5C), nearly 200-fold in the absence and 350-fold in the presence of 10 µM Wy 14,643. When the other PPAR subtypes were tested, co-transfection of PPARgamma in the presence of RXRalpha caused a similar increase in (-4551)UCP1-CAT activity than that of PPARalpha , but no effect was observed due to PPARdelta co-transfection (data not shown).



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Fig. 5.   PPARalpha -dependent induction of (-4551)UCP1-CAT expression in transiently transfected HIB-1B and HepG2 cells: influence of RXR co-transfection. A, HIB-1B cells were transfected with 1 µg of (-4551)UCP1-CAT vector, and included or not 0.3 µg of pSG5-PPARalpha , and/or 0.1 µg of pRSV-RXRalpha . After transfection, cells were exposed (dark bars) or not exposed (open bars) to 10 µM Wy 14,643 for 24 h. Results are shown as relative to the basal expression of (-4551)UCP1-CAT, which is set to 1. Bars are means of at least two independent experiments, each one done in duplicate. B, HepG2 cells were transfected with 1 µg of (-4551)UCP1-CAT vector together with increasing amounts of the expression vector pSG5-PPARalpha . After transfection, cells were exposed () or not exposed (open circle ) to 10 µM Wy 14,643 for 24 h. C, as in B, but 0.1 µg of pRSV-RXRalpha was also co-transfected. Points are means of at least two independent experiments, each one done in duplicate.

CBP and PGC-1 Coactivate the PPARalpha -dependent Activation of the ucp-1 Gene Promoter-- We next analyze whether co-regulators CBP and/or PGC-1 were involved in mediating PPARalpha transcriptional regulation of (-4551)UCP1-CAT. Co-transfection of pCMX-CBP or pSV-PGC1 alone enhanced basal (-4551)UCP1-CAT activity 5- and 7-fold, respectively (Fig. 6). When co-transfected together with pSG5-PPARalpha , an additive effect was observed in the absence of PPARalpha ligand, but when 10 µM Wy 14,643 was added, a synergistic activation was detected (30-fold for CBP and nearly 40-fold for PGC-1). When both coregulators were co-transfected in the presence of PPARalpha , a further increase in (-4551)UCP1-CAT activity was observed. These results point to an involvement of both CBP and PGC-1 in coactivating PPARalpha and further increasing responsiveness of the ucp-1 gene promoter to PPARalpha -ligand.



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Fig. 6.   Effects of CBP and/or PGC-1 co-transfection on the PPARalpha -dependent induction of (-4551)UCP1-CAT expression. HepG2 cells were co-transfected twith 1 µg of (-4551)UCP1-CAT vector, and included or not 0.3 µg of pSG5-PPARalpha . When indicated, 0.1 µg of pCMX-CBP and/or pSV-PGC1 were also co-transfected. After transfection, cells were exposed (dark bars) or not exposed (open bars) to 10 µM Wy 14,643 for 24 h. Results are shown as relative to the basal expression of (-4551)UCP1-CAT, which is set to 1. Bars are means of at least two independent experiments, each one done in duplicate.

PPARalpha - and PPARgamma -dependent Regulation Require the Same Element in the Upstream Region of the ucp-1 Gene Enhancer-- To determine the site in the 5'-region of the rat ucp-1 gene responsible for PPARalpha action, the effects of PPARalpha co-transfection on different deletion and double-point mutants of (-4551)UCP1-CAT were studied in transfected HepG2 and HIB-1B cells (Fig. 7). For comparative purposes, parallel co-transfection experiments were performed with PPARgamma . Results in both cell lines and for each PPAR subtype, indicated that both PPAR subtypes share a responsive site located in the -2494/-2318 enhancer region of the ucp-1 gene. When a double-point mutation, in which the CC at positions -2472 and -2473 were changed to AA (see Fig. 8A), was introduced in both (-3628/-2283)UCP1-CAT and (-2494/-2318)UCP1-CAT vectors, responsiveness to both PPARalpha and PPARgamma was abolished. Furthermore, when the -2494 to -2445 fragment was placed upstream (-172)UCP1-CAT or the HSV thymidine kinase promoter in pBLCAT2, it conferred 6- and 3-fold responsiveness, respectively, to PPARalpha and PPARgamma (data not shown).



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Fig. 7.   Analysis of the PPARalpha - and PPARgamma -dependent regulation of transiently transfected deletion or double-point mutants of the (-4551)UCP1-CAT. HepG2 and HIB-1B cells were transiently transfected with 1 µg of (-4551)UCP1-CAT or equivalent amounts of the deletion or double-point mutants illustrated on the left. Asterisks indicate the double-point mutated versions of (-3628/-2283)UCP1-CAT and (-2494/-2318)UCP1-CAT, containing AA instead of CC at sites -2473 and -2472. Transfections included 0.1 µg of the expression vector pRSV-RXRalpha , and included or not, 0.3 µg of pSG5-PPARalpha (dark bars) or pSG5-PPARgamma (open bars). Results are expressed as the -fold induction caused by PPAR co-transfection on each transfected construct. Bars are means of at least two independent experiments, each one done in duplicate.



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Fig. 8.   Electrophoretic mobility shift assays of the -2485/-2458 region of the rat ucp-1 gene. A, sequence corresponding to the -2485/-2458 region of the rat ucp-1 gene (UCP1-PPRE), was compared with a consensus PPRE (34) and to the analogous regions in the murine (-2499/-2472) and human (-3732/-3705) ucp-1 gene promoters (18, 35). Asterisks indicated the double-point mutant derivative version (mutUCP1-PPRE) in which the CC at positions -2472 and -2473 were changed to AA. The upper arrows show the putative alignments of three motifs closely related to an idealized half-site. B, gel mobility shift assay: the double-stranded oligonucleotide -2485/-2458 was end-labeled and incubated with 5 µl of in vitro transcribed/translated RXRalpha , alone or together with PPARalpha or PPARgamma . Arrows indicate the corresponding heterodimers bound to the probe. Lane 1 showed that the mock lysate produced two nonspecific bands when incubated with the probe. C, super-shift assay: the labeled UCP1-PPRE probe was incubated with 5 µg of nuclear protein extract from differentiated primary brown adipocytes. When indicated, 1 µl of antiserum against RXRalpha , PPARalpha , PPARgamma , or ETS (as negative control) were added. Arrows indicate the super-shifted bands. D, protein extracts from rat brown adipose tissue nuclei (5 µg) were incubated with the labeled UCP1-PPRE probe. Super-shift analysis was performed by incubation with 1 µl of antiserum against PPARalpha , PPARgamma , or ETS. Oligonucleotide competitors, UCP1-PPRE (WT) and mutUCP1-PPRE (MUT), were added at a 100-fold molar excess relative to probe concentration. A nonspecific-binding band was detected (n.s.). Bracket indicates the specific-binding bands and arrows the super-shifted bands.

The -2485/-2458 Site in the ucp-1 Gene Binds PPARalpha and PPARgamma -- Analysis of the sequence required for PPAR responsiveness in the rat ucp-1 gene promoter indicated the presence of a direct repeat with 1-base pair spacing related to a consensus PPRE (34) (Fig. 8A, arrows indicate half-site-related motifs). This -2485/-2458 sequence (UCP1-PPRE) in the rat ucp-1 gene promoter is highly conserved when compared with the previously reported PPARgamma -responsive element in the murine ucp-1 gene (18) and to the corresponding sequence in the human ucp-1 gene promoter (35) (Fig. 8A). Electrophoretic gel mobility shift assays were performed using the UCP1-PPRE as labeled probe. As shown in Fig. 8B, in vitro transcribed/translated RXRalpha alone (lane 2) did not bind significantly to this sequence although two nonspecific bands were detected as with the reticulocyte lysate (lane 1, n.s.). However, incubation with a mixture of PPARalpha or PPARgamma with RXRalpha resulted in the formation of the respective heterodimer complexes (lanes 3 and 4, respectively). To further assess the interaction of UCP1-PPRE with PPARalpha -RXRalpha or PPARgamma -RXRalpha heterodimers found in nuclear extracts from differentiated brown adipocytes in primary culture (Fig. 8C) or from BAT (Fig. 8D), supershift assays were performed using specific antibodies against RXRalpha , PPARalpha , or PPARgamma . Arrows indicate the supershifted complexes formed (that contain RXRalpha and PPARalpha or PPARgamma ). Incubation with an antibody against ETS transcription factors, used as negative control, did not result in any change in the pattern of bands. Competition experiments performed in Fig. 8D with a 100-fold molar excess of specific (UCP1-PPRE) or its mutated version (mutUCP1-PPRE, see legend of Fig. 8A) confirmed the presence of a nonspecific band (n.s.). Taken together, these findings demonstrate that both PPARalpha and PPARgamma are present in brown fat cell nuclei and bind to UCP1-PPRE as heterodimers with RXRalpha .

The PPARalpha Ligand Wy 14,643 Induces ucp-1 Gene Expression in Brown Adipose Tissue in Vivo in Different Physiological Situations-- To assess the in vivo significance of PPAR activators on the expression of the ucp-1 gene, mice at different physiological situations were injected with single doses of the PPARalpha -specific ligand Wy 14,643 or, for comparative purposes, of the PPARgamma activator troglitazone. We have previously reported that sensitivity of gene expression to PPARalpha activators in acute treatments in vivo depends on the status of lipid metabolism able to provide endogenous PPARalpha ligands (36). In adult mice (Fig. 9), Wy 14,643 caused a moderate 1.5-fold increase in UCP-1 mRNA abundance in BAT. When lactating mice were analyzed, Wy 14,643 significantly increased (5-fold) UCP-1 mRNA levels. During lactation, functional atrophy of BAT, including diminished lipolytic and lipoprotein lipase activities, and reduced expression of the ucp-1 gene contribute to energy sparing (37, 38). In contrast, troglitazone only had a moderate effect on brown fat UCP-1 mRNA abundance.



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Fig. 9.   Effects of PPAR activators on UCP-1 mRNA expression in brown fat of adult, lactating, or neonate mice. A, representation of the relative abundance of UCP-1 mRNA in brown fat from adult female control and 15-day lactating dams after 6 h of being injected intraperitoneally with Wy 14,643 (50 µg/g body weight), troglitazone (100 µg/g body weight), or vehicle solution, and neonates that were injected intraperitoneally with Wy 14,643 (50 µg/g body weight), BRL 49653 (50 µg/g body weight), or vehicle solution (see "Experimental Procedures" for details). Data are expressed as relative to the adult female control which was set as 1. Statistical significance of comparisons between groups of treated mice and their respective vehicle treated controls are shown by: *, p <=  0.05; **, p <=  0.01. Comparison between Wy 14,643 and troglitazone treatment is shown by Delta , p <=  0.05. B, representative Northern blot analysis of equal amounts of brown fat RNA (20 µg/lane) hybridized with the UCP-1 and COII probes, as described in the legend to Fig. 1.

When newborn mice at thermoneutrality were analyzed, injection of pups with Wy 14,643 caused a significant 3-fold rise in UCP-1 mRNA levels whereas injection of the PPARgamma -ligand BRL 49653 did not significantly change UCP-1 mRNA expression. The action of PPAR agonists was specific for the ucp-1 gene since COII mRNA levels were essentially unaffected by PPAR activators in BAT (see Fig. 9, bottom). Present results demonstrate an acute regulation of the ucp-1 gene in vivo by the PPARalpha -ligand Wy 14,643 that is more potent than that observed for PPARgamma ligands.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we have established that PPARalpha activators regulate the expression of the ucp-1 gene both in primary brown adipocytes and in BAT in vivo. Brown adipocytes differentiated in primary culture were used since they highly coexpress all PPAR subtypes, equally to BAT (24). In contrast, the HIB-1B brown adipocyte cell line lacks PPARalpha expression (24), and therefore, the results of previous studies using HIB-1B cells to determine the effects of PPAR activators on the expression of the ucp-1 gene must be viewed with caution. Present results also demonstrate that PPARalpha induces the rat ucp-1 gene promoter activity upon treatment with its specific ligand Wy 14,643, but it can also activate transcription in the absence of exogenously added ligand. This has been widely described for other PPARalpha -responsive gene promoters (32, 39), and could be explained by either the presence of endogenous activators, such as fatty acids or their metabolites, or by ligand-independent activity of these nuclear receptors (40). The responsiveness of the ucp-1 gene promoter to PPARalpha -ligand is increased by co-transfection with expression vectors for either coactivator CBP or PGC-1. Furthermore, the synergistic effect observed when adding both coactivators points to the involvement at the same time of CBP and PGC-1 in coactivating PPARalpha . In this way, PPARalpha can interact directly with CBP (41) and also with PGC-1 (42). In addition, CBP can form a complex with PGC-1 (43), thus providing multiple contact points to stabilize the complex assembly. Furthermore, CBP can also interact with other transcription factors, such as CREB and C/EBP, known to regulate transcription of the rat ucp-1 gene through its proximal regulatory region (22, 21).

By deletion and mutation analysis we have identified the PPARalpha -responsive element in the upstream enhancer region of the rat ucp-1 gene. This -2485/-2458 region contains a potential PPRE consensus formed by two direct repeats separated by one nucleotide (DR-1). Highly comparable elements are also found in the human and mouse ucp-1 genes (see Fig. 8A), indicating that these sequences may have an important regulatory role in response to PPARalpha . In fact, the murine element has been described to mediate PPARgamma responsiveness (18). Our present results further demonstrate that the -2485/-2458 element in the rat ucp-1 gene behaves as a promiscuous responsive site to either PPARalpha and PPARgamma activation, but not PPARdelta . From the analysis of various natural PPREs, it has been reported that the binding strength and functional transactivation for each PPAR subtype on the same PPRE was similar (33). Only some significant PPARgamma specificity was described, and it was related to the 5'-flanking sequence with respect to the DR-1 element, which is essential for PPARalpha binding (33). However, present results indicate a similar capacity of PPARalpha and PPARgamma to bind and activate ucp-1 transcription through the UCP1-PPRE. The predominant role of any subtype at any one time may thus depend on: 1) the relative amount of each subtype. For instance, PPARalpha and PPARgamma gene expression in brown adipocytes are under opposite regulation by their ligands and retinoic acid: up-regulation of PPARalpha but down-regulation of PPARgamma (24). 2) Cross-talk with other signaling pathways, like regulation of PPAR transcriptional activity by MAP kinase-dependent phosphorylation, which enhances PPARalpha (44) but decreases PPARgamma activity (45). 3) Ligand availability. Several PPAR ligands have been described to be highly subtype-specific (6), although identification of endogenous ligands and how their synthesis is regulated, is far from being established. 4) Interaction with coregulators. The interaction of PGC-1 with PPARalpha is ligand-dependent whereas that with PPARgamma is not (42, 30). These and other possible events may determine which PPAR subtype activates transcription of ucp-1 in response to brown adipocyte physiological condition, mainly PPARgamma in association with differentiation-dependent events or PPARalpha in coordination with increased lipid catabolism in active BAT.

Other PPAR target genes have been described to be induced by both PPARalpha and gamma  activators through the same PPRE (39, 46). However, since they have been studied in tissues such as liver, which highly expresses PPARalpha but not PPARgamma , or WAT, which predominantly expresses PPARgamma , tissue-specific regulation has been suggested. In contrast, BAT provides a model to study whether PPAR subtypes specifically regulate a PPRE in a target gene or whether a unique element behaves as a common site, as shown by our present findings in the ucp-1 gene promoter. For instance, the lipoprotein lipase (LPL) gene is up-regulated by PPARalpha (in liver) and PPARgamma (in WAT) through the same PPRE (46). During BAT differentiation, induction of LPL allows for increased fatty acids delivery to brown adipocytes, which results in triglyceride accumulation, thus promoting the adipocyte phenotype. However, thermogenic stimulus also up-regulates LPL to increase fatty acids uptake, which increases the supply of substrate for oxidation. Expression of LPL mRNA is increased by PPARalpha and gamma  activators in differentiated brown adipocytes,2 suggesting that LPL gene transcription in BAT could be activated by both PPARalpha and -gamma . Other genes, such as the fatty acid transport protein and the acyl-CoA synthetase genes, which also regulate cell uptake of fatty acids, might be similarly regulated in BAT since they are induced by PPARalpha and -gamma activators (39, 47).

Here we also demonstrate that in vivo activation of PPARalpha by Wy 14,643 up-regulates UCP-1 mRNA expression in BAT. The effects of the acute administration of this synthetic ligand are higher in those physiological situations (lactating dams and newborn pups at thermoneutrality) in which endogenous PPARalpha -ligands are expected to be low, in agreement with previous findings that PPARalpha sensitivity in vivo depends on the status of lipid metabolism (36). Furthermore, the higher ucp-1 gene responsiveness to acute treatments with PPARalpha than PPARgamma agonists underlines the in vivo significance of PPARalpha -dependent regulation of ucp-1 gene expression. In contrast, it has been reported that chronic exposure to PPARalpha or PPARgamma activators led to opposite results: long-term oral treatment of rats with Wy 14,643 did not change UCP-1 mRNA levels and thiazolidinedione administration resulted in a slight up-regulation of UCP-1 mRNA (48). This behavior of ucp-1 is similar to other bona fide PPAR-target genes in BAT, which remain unchanged by chronic exposure to PPARalpha agonists (49). Positive effects of long-term treatment with thiazolidinediones on ucp-1 gene expression may be a consequence of their reported action promoting overall BAT differentiation (17, 48, 50).

Activation of BAT thermogenesis has been classically recognized to be mediated by norepinephrine. Among other regulatory effects, there is a cAMP-dependent activation of hormone sensitive-lipase, which rapidly hydrolyzes the stored triglycerides and releases high concentrations of fatty acids. These fatty acids, in addition to be the major substrate for thermogenesis and the inducers of UCP-1 uncoupling activity, may also act as PPAR activators. Accordingly, cold exposure and beta -adrenergic stimulation of BAT result in activation of the PPAR pathway (51). We have previously reported that norepinephrine directly up-regulates transcription of the ucp-1 gene promoter, mainly through a cAMP responsive region in the proximal promoter region (22). However, the upstream enhancer region of the rat ucp-1 gene is also responsive to norepinephrine, although it lacks a defined cAMP responsive region. Several lines of evidence suggest a role for PPARalpha in mediating this regulation, although the involvement of PPARgamma cannot be ruled out. Mutation of the UCP1-PPRE affects the response of the ucp-1 gene promoter to norepinephrine (17).3 Furthermore, the mitogen-activated protein kinase pathway is activated in BAT by adrenergic stimulation (52). This may result in activation of PPARalpha but inactivation of PPARgamma , as discussed above. The coactivator PGC-1 is rapidly induced by cold-exposure through beta -adrenergic pathways in BAT (30). Present data demonstrate that PGC-1 coactivates PPARalpha and further increases ucp-1 gene responsiveness to PPARalpha -ligand dependent activation. Likewise, PGC-1 cooperates with PPARalpha in the transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes (42), and it also induces mitochondrial gene expression by regulating the nuclear respiratory factor system (53). Taken together, these data point to PGC-1/PPARalpha interaction as playing an important role in mediating changes in gene expression in response to BAT thermogenic requirements. Although basal expression of UCP-1 mRNA in PPARalpha -null mice has been reported to be unaltered (12), as also reported for other bona fide PPAR-target genes in liver (6), further studies are in course to determine whether ucp-1 gene expression is altered in these mice in response to thermogenic stimulus.

In conclusion, PPARalpha directly regulates ucp-1 gene transcription and we propose that this transcriptional regulatory mechanism is a component of the coordinate control of thermogenic and lipid oxidation pathways in active BAT. Recently, PPARalpha has been implicated in obesity (54) and selective PPARalpha activators have been described to improve insulin sensitivity and reduce WAT mass (55). Part of these effects could be due to an increase in energy expenditure in BAT, and the positive action of PPARalpha on ucp-1 gene expression opens new perspectives on the molecular targets of PPARalpha involved in mediating these effects.


    ACKNOWLEDGEMENTS

We thank Dr. Daniel Ricquier for (-4551)UCP1-CAT and Dr. B. Spiegelman for the HIB-1B cell line. We also thank Drs. S. Green, P. Grimaldi, B. Spiegelman, P. Chambon, R. Evans, D. Ricquier, and N. Glaichenhaus for kindly supplying expression vectors and probes.


    FOOTNOTES

* This work was supported by Grants PM98-0188 from Ministerio de Educación y Ciencia, Spain, and SGR99-38 from Generalitat de Catalunya, Spain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. de Bioquímica i Biologia Molecular, Universitat de Barcelona, Avda Diagonal 645, E-08028-Barcelona, Spain. Tel.: 34-93-4034613; Fax: 34-93-4021559; E-mail: giralt@porthos.bio.ub.es.

Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M006246200

2 A. Schlüter, M. Giralt, and F. Villarroya, unpublished observations.

3 M. J. Barberà, F. Villarroya, and M. Giralt, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; UCP-1, uncoupling protein-1; PPRE, PPAR response element; BAT, brown adipose tissue; WAT, white adipose tissue; RXR, retinoid X receptor; CBP, CREB-binding protein; PGC-1, PPARgamma coactivator-1; CAT, chloramphenicol acetyltransferase; LPL, lipoprotein lipase; kb, kilobase(s); COII, cytochrome oxidase subunit II.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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