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W2039992465 abstract "Obesity, i.e. an excess of white adipose tissue (WAT), predisposes to the development of type 2 diabetes and cardiovascular disease. Brown adipose tissue is present in rodents but not in adult humans. It expresses uncoupling protein 1 (UCP1) that allows dissipation of energy as heat. Peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivator 1α (PGC-1α) activate mouse UCP1 gene transcription. We show here that human PGC-1α induced the activation of the human UCP1 promoter by PPARγ. Adenovirus-mediated expression of human PGC-1α increased the expression of UCP1, respiratory chain proteins, and fatty acid oxidation enzymes in human subcutaneous white adipocytes. Changes in the expression of other genes were also consistent with brown adipocyte mRNA expression profile. PGC-1α increased the palmitate oxidation rate by fat cells. Human white adipocytes can therefore acquire typical features of brown fat cells. The PPARγ agonist rosiglitazone potentiated the effect of PGC-1α on UCP1 expression and fatty acid oxidation. Hence, PGC-1α is able to direct human WAT PPARγ toward a transcriptional program linked to energy dissipation. However, the response of typical white adipocyte targets to rosiglitazone treatment was not altered by PGC-1α. UCP1 mRNA induction was shown in vivo by injection of the PGC-1α adenovirus in mouse white fat. Alteration of energy balance through an increased utilization of fat in WAT may be a conceivable strategy for the treatment of obesity. Obesity, i.e. an excess of white adipose tissue (WAT), predisposes to the development of type 2 diabetes and cardiovascular disease. Brown adipose tissue is present in rodents but not in adult humans. It expresses uncoupling protein 1 (UCP1) that allows dissipation of energy as heat. Peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivator 1α (PGC-1α) activate mouse UCP1 gene transcription. We show here that human PGC-1α induced the activation of the human UCP1 promoter by PPARγ. Adenovirus-mediated expression of human PGC-1α increased the expression of UCP1, respiratory chain proteins, and fatty acid oxidation enzymes in human subcutaneous white adipocytes. Changes in the expression of other genes were also consistent with brown adipocyte mRNA expression profile. PGC-1α increased the palmitate oxidation rate by fat cells. Human white adipocytes can therefore acquire typical features of brown fat cells. The PPARγ agonist rosiglitazone potentiated the effect of PGC-1α on UCP1 expression and fatty acid oxidation. Hence, PGC-1α is able to direct human WAT PPARγ toward a transcriptional program linked to energy dissipation. However, the response of typical white adipocyte targets to rosiglitazone treatment was not altered by PGC-1α. UCP1 mRNA induction was shown in vivo by injection of the PGC-1α adenovirus in mouse white fat. Alteration of energy balance through an increased utilization of fat in WAT may be a conceivable strategy for the treatment of obesity. Two types of adipose tissues coexist in mammals. White adipose tissue (WAT) 1The abbreviations used are: WAT, white adipose tissue; BAT, brown adipose tissue; GFP, green fluorescent protein; m.o.i., multiplicity of infection; PGC, peroxisome proliferator-activated receptor γ coactivator; PPAR, peroxisome proliferator-activated receptor; RA, retinoic acid; RXR, retinoic acid X receptor; TZD, thiazolidinedione; UCP, uncoupling protein; MCAD, medium chain acyl-coenzyme A dehydrogenase.1The abbreviations used are: WAT, white adipose tissue; BAT, brown adipose tissue; GFP, green fluorescent protein; m.o.i., multiplicity of infection; PGC, peroxisome proliferator-activated receptor γ coactivator; PPAR, peroxisome proliferator-activated receptor; RA, retinoic acid; RXR, retinoic acid X receptor; TZD, thiazolidinedione; UCP, uncoupling protein; MCAD, medium chain acyl-coenzyme A dehydrogenase. has an essential role for storage of energy in the form of triacylglycerol. In situations of energy deficit such as fasting, lipolysis in WAT controls the supply of energy to the body through the release of fatty acids into the plasma. The uncontrolled expansion of WAT seen in obesity predisposes to the development of an array of metabolic disturbances leading to type 2 diabetes and cardiovascular disease. Although it shares many features with WAT, brown adipose tissue (BAT) is specialized in adaptive thermogenesis (1Himms-Hagen J. FASEB J. 1990; 4: 2890-2898Crossref PubMed Scopus (414) Google Scholar). Differences in gene expression between WAT and BAT, most notably at the mitochondrial level, explain the thermogenic capacity of BAT. Fatty acid oxidation enzymes and respiratory chain components are highly expressed in BAT contributing to a high oxidative capacity. The activity of ATP synthase is low because of a defect in expression of the P1 gene (2Houstek J. Andersson U. Tvrdík P. Nedergaard J. Cannon B. J. Biol. Chem. 1995; 270: 7689-7694Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, the most distinguishing feature of BAT is the expression of uncoupling protein 1 (UCP1) (3Ricquier D. Bouillaud F. J. Physiol. 2000; 529: 3-10Crossref PubMed Scopus (283) Google Scholar). UCP1 is a 32-kDa protein expressed in the inner membrane of the mitochondria. UCP1 allows the dissipation of the proton electrochemical gradient generated by the respiratory chain. Uncoupling between oxygen consumption and ATP synthesis promotes energy dissipation as heat. The mechanism of action of UCP1 is still controversial. One model depicts UCP1 as a true proton transporter, whereas another model states that UCP1 catalyzes a fatty acid protonophoretic cycle (4Garlid K.D. Jaburek M. Jezek P. Varecha M. Biochim. Biophys. Acta. 2000; 1459: 383-389Crossref PubMed Scopus (107) Google Scholar). Fatty acids and retinoids have been shown to activate UCP1 (5Kligenberg M. Huang S.-G. Biochim. Biophys. Acta. 1999; 1415: 271-296Crossref PubMed Scopus (313) Google Scholar, 6Rial E. González-Barroso M. Fleury C. Iturrizaga S. Sanchis D. JiménezJiménez J. Ricquier D. Goubern M. Bouillaud F. EMBO J. 1999; 18: 5827-5833Crossref PubMed Scopus (178) Google Scholar). In neonatal mammals, hibernators and rodents, cold-induced thermogenesis in BAT contributes to the maintenance of body temperature. Fuel is provided as fatty acids derived from BAT and WAT lipolysis. In rodents, BAT also participates in diet-induced thermogenesis and may thereby control the energy efficiency of food (7Rothwell N.J. Stock M.J. Obesity Res. 1997; 5: 650-656Crossref PubMed Scopus (88) Google Scholar). UCP1 biosynthesis is mainly controlled at the transcriptional level. During cold exposure, sympathetic nervous system stimulation of BAT is the primary signal that activates UCP1 gene expression. Retinoic acid (RA) and thyroid hormones are other positive regulators. A critical enhancer has been characterized in rodent UCP1 genes (8Cassard-Doulcier A.-M. Gelly C. Fox N. Schrementi J. Raimbault S. Klaus S. Forest C. Bouillaud F. Ricquier D. Mol. Endocrinol. 1993; 7: 497-506Crossref PubMed Scopus (0) Google Scholar, 9Kozak U.C. Kopecky J. Teisinger J. Enerbäck S. Boyer B. Kozak L.P. Mol. Cell. Biol. 1994; 14: 59-67Crossref PubMed Scopus (166) Google Scholar). This region is required for catecholamine and RA stimulation. The enhancer contains a peroxisome proliferator-activated receptor γ (PPARγ) responsive element that mediates the stimulation induced by thiazolidinediones (TZD) (10Sears I.B. MacGinnitie M.A. Kovacs L.G. Graves R.A. Mol. Cell. Biol. 1996; 16: 3410-3419Crossref PubMed Google Scholar). In cooperation with PPARγ, the PPARγ coactivator PGC-1α, has been shown to induce mouse UCP1 gene transcription (11Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3050) Google Scholar). It also stimulates the expression of electron transport chain genes and mitochondrial biogenesis, through induction of nuclear respiratory factors 1 and 2 (12Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3148) Google Scholar). PGC-1α expression is increased in response to cold exposure and β-adrenergic stimulation (11Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3050) Google Scholar). BAT is present throughout the life in rodents but disappears soon after birth in large mammals. In human fetus and newborn children, it is found in the cervical, axillary, perirenal, and periadrenal depots (13Lean M.E.J. James W.P.T. Trayhurn P. Nicholls D.G. Brown Adipose Tissue. Edward Arnold, London1986: 339-365Google Scholar). There are no BAT depots in adults, and UCP1 mRNA is expressed at very low levels in WAT (14Oberkofler H. Dallinger G. Liu Y.M. Hell E. Krempler F. Patsch W. J. Lipid Res. 1997; 38: 2125-2133Abstract Full Text PDF PubMed Google Scholar). BAT is not thought to contribute to a significant part of thermogenesis (15Astrup A. Bulow J. Madsen J. Christensen N.J. Am. J. Physiol. 1985; 248: E507-E515PubMed Google Scholar). However, UCP1 is expressed in hibernomas and in perirenal WAT of adult patients with phaeochromocytoma and primary aldosteronism revealing that UCP1 expression can be induced in rare tumors and endocrinological disorders (16Garruti G. Ricquier D. Int. J. Obes. 1992; 16: 383-390PubMed Google Scholar, 17Zancanaro C. Pelosi G. Accordini C. Balercia G. Sbabo L. Cinti S. Biol. Cell. 1994; 80: 75-78Crossref PubMed Scopus (23) Google Scholar). Pharmacotherapy targeted at molecular pathways that regulate adaptive thermogenesis provides a plausible and safe means of increasing energy expenditure (18Crowley V.E. Yeo G.S. O'Rahilly S. Nat. Rev. Drug Discov. 2002; 1: 276-286Crossref PubMed Scopus (94) Google Scholar). Reactivation of brown adipocytes is therefore an important goal. Studies on human white adipocytes are mandatory to substantiate the proof of concept. In an attempt to promote a metabolic shift in white fat cells from lipid storage toward fatty acid utilization, human subcutaneous white adipocytes were transduced with an adenovirus expressing PGC-1α. The cells acquire features of brown adipocytes, i.e. an induction of UCP1 and respiratory chain gene expression, and an increased capacity to oxidize fatty acid. Conversion of white into brown adipocytes may therefore constitute a strategy to regulate fat mass in humans. Adenoviral Expression System and Adenofection Experiment in CV-1 Cells—Recombinant adenovirus was generated as described (19He T.-C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3236) Google Scholar). The full-length human PGC-1α cDNA (20Larrouy D. Vidal H. Andreelli F. Laville M. Langin D. Int. J. Obes. 1999; 23: 1327-1332Crossref PubMed Scopus (79) Google Scholar) was cloned into the pAdEasy parent plasmid. Recombination between the pAdEasy and pAdTrack vectors and production of the PGC-1α adenovirus was performed at the Laboratoire de Thérapie Génique de Nantes. The virus contains, in tandem, the green fluorescent protein (GFP) gene and the PGC-1α cDNA downstream of separate cytomegalovirus promoters. An adenovirus containing only the GFP gene was used as control. Viral titers were, respectively, 1.7 × 1011 and 1.4 × 1011 infectious particles per ml. Adenofection experiments were performed in CV-1 cells (ATCC, Manassas, VA) cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Invitrogen, Cergy Pontoise, France). The 6.3-kb UCP1 promoter-chloramphenicol acetyltransferase gene construct (21Del Mar Gonzalez-Barroso M. Pecqueur C. Gelly C. Sanchis D. Alves-Guerra M.-C. Bouillaud F. Ricquier D. Cassard-Doulcier A.-M. J. Biol. Chem. 2000; 275: 31722-31732Abstract Full Text Full Text PDF PubMed Google Scholar) was cotransfected with expression vectors for PPARγ2 (from Bruce Spiegelman, Dana-Farber Cancer Institute, Boston, MA) and retinoic acid X receptor α (RXRα) (from Pierre Chambon, IGBMC, Strasbourg, France) and a cytomegalovirus promoter-β-galactosidase gene vector to check for transfection efficiency. The PGC-1α or the GFP adenoviruses were added to the LipofectAMINE (Invitrogen) transfection mixture at a multiplicity of infection (m.o.i.) of 200. Cells were exposed for 6 h to the transfection mixture. Chloramphenicol acetyltransferase activity was assayed on cell extracts 72 h post-adenofection. Differentiation of Human Preadipocytes, Adenovirus Infection, and Flow Cytometry—Subcutaneous abdominal adipose tissue was obtained from female subjects undergoing plastic surgery in agreement with French laws on biomedical research. Human adipocytes in primary culture were differentiated as described by Hauner et al. (22Hauner H. Entenmann G. Wabitsch M. Gaillard D. Ailhaud G. Negrel R. Pfeiffer E.F. J. Clin. Invest. 1989; 84: 1663-1670Crossref PubMed Scopus (557) Google Scholar) with modifications (23Smih F. Rouet P. Lucas S. Mairal A. Sengenes C. Lafontan M. Vaulont S. Casado M. Langin D. Diabetes. 2002; 51: 293-300Crossref PubMed Scopus (63) Google Scholar). Stromal cells prepared from WAT were cultured for 13 days in a chemically defined medium. At day 13, 60–80% of cells were differentiated into lipid droplet-containing adipocytes. UCP1 mRNA levels in differentiated cells were similar to the levels found in native subcutaneous adipose tissue. 2C. Tiraby and D. Langin, unpublished data. Hence, differentiation of preadipocytes in primary culture did not result in the dysregulation of UCP1 gene expression. The cells were infected at a m.o.i. of 200 for 6 h. The day after infection, cells were treated as indicated in text and figure legends with the following drugs at 1 μm unless otherwise indicated: rosiglitazone (BRL49653, Smith Kline and French, Harlow, UK), Wy14643 (Cayman Chemical, Ann Arbor, MI), L165041 (Merck), and 9-cis-RA (Sigma). Cells were harvested after 48 h of treatment for mRNA assays and 72 h of treatment for protein and fatty acid oxidation assays. To isolate adenovirus-transduced cells, GFP-positive cells were sorted, after trypsinization, using an EPICS Altra Hypersort System (Beckman Coulter, Roissy, France). Quantitative Reverse Transcriptase-PCR Analysis—Total RNA was isolated using RNeasy kit (Qiagen, Courtaboeuf, France). Total RNA (1 μg) was treated with DNase I (DNase I amplification grade, Invitrogen), then retrotranscribed using random hexamers and Thermoscript reverse transcriptase (Invitrogen). Real time quantitative PCR was performed on GeneAmp 7000 Sequence Detection System using SYBR green chemistry (Applied Biosystems, Courtaboeuf, France) as described (24Clément K. Viguerie N. Diehn M. Alizadeh A. Barbe P. Thalamas C. Storey J.D. Brown P.O. Barsh G.S. Langin D. Genome Res. 2002; 12: 281-291Crossref PubMed Scopus (140) Google Scholar). Primers were designed using the Primer Express 1.5 software (Table I). Some mRNAs were quantified using Assay-on-Demand gene expression assays (Applied Biosystems). 18 S ribosomal RNA was used as control to normalize gene expression using the ribosomal RNA control Taqman assay kit (Applied Biosystems). Similar results were obtained using SYBR green- and Assay-on-Demand-based detections for the quantification of PGC-1α and UCP1 mRNA levels.Table IPrimers used in real time quantitative PCRmRNAAccession numberSense primerAntisense primerAmplicon sizebpALBPBC0036725′-GCATGGCCAAACCTAACATGA-3′5′-CCTGGCCCAGTATGAAGGAAA-3′105Cyt.cBC0084775′-AGGCCCCTGGATACTCTTACACAG-3′5′-TCAGTGTATCCTCTCCCCAGATG-3′69M-CPT1D878125′-TACAACAGGTGGTTTGACA-3′5′-CAGAGGTGCCCAATGATG-3′105GKNT_0163545′-GCAGAAGGAGTCGGCGTATG-3′5′-CCCAACCCATTGACTTCATCA-3′145MCADAF2510435′-AGCTCCTGCTAATAAAGCCTTTACTG-3′5′-CATGTTTAATTCCTTTCTCCCAATC-3′85hPGC-1αAF1597145′-CTGTGTCACCACCCAAATCCTTAT-3′5′-TGTGTCGAGAAAAGGACCTTGA-3′78hmPGC-1αNM_0089045′-AAAGGATGCGCTCTCGTTCA-3′5′-GGAATATGGTGATCGGGAACA-3′65hUCP1NM_0218335′-TGCCCAACTGTGCAATGAA-3′5′-TCGCAAGAAGGAAGGTACCAA-3′80mUCP1NM_0094635′-CCTGCCTCTCTCGGAAACAA-3′5′-TGTAGGCTGCCCAATGAACA-3′75 Open table in a new tab Western Blot Analysis—Mitochondria from mouse BAT and human adipocytes were prepared by differential centrifugation in 10 mm Tris, pH 8, 1 mm EDTA, 250 mm sucrose supplemented with a mixture of protease inhibitors (Sigma). Mitochondrial proteins (5 μg for human adipocytes and 0.2 μg for BAT) were subjected to 10% SDS-PAGE, transferred onto nitrocellulose membrane (Hybond ECL, Amersham Biosciences, Orsay, France), and probed with a polyclonal anti-rat UCP1 antibody (25Ricquier D. Barlet J.P. Garel J.M. Combes-George M. Dubois M.P. Biochem. J. 1983; 210: 859-866Crossref PubMed Scopus (57) Google Scholar) and an anti-cytochrome c antibody (Pharmingen, Le Pont de Claix, France). Immunoreactive protein was determined by enhanced chemiluminescence reagent (Amersham Biosciences). In Vivo Adenovirus Injection in Mouse Fat Pad—Studies with mice followed the INSERM and Louis Bugnard Institute Animal Care Facility guidelines. Male B6D2/JIco mice (24–30 weeks old, IFFA-CREDO, L'Arbresle, France) were anesthetized with avertin (Sigma). Following dissection of the skin and body wall, one testis with attached epididymal fat pad was pulled out. The adenoviral preparation (1.7 × 108 infectious particles) was injected to 6 points in the fat pad. A fat pad was injected with PGC-1α adenovirus and the contralateral fat pad with GFP adenovirus. After 5 days, total RNA was prepared from the fat pads for quantitative reverse transcriptase-PCR analyses. Palmitate Oxidation Experiment—Differentiated human adipocytes were incubated for 3 h in a medium containing Dulbecco's modified Eagle's medium without glucose, 50 mm Hepes, pH 7.8, 1% fatty acid-free bovine serum albumin, 2 mm l-carnitine, 50 μm palmitate, and 118 nm [14C]palmitate (850 μCi/μmol, Amersham Biosciences). Medium was transferred in a flask with a center well containing Carbosorb E (PerkinElmer Life Sciences, Courtaboeuf, France). 14CO2 was liberated by acidification with 5 n HCl and collected overnight on Carbosorb. 14CO2 was measured by scintillation counting. The acid-soluble fraction of the medium containing 14C-labeled β-oxidation metabolites were measured by scintillation counting after 1-butanol extraction of palmitate. To inhibit fatty acid oxidation, we used 50 μm etomoxir (from Wolfgang Langhans, Swiss Federal Institute of Technology, Zurich, Switzerland). To study the effect of mitochondrial uncoupling, m-chlorocarbonylcyanide phenylhydrazone (Sigma) was added at 10 μm during the 3-h incubation period. Statistical Analysis—Data are expressed as mean ± S.E. Statistical analyses were performed using analysis of variance with least-square difference post-hoc analysis or Student's t test. The Nuclear Receptor PPARγ2 and Its Coactivator PGC-1α Transactivate the Human UCP1 Promoter—To test the transcriptional coactivation of the human UCP1 promoter by PGC-1α, we utilized an adenovirus expressing human PGC-1α in combination with expression vectors for PPARγ2 and its partner, RXRα. PGC-1α functions as a transcriptional coactivator for the two nuclear receptors (11Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3050) Google Scholar, 26Delerive P. Wu Y. Burris T.P. Chin W.W. Suen C.S. J. Biol. Chem. 2002; 277: 3913-3917Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The 6300-bp human UCP1 promoter region mediates the stimulation of transcription by TZD in a murine brown adipocyte cell line (21Del Mar Gonzalez-Barroso M. Pecqueur C. Gelly C. Sanchis D. Alves-Guerra M.-C. Bouillaud F. Ricquier D. Cassard-Doulcier A.-M. J. Biol. Chem. 2000; 275: 31722-31732Abstract Full Text Full Text PDF PubMed Google Scholar). In simian CV-1 cells, the PPARγ2/RXRα combination had no transactivation potency (Fig. 1). The expression of PGC-1α alone induced a modest rise of UCP1 gene transcription. However, the addition of PGC-1α to the PPARγ2/RXRα combination led to a marked increase in activity. PGC-1α Induces UCP1 Expression in Human White Adipocyte—Human WAT express low levels of PGC-1α (20Larrouy D. Vidal H. Andreelli F. Laville M. Langin D. Int. J. Obes. 1999; 23: 1327-1332Crossref PubMed Scopus (79) Google Scholar). We used a human PGC-1α adenovirus to increase the expression of the coactivator in primary culture of human subcutaneous adipocytes (Fig. 2A). Unlike retroviral vector systems that must be used on proliferating preadipocytes, adenoviruses can transduce quiescent mature adipocytes (27Hertzel A.V. Sanders M.A. Bernlohr D.A. J. Lipid Res. 2000; 41: 1082-1086Abstract Full Text Full Text PDF PubMed Google Scholar). This method permits to avoid the effect of continuous PGC-1α expression during adipogenesis. The PGC-1α adenovirus efficiently transduced differentiated adipocytes as revealed by GFP labeling of 40–50% fat cells. Remarkably, adenoviral infection occurred exclusively in differentiated cells (Fig. 2B). There was no GFP staining in undifferentiated fibroblasts. To ascertain that preadipocytes were resistant to adenoviral infection, human fibroblasts at day 3 of culture were infected with PGC-1α adenovirus at various multiplicity of infections. No increase in PGC-1α mRNA was observed at an m.o.i. of 500. At m.o.i. of 1000 and 2000, PGC-1α mRNA were only increased by 3- and 5-fold, respectively, whereas the induction was 100–150-fold in day 13 differentiated adipocytes at a m.o.i. of 200. The data reveal that human adipocytes possess much more efficient plasma membrane binding and internalization components for human serotype 5 adenovirus than preadipocytes. The effects mediated by PGC-1α can thereby be ascribed to its selective overexpression in adipocytes. The robust overexpression of PGC-1α in human adipocytes was accompanied by an induction of UCP1 mRNA expression (Fig. 3A). UCP1 mRNA was barely detectable in cells infected with control GFP adenovirus. The marked increase associated with PGC-1α expression was amplified in the presence of PPARγ and RXRα ligands (p < 0.01). As shown on Fig. 3B, the mRNA level of UCP1 paralleled that of PGC-1α when cells were transduced at different multiplicity of infections. The expression of GFP allowed us to select the transduced cells using flow cytometry (Fig. 3C). Cells infected with PGC-1α adenovirus and treated with TZD and RA co-expressed PGC-1α and UCP1 mRNA. These data show that the induction of UCP1 is restricted to adipocytes expressing PGC-1α. Western blot analysis showed an increase of a 32-kDa immunoreactive band corresponding to UCP1 (Fig. 4). Similar results were obtained using an antibody raised against the whole rat protein (25Ricquier D. Barlet J.P. Garel J.M. Combes-George M. Dubois M.P. Biochem. J. 1983; 210: 859-866Crossref PubMed Scopus (57) Google Scholar) or an antibody directed against a 19-amino acid C-terminal UCP1 peptide.2 As for the mRNA level, the addition of PPARγ/RXRα ligands further increased UCP1 protein expression. The amount of UCP1 mRNA and protein in transduced and treated human adipocyte cultures represented 1.0 ± 0.4 and 1.6 ± 0.6% (n = 4) of the corresponding levels in mouse brown fat.Fig. 4Western blot analysis of UCP1 and cytochrome c expression. Mitochondrial proteins were prepared from mouse BAT (0.2 μg) and human white adipocytes (5 μg). Adipocytes were treated with rosiglitazone (Rosi) and 9-cis-retinoic acid (RA).View Large Image Figure ViewerDownload Hi-res image Download (PPT) PPARγ Cooperates with PGC-1α to Induce UCP1 Expression—To determine which PPAR was involved in PGC-1α coactivation of UCP1 expression, we tested ligands for the three subtypes of nuclear receptors (Fig. 3D). Rosiglitazone, a PPARγ agonist, was a potent stimulator of UCP1 expression. Agonists for PPARα and PPARβ and 9-cis-RA had poor inducing potency. The data show the pre-eminent role of the PPARγ/PGC-1α association for the induction of UCP1 in human white adipocytes. PGC-1α-expressing Adipocytes Show Increased Expression of Mitochondrial Proteins and Brown Adipocyte Markers—We wished to determine whether the up-regulation of UCP1 in TZD-treated PGC-1α-overexpressing human adipocytes was associated with other metabolically relevant adaptations in gene expression. PGC-1α induces expression of components of the mitochondrial respiratory chain in several cell types (11Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3050) Google Scholar, 12Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3148) Google Scholar). The mRNAs for cytochrome c and cytochrome oxidase 4 were increased (Fig. 5). An induction of cytochrome c was found at the protein level (Fig. 4). We also observed an induction in the mRNA expression of mitofusin 2, a mitochondrial protein essential for mitochondrial network architecture highly expressed in brown adipose tissue (28Bach D. Pich S. Soriano F.X. Vega N. Baumgartner B. Oriola J. Daugaard J.R. Lloberas J. Camps M. Zierath J.R. Rabasa-Lhoret R. Wallberg-Henriksson H. Laville M. Palacin M. Vidal H. Rivera F. Brand M. Zorzano A. J. Biol. Chem. 2003; 278: 17190-17197Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar). Ectopic expression of PGC-1α and PPARα in 3T3-L1 murine fibroblasts leads to an increase in mitochondrial fatty acid oxidation enzyme gene expression (29Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Crossref PubMed Scopus (931) Google Scholar). We therefore determined the mRNA levels for muscle carnitine palmitoyltransferase I, the isoform of CPT1 expressed in human white adipocytes (30Brown N.F. Hill J.K. Esser V. Kirkland J.L. Corkey B.E. Foster D.W. McGarry J.D. Biochem. J. 1997; 327: 225-231Crossref PubMed Scopus (95) Google Scholar), and medium chain acyl-coenzyme A dehydrogenase (MCAD). Treated PGC-1α-expressing adipocytes showed higher muscle carnitine palmitoyltransferase I and MCAD mRNA levels than control cells. Glycerol kinase activity is very low in WAT while relatively high levels are found in BAT (31Chakrabarty K. Chaudhuri B. Jeffay H. J. Lipid Res. 1983; 24: 381-390Abstract Full Text PDF PubMed Google Scholar). PGC-1α in the presence of TZD and RA led to a 7-fold induction of glycerokinase mRNA levels. PGC-1α Does Not Alter TZD Response of Typical White Adipocyte Target Genes—We next investigated the effect of PGC-1α on the response of WAT genes regulated by TZD (Fig. 6). Adipocyte lipid-binding protein and cytosolic phosphoenolpyruvate carboxykinase are direct targets of TZD in white adipocytes with identified PPARγ responsive elements (32Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (1990) Google Scholar, 33Glorian M. Duplus E. Beale E.G. Scott D.K. Granner D.K. Forest C. Biochimie (Paris). 2001; 83: 933-943Crossref PubMed Scopus (55) Google Scholar). Rosiglitazone increased adipocyte lipid-binding protein and phosphoenolpyruvate carboxykinase 1 mRNA levels in cells transduced with PGC-1α or GFP adenovirus. Hence, the expression of PGC-1α was not accompanied by a loss of TZD response of target genes. PGC-1α Stimulates UCP1 mRNA Expression in Mouse White Fat Pads in Vivo—We then asked whether overexpression of PGC-1α could induce UCP1 in vivo. The PGC-1α adenovirus was injected in the epididymal fat pad of male mice. This fat pad was selected because it contains very few brown fat cells (34Commins S.P. Watson P.M. Padgett M.A. Dudley A. Argyropoulos G. Gettys T.W. Endocrinology. 1999; 140: 292-300Crossref PubMed Scopus (136) Google Scholar). Compared with the contralateral side injected with GFP adenovirus, the increase in PGC-1α mRNA level was accompanied by a more than 10-fold induction of UCP1 mRNA expression (Fig. 3E). The experiment reveals that PGC-1α is able to induce UCP1 gene expression in WAT in vivo. PGC-1α Increases Fat Oxidation in Human White Adipocytes—We hypothesized that an induction of UCP1, proteins of the respiratory chain, and enzymes of fatty acid oxidation may lead to an increased capacity of fat oxidation. Fig. 7 shows that PGC-1α-expressing human adipocytes had higher total fatty acid oxidation than cells infected with the GFP adenovirus. The effect was mimicked by the chemical mitochondrial uncoupler, m-chlorocarbonylcyanide phenylhydrazone. This data reveals that, in human adipocytes, uncoupling of the respiratory chain is associated with an increase in fatty acid oxidation. There was both an increase in the production of CO2 and β-oxidation metabolites.2 Addition of rosiglitazone potentiated the effect of PGC-1α (p < 0.01). Compared with basal conditions, total oxidation was doubled. Addition of etomoxir, a CPT1 inhibitor, abolished palmitate oxidation. The transcriptional coactivator PGC-1α may play an important role in adaptive thermogenesis in rodents through a positive regulation of mitochondrial proteins (11Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3050) Google Scholar, 35Puigserver P. Adelmant G. Wu Z. Fan M. Xu J. O'Malley B. Spiegelman B.M. Science. 1999; 286: 1368-1371Crossref PubMed Scopus (493) Google Scholar, 36Knutti D. Kralli A. Trends Endocrinol. Metab. 2001; 12: 360-365Abstract Full Text Full Text PDF PubM" @default.
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W2039992465 date "2003-08-01" @default.
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W2039992465 title "Acquirement of Brown Fat Cell Features by Human White Adipocytes" @default.
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W2039992465 doi "https://doi.org/10.1074/jbc.m305235200" @default.
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