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• W2031551101 abstract "Brown adipose tissue (BAT) hyperplasia is a fundamental physiological response to cold; it involves an acute phase of mitotic cell growth followed by a prolonged differentiation phase. Peroxisome proliferator-activated receptors (PPARs) are key regulators of fatty acid metabolism and adipocyte differentiation and may therefore mediate important metabolic changes during non-shivering thermogenesis. In the present study we have investigated PPAR mRNA expression in relation to peroxisome proliferation in rat BAT during cold acclimatization. By immunoelectron microscopy we show that the number of peroxisomes per cytoplasmic volume and acyl-CoA oxidase immunolabeling density remained constant (thus increasing in parallel with tissue mass and cell number) during the initial proliferative phase and the acute thermogenic response but increased after 14 days of cold exposure, correlating with terminal differentiation of BAT. A pronounced decrease in BAT PPARα and PPARγ mRNA levels was found within hours of exposure to cold, which was reversed after 14 days, suggesting a role for either or both of these subtypes in the proliferation and induction of peroxisomes and peroxisomal β-oxidation enzymes. In contrast, PPARδ mRNA levels increased progressively during cold exposure. Transactivation assays in HIB 1B and HEK-293 cells demonstrated an adrenergic stimulation of peroxisome proliferator response element reporter activity via PPAR, establishing a role for these nuclear receptors in hormonal regulation of gene transcription in BAT. Brown adipose tissue (BAT) hyperplasia is a fundamental physiological response to cold; it involves an acute phase of mitotic cell growth followed by a prolonged differentiation phase. Peroxisome proliferator-activated receptors (PPARs) are key regulators of fatty acid metabolism and adipocyte differentiation and may therefore mediate important metabolic changes during non-shivering thermogenesis. In the present study we have investigated PPAR mRNA expression in relation to peroxisome proliferation in rat BAT during cold acclimatization. By immunoelectron microscopy we show that the number of peroxisomes per cytoplasmic volume and acyl-CoA oxidase immunolabeling density remained constant (thus increasing in parallel with tissue mass and cell number) during the initial proliferative phase and the acute thermogenic response but increased after 14 days of cold exposure, correlating with terminal differentiation of BAT. A pronounced decrease in BAT PPARα and PPARγ mRNA levels was found within hours of exposure to cold, which was reversed after 14 days, suggesting a role for either or both of these subtypes in the proliferation and induction of peroxisomes and peroxisomal β-oxidation enzymes. In contrast, PPARδ mRNA levels increased progressively during cold exposure. Transactivation assays in HIB 1B and HEK-293 cells demonstrated an adrenergic stimulation of peroxisome proliferator response element reporter activity via PPAR, establishing a role for these nuclear receptors in hormonal regulation of gene transcription in BAT. peroxisome proliferator-activated receptor(s) acyl-CoA oxidase brown adipose tissue lipoprotein lipase thymidine kinase luciferase human embryonic kidney polymerase chain reaction base pair cytomegalovirus rapid amplification of cDNA ends untranslated region electrophoretic mobility shift assay peroxisome proliferator response element retinoid X receptor N -[1-(2,3-dioleoyloxy)propyl]-N ,N ,N -trimethylammonium methyl sulfate, C/EBP, CCAAT/enhances binding protein Peroxisome proliferator-activated receptors (PPARs)1 are ligand-activated transcription factors that control the expression of genes involved in lipid metabolism. These genes include the P450 IVA1 (1Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1992; 267: 19051-19053Abstract Full Text PDF PubMed Google Scholar), acyl-CoA oxidase (AOx) (2Tugwood J.D. Issemann I. Anderson R.G. Bundell K.R. McPheat W.L. Green S. EMBO J. 1992; 11: 433-439Crossref PubMed Scopus (804) Google Scholar, 3Zhang B. Marcus S.L. Sajjadi F.G. Alvares K. Reddy J.K. Subramani S. Rachubinski R.A. Capone J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7541-7545Crossref PubMed Scopus (235) Google Scholar), the enoyl-CoA/3-hydroxyacyl-CoA hydratase/dehydrogenase multifunctional enzyme (3Zhang B. Marcus S.L. Sajjadi F.G. Alvares K. Reddy J.K. Subramani S. Rachubinski R.A. Capone J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7541-7545Crossref PubMed Scopus (235) Google Scholar, 4Bardot O. Aldridge T.C. Latruffe N. Green S. Biochem. Biophys. Res. Commun. 1993; 192: 37-45Crossref PubMed Scopus (233) Google Scholar), fatty acid transporters (5Motojima K. Passilly P. Peters J.M. Gonzalez F.J. Latruffe N. J. Biol. Chem. 1998; 273: 16710-16714Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar), apolipoprotein A-I (6Vu-Dac N. Schoonjans K. Laine B. Fruchart J.C. Auwerx J. Staels B. J. Biol. Chem. 1994; 269: 31012-31018Abstract Full Text PDF PubMed Google Scholar), as well as mitochondrial β-oxidation enzymes (7Gulick T. Cresci S. Caira T. Moore D.D. Kelly D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11012-11016Crossref PubMed Scopus (491) Google Scholar, 8Aoyama T. Peters J.M. Iritani N. Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar). Three different PPAR subtypes, α, γ, and δ, are present in rat, mouse, human, and Xenopus tissues. These subtypes exhibit tissue-specific expression and selectivity of ligand activation. PPARα is the predominant subtype in rodent liver. Disruption of the mouse PPARα gene by homologous recombination demonstrated the important role for this PPAR subtype in the transcriptional regulation of peroxisome proliferator-responsive genes and in hepatic peroxisome proliferation (5Motojima K. Passilly P. Peters J.M. Gonzalez F.J. Latruffe N. J. Biol. Chem. 1998; 273: 16710-16714Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 8Aoyama T. Peters J.M. Iritani N. Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar, 9Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1506) Google Scholar, 10Peters J.M. Hennuyer N. Staels B. Fruchart J.-C. Fievet C. Gonzalez F.J. Auwerx J. J. Biol. Chem. 1997; 272: 27307-27312Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). PPARγ expression is highest in adipose tissue. Ectopic expression of PPARγ (and to a lesser extent PPARα) can effectively induce adipose phenotype such as fat accumulation and induction of adipose markers in fibroblasts and premyotic cells (11Brun R.P. Tontonoz P. Forman B.M. Ellis R. Chen J. Evans R.M. Spiegelman B.M. Genes Dev. 1996; 10: 974-984Crossref PubMed Scopus (411) Google Scholar, 12Hu E. Tontonoz P. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9856-9860Crossref PubMed Scopus (583) Google Scholar, 13Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2005) Google Scholar, 14Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar). PPARδ is expressed ubiquitously. Its physiological function is not yet clear, but like the other PPAR subtypes PPARδ has also been implicated in adipocyte development (15Amri E.Z. Bonino F. Ailhaud G. Abumrad N.A. Grimaldi P.A. J. Biol. Chem. 1995; 270: 2367-2371Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Brown adipose tissue (BAT) is a major site for non-shivering thermogenesis because of a very high capacity for uncoupled oxidation of fatty acids. Norepinephrine released from sympathetic nerves innervating the tissue acts both as an acute inducer of the thermogenic function and as a promoter of tissue recruitment (for review, see Ref.16Cannon B. Jacobsson A. Rehnmark S. Nedergaard J. Int. J. Obes. 1996; 20: S36-S42Google Scholar). Increased BAT metabolic activity occurs in concert with increased expression of some adipocyte genes (i.e. lipoprotein lipase (LPL), uncoupling protein, and C/EBPβ) (17Mitchell J.R.D. Jacobsson A. Kirchgessner T.G. Schotz M.C. Cannon B. Nedergaard J. Am. J. Physiol. 1992; 263: E500-E506PubMed Google Scholar, 18Jacobsson A. Stadler U. Glotzer M.A. Kozak L.P. J. Biol. Chem. 1985; 260: 16250-16254Abstract Full Text PDF PubMed Google Scholar, 19Rehnmark S. Antonson P. Xanthopoulos K.G. Jacobsson A. FEBS Lett. 1993; 318: 235-241Crossref PubMed Scopus (37) Google Scholar) and decreased expression of other genes (i.e. C/EBPα and β3-adrenergic receptor) (19Rehnmark S. Antonson P. Xanthopoulos K.G. Jacobsson A. FEBS Lett. 1993; 318: 235-241Crossref PubMed Scopus (37) Google Scholar, 20Bengtsson T. Redegren K. Strosberg A.D. Nedergaard J. Cannon B. J. Biol. Chem. 1996; 271: 33366-33375Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Additionally, tissue and cell morphologies are altered within days of cold exposure (21Cameron I.L. Smith R.E. J. Cell Biol. 1964; 23: 89-100Crossref PubMed Scopus (80) Google Scholar,22Thomson J.F. Habeck D.A. Nance S.L. Beetham K.L. J. Cell Biol. 1969; 41: 312-334Crossref PubMed Scopus (61) Google Scholar). The mitochondrial ultrastructure changes (23Barnard T. J. Ultrastruct. Res. 1969; 29: 311-332Crossref PubMed Scopus (50) Google Scholar), and the tissue mass increases about 3–4-fold after 2 weeks of cold exposure (21Cameron I.L. Smith R.E. J. Cell Biol. 1964; 23: 89-100Crossref PubMed Scopus (80) Google Scholar, 24Page E. Babineau L.M. Rev. Can. Biol. Exp. 1950; 9: 202-206Google Scholar). This increase is caused by activation of cellular proliferation within the tissue, which reaches its maximum after 1 week of cold exposure (25Hunt T.E. Hunt E.A. Anat. Rec. 1967; 157: 537-546Crossref Scopus (26) Google Scholar,26Rehnmark S. Nedergaard J. Exp. Cell Res. 1989; 180: 574-579Crossref PubMed Scopus (54) Google Scholar). The cessation of mitotic cell growth in the tissue is followed by differentiation of the cells into brown adipocytes (27Lean M. Branch W.J. James W. Jennings G. Ashwell M. Biosci. Rep. 1983; 3: 61-71Crossref PubMed Scopus (51) Google Scholar, 28Arnold J. Patel H.V. Ridley R.G. Freeman K.B. Biosci. Rep. 1985; 5: 57-62Crossref PubMed Scopus (2) Google Scholar). The heat is produced mainly by mitochondrial fatty acid oxidation, which is increased dramatically through the action of the BAT-specific mitochondrial uncoupling protein that uncouples cellular respiration from oxidative phosphorylation (29Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1353) Google Scholar). Cold acclimatization of rats results in proliferation of peroxisomes (30Ahlabo I. Barnard T. J. Histochem. Cytochem. 1971; 19: 670-675Crossref PubMed Scopus (50) Google Scholar) and in a 10-fold induction of peroxisomal β-oxidation enzyme activity (31Nedergaard J. Alexson S. Cannon B. Am. J. Physiol. 1980; 239: C208-C216Crossref PubMed Google Scholar). Because PPAR is a known mediator of peroxisome proliferation and PPAR subtypes have been implicated in adipogenesis, physiological stimulation of BAT offers an excellent in vivo model for studying PPARs in relation to adipogenesis and peroxisome proliferation under physiological conditions. In an attempt to unravel the biological functions of PPARα, PPARγ, and PPARδ in a physiological context, we have investigated the expression of these receptors in rat BAT during cold acclimatization and correlated the expression of the different receptor subtypes with the particular metabolic and developmental state of the tissue. Quantitative analysis of peroxisome proliferation in BAT was performed by immunoelectron microscopy. The data indicate that peroxisome proliferation correlates with the expression of PPARα and γ. In a transactivation assay utilizing the brown preadipose cell line HIB 1B, it was found that norepinephrine, 8-bromo-cAMP, and forskolin induced AOx-tk-Luc reporter gene activity, suggesting that the β-adrenergic signaling pathway can activate endogenous PPARs. The PPAR dependence of this activation was confirmed in a cell line (HEK-293) that is devoid of detectable endogenous PPARs, suggesting that PPARs are involved in the thermogenic activation of BAT and proliferation of peroxisomes in this tissue. BAT may thus serve as a promising in vivo system for identification of the physiological ligands for PPARs and elucidation of the biological functions of these receptors. PCR template cDNA was synthesized from 10 μg of BAT RNA utilizing a cDNA synthesis system (Life Technologies, Inc.). Two sets of degenerate oligonucleotide primers were designed to amino acid regions conserved in mouse and Xenopus PPARγ. The 5′-primer corresponds to amino acids TVDFSSI near the amino terminus (5′-GCGGATCCAC(A/T)GT(A/T)GA(T/C)TT(T/C)TCCAGCAT-3′), and the 3′-primer corresponds to amino acids QEIY(R/K)DMY at the carboxyl terminus (5′-GCGGTACCTACATGTC(T/C)TTGTA(A/G)ATCTC(T/C)TG-3′). Bam HI and Kpn I recognition sites were included at the 5′-end of the oligonucleotide primers for cloning purposes. PCRs performed as described previously (32Wong C.W. Privalsky M.L. Mol. Endocrinol. 1995; 9: 551-562PubMed Google Scholar) yielded a 1,325-bp cDNA fragment. This fragment was digested with Bam HI and Kpn I resulting in a 980-bp fragment that was cloned into the pBluescript II KS cloning vector (Stratagene) and partially sequenced in the core facility (CyberGene) by cycle sequencing. To generate a rat PPARδ probe, two oligonucleotides were designed to amino acid regions conserved in mouse and human PPARδ (NUC1). The 5′-primer corresponds to the amino-terminal amino acids MEQPQEEAPE (5′-ATGGACAGCCACAGGAGGACCCTGAGG-3′), and the 3′-primer corresponds to the carboxyl-terminal amino acids LLQE- IYKDMY (5′-GTCCTTGTAGATTCCTGGAGCAGGGGTGC-3′). PCRs utilizing BAT cDNA and the Expand™ Long Template PCR system according to the manufacturer's instructions (Roche Molecular Biochemicals) resulted in amplification of a 1,323-bp PPARδ. This 1,323-bp PCR product was cloned into the pCRII cloning vector (Invitrogen) and sequenced. Rat interscapular BAT mRNA was purified utilizing Ultraspec™ (Biotecx) and oligo(dT)-cellulose (Stratagene) and utilized to construct a cDNA library in the pBKCMV phagemid according to the manufacturer's instructions (Stratagene). The 980-bp PPARγBam HI/Kpn I cDNA fragment and the 1,323-bp PPARδ cDNA fragment were labeled by random priming and used independently to screen the rat BAT cDNA library. Four independent PPARγ plaques were purified and the cDNA rescued into pBKCMV plasmid and sequenced (CyberGene). Three of the isolated clones corresponded to the full-length rPPARγ2 subtype (clones 9, 10, and 11) and one to the full-length PPARγ subtype (clone 7). Two independent clones containing partial PPARδ coding sequences were purified and the cDNA rescued as pBKCMV plasmid. RACE (rapid amplification of cDNA ends) was employed to obtain the 5′-UTR and amino-terminal sequences of the PPARδ subtype utilizing the Marathon-Ready cDNA system (CLONTECH) and the gene-specific primer (5′-CGCTTCCAGAAGTGCCTGGCACTCGGCATG-3′). A 654-bp cDNA fragment was cloned into the pCR2.1 cloning vector (Invitrogen) and sequenced. Male Harlan Sprague-Dawley rats (B&K Universal AB, Stockholm) weighing about 150 g were preacclimated to standard animal house conditions for 1 week before handling. For thermoregulatory studies, rats were housed individually at 28 °C (controls) or in the cold (4 °C) for the periods of time indicated. Two separate time course experiments were carried out, one with three rats in each time point (up to 21 days in the cold) and another experiment with two animals in each time point (up to 28 days in the cold). Interscapular BAT was dissected out and processed individually for RNA purification utilizing the Ultraspec system (Biotecx). This method was also utilized to isolate RNA from HIB 1B and HEK-293 cells. The RNA samples were resolved in 1.2% agarose gels containing 20 mm HEPES, 1 mm EDTA, and 6% formaldehyde. The gels, containing 10 μg of total RNA/well, were blotted onto Hybond-N nylon membranes (Amersham Pharmacia Biotech) by capillary transfer. Membranes were hybridized with32P-labeled cDNA probes in a hybridization buffer containing 5% SDS, 400 mm NaPO4, 1 mm EDTA, 1 mg/ml bovine serum albumin, and 50% formamide. PPAR Northern analyses were carried out with full-length probes to PPARα (accession number M88592 (33Göttlicher M. Widmark E. Li Q. Gustafsson J.-Å. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4653-4657Crossref PubMed Scopus (800) Google Scholar)), PPARγ, and δ described here. Full-length AOx (34Miyazawa S. Hayashi H. Hijikata M. Ishii N. Furuta S. Kagamiyama H. Osumi T. Hashimoto T. J. Biol. Chem. 1987; 262: 8131-8137Abstract Full Text PDF PubMed Google Scholar), Δ3,Δ2-enoyl-CoA isomerase (35Palosaari P.M. Vihinen M. Mäntsälä P.I. Alexson S.E.H. Pihlajaniemi T. Hiltunen J.K. J. Biol. Chem. 1991; 266: 10750-10753Abstract Full Text PDF PubMed Google Scholar), LPL (36Kirchgessner T.G. Svenson K.L. Lusis A.J. Schotz M.C. J. Biol. Chem. 1987; 262: 8463-8466Abstract Full Text PDF PubMed Google Scholar), and C/EBPα and β (37Xanthopoulos K.G. Mirkovitch J. Friedman J.M. Darnell Jr., J.E. Cytogenet. Cell Genet. 1989; 50: 174-175Crossref PubMed Google Scholar, 38Chang C.-J. Chen T.-T. Lei H.-Y. Chen D.-S. Lee S.-C. Mol. Cell. Biol. 1990; 10: 6642-6653Crossref PubMed Scopus (200) Google Scholar) were those earlier described. After 16-h hybridizations at 42 °C, blots were washed at 53 °C in 0.1 × SSC, 0.1% SDS, and 1 mmEDTA and exposed to a PhosphorImaging plate (Fuji). Female Harlan Sprague-Dawley rats weighing approximately 150 g were housed individually at 5 °C or at standard animal house conditions (23 °C). All rats had free access to food (rat pellets, Oriental M, Tokyo) and water. Tissue processing and immunohistochemical staining of Lowicryl-K4membedded specimens, using the protein A-gold technique, were performed as described previously (39Usuda N. Reddy M.K. Hashimoto T. Rao M.S. Reddy J.K. Lab. Invest. 1988; 58: 100-111PubMed Google Scholar). The infiltration of the resin into BAT isolated from control rats was highly inefficient, probably because of high fat content. Therefore, tissues isolated from rats housed in the cold for 4 days represent the shortest time point available for analysis. After the immunostaining, the sections were stained with 1% uranyl acetate solution and were examined in a Hitachi H700 electron microscope at an accelerated voltage of 150 kV. The morphometric analysis of changes in the peroxisome volume and labeling densities was performed as described previously (39Usuda N. Reddy M.K. Hashimoto T. Rao M.S. Reddy J.K. Lab. Invest. 1988; 58: 100-111PubMed Google Scholar), using 20 electron micrographs of each period of cold acclimatization selected from 200 pictures. The immunostaining was performed with all samples at the same time to equalize the staining conditions. The analysis was performed with a computer-assisted image analyzer, Digigramer G (Muto Co., Tokyo, Japan). The peroxisome volume density was calculated as the ratio of the area of peroxisomes to the cytoplasmic area excluding the lipid droplets. The labeling density was expressed as the number of gold particles/μm2 of peroxisome area, and the immunolabel concentration was expressed as the number of gold particles/unit of cytoplasmic volume by multiplying the peroxisome volume density with the labeling density. The statistical analyses were done using analysis of variance and Student's t tests. The PPRE probe in our EMSA was a double-stranded oligonucleotide, 5′-TCGAGACGTGACCTTTGTCCTGGTC-3′, derived from the AOx PPRE, which was labeled by a Klenow fill-in reaction in the presence of [α-32P]dCTP. Nuclear protein extracts were obtained from cells harvested in TEN buffer (40 mm Tris, pH 7.9, 10 mm EDTA, 150 mm NaCl) by extraction in the presence of complete TM (EDTA-free) protease inhibitor mixture from Roche Molecular Biochemicals. PPARγ (6 × His-tagged full-length) and RXRα (6 × His-tagged full-length) were translated utilizing the Promega TNT Coupled Reticulocyte Lysate system. 25-μl incubation mixes contained 2–5 μg of protein (nuclear extract), 20,000 cpm of probe, 20 mm KCl in 10 mm Tris, pH 7.8, 10% glycerol, 500 ng of poly(dI-dC)·poly(dI-dC), and 500 ng of sonicated salmon sperm DNA. Samples were resolved on a 5% polyacrylamide gel. Antibodies used were RXRα mouse monoclonal antibodies directed against the ligand binding domain, which were a kind gift from Dr. Pierre Chambon, and PPARy2 mouse monoclonal antibody (sc 7273x) from Santa Cruz Biotech Inc. Specific competitors corresponding to DR2 (5′-CGACCCCAGTTCACCAGGTCAGGGCT-3′) and DR5 (5′-TCGACTGGGTTCACCGAAAGTTCACAGTC-3′) and an unspecific competitor were used as indicated. The dried gels were exposed to film overnight. For cell culture experiments, all reagents were purchased from Life Technologies unless specified otherwise. HEK-293 cells were purchased from American Type Culture Collection and were maintained in a 50:50 mix of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% heat-inactivated fetal calf serum and gentamycin (25 μg/ml). HIB 1B cells, kindly provided by Dr. Bruce Spiegelman, were maintained in preadipocyte medium, a 50:50 mix of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% heat-inactivated fetal calf serum and gentamycin (25 μg/ml). The luciferase reporter plasmids utilized in our experiments contain the herpesvirus thymidine kinase (tk) basal promoter linked upstream of the luciferase gene (tk-Luc). PPAR activity was monitored by transfection of a AOx-tk-Luc plasmid, which contains two copies of the AOx PPRE upstream of the basal tk promoter in the tk-Luc plasmid. The CMV-α, CMV-γ2, and CMV-δ plasmids contain the open reading frames of the PPARα cDNA (accession number M88592 (33Göttlicher M. Widmark E. Li Q. Gustafsson J.-Å. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4653-4657Crossref PubMed Scopus (800) Google Scholar)) and PPARγ2 and δ cDNAs described here, respectively. For transient transfections, cells were plated at a density of 100,000 cells/30-mm plate 24 h before transfection. DNA:DOTAP mixes were prepared as recommended by the manufacturer (Roche Molecular Biochemicals). Cells were incubated for 6 h with 1.25 μg of reporter (HIB 1B and HEK-293 cells) and 125 ng of CMV expression plasmid (HEK-293 cells only)/plate and lysed 36 h later. Regulators were prepared as 1,000 × concentrated stocks. Cells were treated for 10–12 h with the indicated compounds. Luciferase assays were performed according to the manufacturer's specifications (Promega). Duplicate plates were used in all experiments for both control and treated conditions. All experiments were repeated several times with consistent results. Unless specified otherwise, data shown represent the mean ± S.E. of the mean for three or four independent experiments. cDNA clones encoding the PPARγ and δ subtypes were isolated from a rat interscapular BAT cDNA library. Two rat PPARγ isoforms, γ1 and γ2, were obtained which, as has been reported for the mouse PPARγ isoforms, probably result from differential splicing of PPARγ RNA. The nucleotide and deduced amino acid sequences of the rat PPARγ and γ2 subtypes are shown in Fig.1. Of the four positive clones isolated, only one encoded PPARγ1. The rat PPARγ1 cDNA clone contains an open reading frame that encodes a protein of 475 amino acids with a calculated molecular mass of 54.5 kDa (Fig. 1 A ). The PPARγ2 cDNA clone encodes a 57.6-kDa protein containing 30 additional amino acids at the amino terminus (Fig. 1 B ) compared with PPARγ1. A partial rat PPARδ cDNA clone was also isolated. This clone contained a sequence encoding amino acids 85–440 and approximately 2 kilobases of 3′-UTR. The 5′-end of the rat PPARδ cDNA was isolated by RACE, resulting in amplification and cloning of a 654-bp cDNA fragment that contains part of the 5′-UTR and the nucleotides encoding the amino-terminal 137 amino acids of the protein. The combined sequence information obtained from the RACE, PCR, and library screen products results in a PPARδ cDNA encoding a predicted protein of 440 amino acids with a calculated molecular mass of 49.7 kDa. The published PPARδ sequence (40Xing G. Zhang L. Zhang L. Heynen T. Yoshikawa T. Smith M. Weiss S. Detera-Wadleigh S. Biochem. Biophys. Res. Commun. 1995; 217: 1015-1025Crossref PubMed Scopus (71) Google Scholar) differs slightly from the sequence obtained here. In the sequence published previously, the nucleotide differences in the coding region which would encode different amino acids are: C305 (Thr instead of Met21), T436 (Ser instead of Pro65), and T1271 (Val instead of Asp343). The predicted rat PPAR sequences contain the expected DNA binding domain typical of nuclear receptors and a highly homologous ligand binding domain at the carboxyl terminus which may mediate activation of these receptors. Alignment of the deduced amino acid sequences of the rat PPAR subtypes to rat PPARα demonstrated a high conservation in the DNA binding domain and ligand binding domain relative to the highly divergent amino termini (not shown). In vitro transcription and translation of these PPAR cDNA clones resulted in proteins of the expected molecular masses (data not shown). The PPAR subtype mRNAs are expressed in a tissue-specific manner. As reported recently, the PPARγ subtype is expressed predominantly in adipose tissue where it is thought to play a crucial role in adipogenesis (14Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar). Because this process is pivotal to BAT function and the mammalian adaptation to cold, we hypothesized that the PPARα, γ, and δ subtypes may be expressed in BAT and that the mRNA levels could be affected during adipogenesis triggered by cold exposure. We have tested this hypothesis by analyzing expression of these PPAR subtypes in rat BAT as a function of time in the cold (4 °C). Two independent time course experiments were performed. In the first experiment rats were exposed to cold for up to 21 days, and in the second time course experiment rats were exposed to the cold for up to 28 days. RNA samples were analyzed from each animal by Northern analysis. Our results demonstrate that the PPAR subtype mRNAs are regulated differentially during cold exposure (Fig.2, A and B ). PPARα mRNA was decreased markedly after 5 h of cold exposure and was almost undetectable after 1 day in the cold. After about 10 days of cold exposure, PPARα mRNA increased gradually but remained lower than in the controls. PPARγ mRNA levels were also repressed profoundly within hours of cold exposure and remained decreased for 5–10 days in the cold (30–35% of control levels). After 10 days in the cold PPARγ levels increased to control levels after 28 days in the cold. In contrast, the PPARδ mRNA level increased progressively during acclimatization to cold. Fig.2 B shows PhosphorImager quantitation of Northern blots on RNA samples obtained at various time points of cold exposure, analyzed from three different rats at each time point. In another cold exposure experiment, RNA samples were analyzed from two rats at each time point with essentially the same results (as shown in Fig. 2 A ). C/EBPα, C/EBPβ, and PPARs are expressed sequentially and seem to determine the adipocyte phenotype in concert (14Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar, 41Wu Z. Bucher N.L.R. Farmer S.R. Mol. Cell. Biol. 1996; 16: 4128-4136Crossref PubMed Google Scholar, 42Wu Z. Xie Y. Bucher N.L.R. Farmer S.R. Genes Dev. 1995; 9: 2350-2356Crossref PubMed Scopus (481) Google Scholar, 43Yeh W.-C. Cao M. Classen M. McKnight S.L. Genes Dev. 1995; 9: 169-191Crossref Scopus (813) Google Scholar), therefore we also analyzed the expression of these mRNAs in BAT during cold exposure. RNA from two animals at each time point were analyzed, and the results demonstrated that the expression of C/EBPα and C/EBPβ mRNAs is rapidly and differentially regulated during the cold exposure of rats (Fig. 2 A ). PhosphorImager quantitation showed that the C/EBPα mRNA level was decreased transiently within 5 h of cold exposure, to less than 50% of the control levels, then returned to control level after 5–10 days and remained at about this level throughout the experiment (not shown). In contrast, the C/EBPβ mRNA level increased rapidly, almost 3-fold within 1 h of cold exposure, after which the expression returned to near control level at 24 h and remained slightly elevated for the remaining experimental period. Because the AOx gene is transcriptionally regulated by PPARα in the liver (2Tugwood J.D. Issemann I. Anderson R.G. Bundell K.R. McPheat W.L. Green S. EMBO J. 1992; 11: 433-439Crossref PubMed Scopus (804) Google Scholar, 3Zhang B. Marcus S.L. Sajjadi F.G. Alvares K. Reddy J.K. Subramani S. Rachubinski R.A. Capone J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7541-7545Crossref PubMed Scopus (235) Google Scholar), and peroxisomal β-oxidation is induced about 10-fold in BAT (31Nedergaard J. Alexson S. Cannon B. Am. J. Physiol. 1980; 239: C208-C216Crossref PubMed Google Scholar) during acclimatization to cold, BAT AOx mRNA levels were analyzed. AOx mRNA steady-state levels increased immediately upon cold exposure, peaking at 5 h. The mRNA amount was near the control at 1 and 5 days and increased thereafter at 10–14 days, ultimately resulting in a 4.2-fold (±0.4) increase in AOx mRNA in BAT afte" @default.
• W2031551101 created "2016-06-24" @default.
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• W2031551101 date "1999-08-01" @default.
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• W2031551101 title "Rat Peroxisome Proliferator-activated Receptors and Brown Adipose Tissue Function during Cold Acclimatization" @default.
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