Energy Metabolism in Uncoupling Protein 3 Gene Knockout Mice
2000; Elsevier BV; Volume: 275; Issue: 21 Linguagem: Inglês
10.1074/jbc.m910179199
ISSN1083-351X
AutoresAntónio Vidal-Puig, Danica Grujić, Chenyu Zhang, Thilo Hagen, Olivier Boss, Yasuo Ido, Alicja Szczepanik, Jennifer M. Wade, Vamsi K. Mootha, Ronald Cortright, Deborah M. Muoio, Bradford B. Lowell,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoUncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily. Based upon its high homology with UCP1 and its restricted tissue distribution to skeletal muscle and brown adipose tissue, UCP3 has been suggested to play important roles in regulating energy expenditure, body weight, and thermoregulation. Other postulated roles for UCP3 include regulation of fatty acid metabolism, adaptive responses to acute exercise and starvation, and prevention of reactive oxygen species (ROS) formation. To address these questions, we have generated mice lacking UCP3 (UCP3 knockout (KO) mice). Here, we provide evidence that skeletal muscle mitochondria lacking UCP3 are more coupled (i.e. increased state 3/state 4 ratio), indicating that UCP3 has uncoupling activity. In addition, production of ROS is increased in mitochondria lacking UCP3. This study demonstrates that UCP3 has uncoupling activity and that its absence may lead to increased production of ROS. Despite these effects on mitochondrial function, UCP3 does not seem to be required for body weight regulation, exercise tolerance, fatty acid oxidation, or cold-induced thermogenesis. The absence of such phenotypes in UCP3 KO mice could not be attributed to up-regulation of other UCP mRNAs. However, alternative compensatory mechanisms cannot be excluded. The consequence of increased mitochondrial coupling in UCP3 KO mice on metabolism and the possible role of yet unidentified compensatory mechanisms, remains to be determined. Uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily. Based upon its high homology with UCP1 and its restricted tissue distribution to skeletal muscle and brown adipose tissue, UCP3 has been suggested to play important roles in regulating energy expenditure, body weight, and thermoregulation. Other postulated roles for UCP3 include regulation of fatty acid metabolism, adaptive responses to acute exercise and starvation, and prevention of reactive oxygen species (ROS) formation. To address these questions, we have generated mice lacking UCP3 (UCP3 knockout (KO) mice). Here, we provide evidence that skeletal muscle mitochondria lacking UCP3 are more coupled (i.e. increased state 3/state 4 ratio), indicating that UCP3 has uncoupling activity. In addition, production of ROS is increased in mitochondria lacking UCP3. This study demonstrates that UCP3 has uncoupling activity and that its absence may lead to increased production of ROS. Despite these effects on mitochondrial function, UCP3 does not seem to be required for body weight regulation, exercise tolerance, fatty acid oxidation, or cold-induced thermogenesis. The absence of such phenotypes in UCP3 KO mice could not be attributed to up-regulation of other UCP mRNAs. However, alternative compensatory mechanisms cannot be excluded. The consequence of increased mitochondrial coupling in UCP3 KO mice on metabolism and the possible role of yet unidentified compensatory mechanisms, remains to be determined. uncoupling protein 3 reactive oxygen species polymerase chain reaction respiratory exchange ratio wild type lucigenin-derived chemiluminescence carbonyl cyanidep-(trifluoromethoxy) phenylhydrazone knockout mice base pair(s) high fat diet Uncoupling protein 3 (UCP3)1 (1.Vidal-Puig A. Solanes G. Grujic D. Flier J.S. Lowell B.B. Biochem. Biophys. Res. Commun. 1997; 235: 79-82Crossref PubMed Scopus (677) Google Scholar, 2.Boss O. Samec S. Paoloni-Giacobino A. Rossier C. Dulloo A. Seydoux J. Muzzin P. Giacobino J.P. FEBS Lett. 1997; 408: 39-42Crossref PubMed Scopus (990) Google Scholar, 3.Gong D.-W. He Y. Karas M. Reitman M. J. Biol. Chem. 1997; 272: 24129-24132Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar) is a member of the mitochondrial anion carrier superfamily with high homology (57%) to UCP1, a well characterized uncoupling protein (4.Lin C.S. Klingenberg M. FEBS Lett. 1980; 113: 299-303Crossref PubMed Scopus (208) Google Scholar, 5.Lin C.S. Hackenberg H. Klingenberg E.M. FEBS Lett. 1980; 113: 304-306Crossref PubMed Scopus (94) Google Scholar). UCP3 together with UCP1, UCP2 (6.Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.F. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-272Crossref PubMed Scopus (1548) Google Scholar, 7.Gimeno R.E. Dembski M. Weng X. Andrew W. Shyjan A.W. Gimeno C.J. Iris F. Ellis S.J. Deng N. Woolf E.A. Trataglia L.A. Diabetes. 1997; 46: 900-906Crossref PubMed Scopus (0) Google Scholar), and possibly BMCP1 (brain mitochondrial carrier protein) (8.Sanchis D. Fleury C. Chomiki N. Goubern M. Huang Q. Neverova M. Gregoire F. Easlick J. Raimbault S. Levi-Meyrueis C. Miroux B. Collins S. Seldin M. Richard D. Warden C. Bouillaud F. Ricquier D. J. Biol. Chem. 1998; 273: 34611-34615Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) and UCP4 (9.Mao W., Yu, X.X. Zhong A. Li W. Brush J. Sherwood S.W. Adams S.H. Pan G. FEBS Lett. 1999; 443: 326-330Crossref PubMed Scopus (317) Google Scholar), form a family of uncoupling proteins located in the inner mitochondrial membrane. The evidence supporting the uncoupling activity of these proteins comes from studies where UCPs have been heterologously expressed in yeast or reconstituted into proteoliposomes. The expression of UCP2 and -3 decreases the mitochondrial membrane potential, as assessed by uptake of fluorescent membrane potential-sensitive dyes in whole yeast. They also increase state 4 respiration in isolated mitochondria, which serves as an indicator of inner membrane proton leak (3.Gong D.-W. He Y. Karas M. Reitman M. J. Biol. Chem. 1997; 272: 24129-24132Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar, 6.Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.F. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-272Crossref PubMed Scopus (1548) Google Scholar, 10.Zhang C.Y. Hagen T. Mootha V.K. Slieker L.J. Lowell B.B. FEBS Lett. 1999; 449: 129-134Crossref PubMed Scopus (102) Google Scholar). More recently, reconstitution of UCPs into liposomes has shown that UCP2 and UCP3, like UCP1, mediate proton transport across bilipid layers (11.Jaburek M. Varecha M. Gimeno R. Dembski M. Jezek P. Zhang M. Burn P. Trataglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). It is well established that UCP1 is exclusively expressed in brown fat, where it plays a key role in facultative thermogenesis in rodents. Although there is controversy about the molecular mechanisms involved (12.Klingenberg M. Echtay K.S. Bienengraeber M. Winkler E. Huang S.G. Int. J. Obes. 1999; 23: 24-29Crossref PubMed Scopus (60) Google Scholar, 13.Klingenberg M. Huang S.G. Biochim. Biophys. Acta. 1999; 1415: 271-296Crossref PubMed Scopus (313) Google Scholar, 14.Jezek P. Hanus J. Semrad C. Garlid K.D. J. Biol. Chem. 1996; 271: 6199-6205Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 15.Garlid K.D. Orosz D.E. Modriansky M. Vassanelli S. Jezek P. J. Biol. Chem. 1996; 271: 2615-2620Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 16.Garlid K.D. Jaburek M. Jezek P. FEBS Lett. 1998; 438: 10-14Crossref PubMed Scopus (132) Google Scholar), it is clear that activated UCP1 catalyzes a proton leak across the mitochondrial inner membrane leading to thermogenesis. The activity of UCP1 is highly regulated, facilitated by fatty acids and inhibited by purine ribose di- and trinucleotides (ATP, ADP, GTP, GDP) (17.Brand M.D. Brindle K.M. Buckingham J.A. Harper J.A. Rolfe D.F.S. Stuart J.A. Int. J. Obes. 1999; 23 (suppl.): 4-11Crossref Scopus (129) Google Scholar). UCP1 is also highly regulated at the transcriptional level (18.Silva J.E. Rabelo R. Eur. J. Endocrinol. 1997; 136: 251-264Crossref PubMed Scopus (187) Google Scholar) by catecholamines, thyroid hormone, retinoids, and thiazolidinediones. The characterization of the new uncoupling proteins (UCP2, UCP3, BMCP1, and UCP4) is in its early phases. UCP2 and UCP3 are of particular interest because they share the highest homology with UCP1 (57%) and are expressed in tissues that may be important for energy expenditure (see below). The tissue distribution of UCP1, UCP2, and UCP3 is markedly different. UCP2 is widely distributed (6.Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.F. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-272Crossref PubMed Scopus (1548) Google Scholar, 7.Gimeno R.E. Dembski M. Weng X. Andrew W. Shyjan A.W. Gimeno C.J. Iris F. Ellis S.J. Deng N. Woolf E.A. Trataglia L.A. Diabetes. 1997; 46: 900-906Crossref PubMed Scopus (0) Google Scholar), whereas UCP3 expression is restricted to skeletal muscle and brown fat (1.Vidal-Puig A. Solanes G. Grujic D. Flier J.S. Lowell B.B. Biochem. Biophys. Res. Commun. 1997; 235: 79-82Crossref PubMed Scopus (677) Google Scholar, 2.Boss O. Samec S. Paoloni-Giacobino A. Rossier C. Dulloo A. Seydoux J. Muzzin P. Giacobino J.P. FEBS Lett. 1997; 408: 39-42Crossref PubMed Scopus (990) Google Scholar, 3.Gong D.-W. He Y. Karas M. Reitman M. J. Biol. Chem. 1997; 272: 24129-24132Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar). It is well established that brown adipose tissue is an important tissue for thermogenesis in rodents. However, in large mammals in which brown fat is less common, skeletal muscle may be more important for thermogenesis (19.Rolfe D.F.S. Brand M.D. Am. J. Physiol. 1996; 271: C1380-C1389Crossref PubMed Google Scholar). Thus, based upon the high homology with UCP1 and its expression in skeletal muscle, UCP3 has been suggested to play an important role in regulating energy expenditure, thereby influencing body weight regulation (1.Vidal-Puig A. Solanes G. Grujic D. Flier J.S. Lowell B.B. Biochem. Biophys. Res. Commun. 1997; 235: 79-82Crossref PubMed Scopus (677) Google Scholar, 2.Boss O. Samec S. Paoloni-Giacobino A. Rossier C. Dulloo A. Seydoux J. Muzzin P. Giacobino J.P. FEBS Lett. 1997; 408: 39-42Crossref PubMed Scopus (990) Google Scholar, 3.Gong D.-W. He Y. Karas M. Reitman M. J. Biol. Chem. 1997; 272: 24129-24132Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar, 20.Ravussin E. Lilloja S. Knowler W.C. Christin L. Freymond D. Abbot W.G. Boyce V. Howard B.V. Bogardus C. N. Engl. J. Med. 1988; 318: 467-472Crossref PubMed Scopus (983) Google Scholar). UCP3, like UCP1, is highly regulated at the transcriptional level. Factors such as fatty acids (21.Weigle D.S. Selfridge L.E. Schwartz M.W. Seeley R.J. Cummings D.E. Havel P.J. Kuijper J.L. Beltradelrio H. Diabetes. 1998; 47 (272): 298Crossref PubMed Google Scholar, 22.Hwang C.S. Lane D. Biochem. Biophys. Res. Commun. 1999; 258: 464-469Crossref PubMed Scopus (64) Google Scholar, 23.Brun S. Carmona S.C. Mampel T. Vinas O. Giralt M. Iglesias R. Villarroya F. FEBS Lett. 1999; 453: 205-209Crossref PubMed Scopus (55) Google Scholar), diet (24.Gong D.-W. He Y. Reitman M. Biochem. Biophys. Res. Commun. 1999; 256: 27-32Crossref PubMed Scopus (57) Google Scholar), exercise (25.Boss O. Samec C. Desplanches D. Mayet M.H. Seydoux J. Muzzin P. Giacobino J.P. FASEB J. 1998; 12: 335-339Crossref PubMed Scopus (132) Google Scholar, 26.Tsuboyama- Kasaoka N. Tsunoda N. Maruyama K. Takahashi M. Kim H. Ikemoto S. Esaki O. Biochem. Biophys. Res. Commun. 1998; 247: 498-503Crossref PubMed Scopus (116) Google Scholar), and fasting (27.Millet L. Vidal H. Andrealli F. Larrouy D. Riou J.P. Risquier D. Laville M. Langin D. J. Clin. Invest. 1997; 100: 26654-26670Crossref Scopus (336) Google Scholar) markedly induce UCP3 expression in skeletal muscle. The induction of UCP3 during starvation, at a time when energy expenditure is decreased (28.Leibel R.L. Rosenbaum M. Hirsch J. N. Engl. J. Med. 1995; 332: 621-628Crossref PubMed Scopus (1498) Google Scholar), does not support a primary role for UCP3 in energy dissipation. However, data showing increases in circulating fatty acid levels associated with starvation, together with several findings linking UCP3 mRNA levels to fatty acid metabolism, suggest that UCP3 could be required for fatty acid metabolism. Thus, it is conceivable that UCP3 could facilitate the oxidation of fatty acids (29.Argyropulos G. Brown A.M. Willi S.M. Zhu J. He Y. Reitman M. Gevao S.M. Spruill I. Garwey W.T. J. Clin. Invest. 1998; 102: 1345-1351Crossref PubMed Scopus (196) Google Scholar). As is true for UCP1, it is likely that UCP3 activity is modulated by allosteric regulators. Based upon studies employing reconstituted proteoliposomes, it has been suggested that fatty acids are required for the uncoupling activity of UCP2 and UCP3 (11.Jaburek M. Varecha M. Gimeno R. Dembski M. Jezek P. Zhang M. Burn P. Trataglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) and that UCP2 and UCP3 activities are inhibited by purine nucleotides. However, in contrast to UCP1, much higher concentrations of purine nucleotides are required for inhibition, and the maximal degree of inhibition is significantly less (11.Jaburek M. Varecha M. Gimeno R. Dembski M. Jezek P. Zhang M. Burn P. Trataglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Other strategies have been used to clarify the function of UCP3 in humans. Using a genetic approach, linkage analysis studies suggest that UCP2 and UCP3 may influence resting metabolic rate (30.Bouchard C. Perusse L. Chagnon L. Warden C. Ricquier D. Hum. Mol. Genet. 1997; 6: 1887-1889Crossref PubMed Scopus (229) Google Scholar). However, association studies indicate that variants of UCP2/3 are unlikely to contribute to the development of obesity (31.Urhammer S.A. Dalgaard L.T. Sorensen T.I. Tybjaerg-Hansen A. Echwald S.M. Andersen T. Clausen J.O. Pedersen O. Diabetologia. 1998; 41: 241-244Crossref PubMed Scopus (50) Google Scholar). Of note, association studies in African-Americans report the existence of a mutation of the splice donor junction at exon 6 that results in exclusive production of UCP3S (29.Argyropulos G. Brown A.M. Willi S.M. Zhu J. He Y. Reitman M. Gevao S.M. Spruill I. Garwey W.T. J. Clin. Invest. 1998; 102: 1345-1351Crossref PubMed Scopus (196) Google Scholar, 32.Chung W.K. Luke A. Cooper R.A. Rotini C. Vidal-Puig A. Rosenbaum M. Chua M. Solanes G. Zheng M. Zhao L. LeDuc C. Eisberg A. Chu F. Murphy E. Schereier M. Arrone L. Caprio S. Kahle B. Gordon D. Leal S. Goldsmith R. Andreu A.L. Bruno C. DiMauro S. Heo M. Lowe J. Lowell B.B. Allison D.B. Leibel R.L. Diabetes. 1999; 48: 1890-1895Crossref PubMed Scopus (50) Google Scholar) a truncated version of UCP3 lacking the last 37 C-terminal residues. Argyropulos et al. (29.Argyropulos G. Brown A.M. Willi S.M. Zhu J. He Y. Reitman M. Gevao S.M. Spruill I. Garwey W.T. J. Clin. Invest. 1998; 102: 1345-1351Crossref PubMed Scopus (196) Google Scholar) find that individuals heterozygous for this mutation showed reduced fatty acid oxidation and increased respiratory quotients (29.Argyropulos G. Brown A.M. Willi S.M. Zhu J. He Y. Reitman M. Gevao S.M. Spruill I. Garwey W.T. J. Clin. Invest. 1998; 102: 1345-1351Crossref PubMed Scopus (196) Google Scholar). In contrast, a recent study including individuals homozygous for this mutation have not found abnormalities in the respiratory quotient (32.Chung W.K. Luke A. Cooper R.A. Rotini C. Vidal-Puig A. Rosenbaum M. Chua M. Solanes G. Zheng M. Zhao L. LeDuc C. Eisberg A. Chu F. Murphy E. Schereier M. Arrone L. Caprio S. Kahle B. Gordon D. Leal S. Goldsmith R. Andreu A.L. Bruno C. DiMauro S. Heo M. Lowe J. Lowell B.B. Allison D.B. Leibel R.L. Diabetes. 1999; 48: 1890-1895Crossref PubMed Scopus (50) Google Scholar). It has also been proposed that UCP3 could prevent the formation of oxygen-free radicals in skeletal muscle. This hypothesis is based upon the observation that mitochondrial membrane potential regulates the production of reactive oxygen species (ROS) (33.Bodrova M.E. Dedukhova V.I. Mokhova E.N. Skulachev V.P. FEBS Lett. 1998; 435: 269-274Crossref PubMed Scopus (44) Google Scholar, 34.Skulachev V.P. Biosci. 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In summary, the information available about the physiological role of UCP3 is still controversial and incomplete. In the present study, we attempt to address these questions by using homologous recombination to create gene knockout mice lacking UCP3. Two UCP3 genomic clones were obtained after PCR screening of a P1 C129/SvJ genomic library (Genome System). Both clones were mapped using Southern blot analysis and end-labeled oligonucleotide probes designed according to mouse UCP3 cDNA sequence (GenBank™ accession number AF053352). A replacement targeting vector was prepared in which a segment of the UCP3 gene between exons 2 and 3, including the start codon, was removed and replaced with a PGK-NEO-poly(A) expression cassette (38.Susulic V.S. Frederic R.C. Lawitts J. Tozzo E. Kahn B.B. Harper M.E. Himms-Hagen J. Flier J.S. Lowell B.B. J. Biol. Chem. 1995; 270: 29483-29492Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). The targeting plasmid was linearized withNotI and electroporated into J1 embryonic stem cells provided by E. Li, A. Sharp, and R. Jaenisch (39.Li E. Sucov H.M. Lee K.F. Evans R.M. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1590-1594Crossref PubMed Scopus (182) Google Scholar). Selection with G418 was done as described (38.Susulic V.S. Frederic R.C. Lawitts J. Tozzo E. Kahn B.B. Harper M.E. Himms-Hagen J. Flier J.S. Lowell B.B. J. Biol. Chem. 1995; 270: 29483-29492Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). Drug-resistant clones were isolated and expanded followed by genomic DNA extraction for Southern blot analysis (Fig. 1). Three targeted clones were injected into C57B1/6 embryos at the blastocyst stage. Chimeric offspring were mated with C57B1/6 mice. Germline transmission of the mutant allele was determined by Southern analysis of mouse tail genomic DNA. Three lines of mice carrying the disrupted UCP3 were generated. Genotyping was performed by mutiplex PCR. Specific primers used to detect the KO allele were sense 5′-CCT CCA CTC ATG ATC TAT AGA TC-3′, located in the neo cassette, and antisense 5′-ACC CTC TGT CGC CAC CAT AGT CA-3′, located in the UCP3 coding sequence. This set of primers amplified a 300-bp PCR product. Primers used to detect the wild type allele were sense 5′-GCA CTG CGG CCT GTT TTG-3′ and antisense 5′-ACC CTC TGT CGC CAC CAT AGT CA-3′. This set of primers amplified a 600-bp PCR product. Standard protocols were used for PCR. Animals were housed four per cage in a temperature-controlled room with a 12-h light/dark cycle. Food and water were availablead libitum unless noted. All experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Isolation of nucleic acids (RNA, DNA) and Southern, Northern, and Ribonuclease protection assays were carried out as described previously (40.Boss O. Bachman E. Vidal-Puig A. Zhang C.Y. Peroni O. Lowell B.B. Biochem. Biophys. Res. Commun. 1999; 261: 870-876Crossref PubMed Scopus (95) Google Scholar, 41.Vidal-Puig A. Jimenez-Linan M. Lowell B.B. Hamann A. Hu E. Spiegelman B. Flier J.S. Moller D.E. J. Clin. Invest. 1996; 97: 2553-2561Crossref PubMed Scopus (572) Google Scholar). Most of the probes were PCR-amplified using specific primers listed in Table I. A full-length rat UCP1 cDNA including the whole coding sequence was used as a probe. Isotopic bands were visualized by autoradiography and quantitated by PhosphoImager analysis using ImageQuant software (Molecular Dynamics, Sunnvale, CA).Table IList of primers for Northern and RNase protection assay probesmUCP3 (10–300 bp)5′-GCT GCT ACC TAA TGG AGT GG-3′ 5′-GTT CTC CCC TTG GAT CTG CAG-3′mUCP3 (581–775 bp) 5′-AGG TCC GAT TTC AAG CCA TG-3′ 5′-CAG GTG AGA CTC CAG CAA-3′mUCP3 (740–1054 bp) 5′-GAT GGT GAC CTA CGA CAT CAT CAA GGA-3′ 5′-AGG CCC TCT TCA GTT GCT CAT A-3′mUCP3 (1015–1190 bp) 5′-CAT ATG AGC AAC TGA AGA GG-3′ 3′-CGT GTC AGC AGC AGT GCA GGG-3′mUCP2 (709–904 bp) 5′-CTG GTC GCC GGC CTG CAG CGC-3′ 5′-GGG CAC CTG TGG TGC TAC CTG-3′ Open table in a new tab Rabbit polyclonal antibody against mouse cytochrome c was purchased from Santa Cruz Biotechnology, Santa Cruz, CA (SC-7159). Mouse UCP3 antibody was obtained after rabbit immunization against a peptide (MIRLGTGGERKYRGTMDAYRC), corresponding to mouse and rat UCP3 amino acids 147–166 encoded by exon 4 (Covance Research Products, Denver, PA). Antisera was affinity-purified on a peptide column generated from the respective peptide coupled to a Sulfo-Link column (Pierce). UCP3 antibody was eluted with 0.1m glycine, pH 2.5, and neutralized with 1 mTris, pH 10. Western blot analyses were performed on isolated skeletal muscle mitochondria (isolation of mitochondria describe below). Western blot analysis was performed as described previously (41.Vidal-Puig A. Jimenez-Linan M. Lowell B.B. Hamann A. Hu E. Spiegelman B. Flier J.S. Moller D.E. J. Clin. Invest. 1996; 97: 2553-2561Crossref PubMed Scopus (572) Google Scholar). Mitochondria were isolated from skeletal muscle of wild type (n = 15) and UCP3 KO mice (n = 15). Tissue was ground and homogenized in 10–20 ml of cold buffer (250 mm sucrose, 10 mm Hepes, 0.5 mm EDTA, pH 7.2 with KOH, 0.1% bovine serum albumin) and kept on ice. Homogenate was centrifuged at 600 × gfor 5 min. The pellet was discarded, and the supernatant was centrifuged at 8000 × g for 10 min. The mitochondrial pellet was washed twice and finally resuspended in buffer without bovine serum albumin. The mitochondria were then used for immunoblotting, polarography, and assays of aconitase activity. Mitochondrial respiration was measured in a Clark-type oxygen electrode at 37 °C using the following incubation conditions: 250 mm sucrose, 10 mm Hepes, pH 7.2, 5 mmKH2PO4/K2HPO4, 0.5 mm EDTA, 5 mm malate, 10 mmglutamate, and approximately 0.5 mg of mitochondrial protein/ml. The data were channeled into an A/D converter and recorded on a Pentium-based PC using the DataShuttle A/D converter and the Quicklog software package. The oxygen electrode was calibrated with experimental buffer saturated with room air assuming a solubility coefficient of 199 nmol O2/ml at 30 °C. Perchloric acid extracts of frozen muscle were prepared as follows. 100–200 mg of skeletal muscle (gastrocnemius) tissue were homogenized using a Polytron in 1.0 ml of 6% perchloric acid on ice. The samples were centrifuged for 1 min at 8000 × g. 600 μl of the supernatant were removed and neutralized by adding 300 μl of 0.4m triethanolamine HCl, 1.6 mK2CO3. ADP, ATP, and phosphocreatine were measured enzymatically as described previously by Jaworek et al. (42.Jaworek D. Gruber W. Bergmeyer H.U. Bergmeyer H.U. Methods of Enzymatic Analysis. 4. Verlag- Chemie, Winheim/Academic Press, New York1974: 2127-2131Crossref Google Scholar). Oxygen consumption and respiratory exchange ratio were measured in 10-week-old UCP3 knockout and wild type mice in basal and fasted conditions (5 mice per group and treatment) using the OXYMAX System 4.93 (Columbus Instruments, Columbus, OH) and conditions of a settling time of 100 s, measuring time of 50 s, and room air as a reference. Animals were placed individually in 4 0.3-liter chambers. Results are expressed as ml/kg/min. To correct for body size, results are also expressed as ml/kg2/3/min. UCP3 KO and wild type mice body weights were recorded weekly for 18–22 weeks (n = 9–12 per group and treatment). The effect of chow diet (Richmond Stenolard #5008) and a high fat diet (Research Diets Inc #D12451) on the evolution of body weight was assessed. Chow diet = 10% fat, 70% carbohydrate, and 20% protein (as percent o total calories); high fat diet = 45% fat, 35% carbohydrate, and 20% protein (as % of total calories). Both genders and the three possible genotypes (+/+, +/−, −/−) were studied. Tibialis anterior, soleus, and heart were carefully dissected and weighed. To correct for differences in muscle weight due to differences in age and body weight, results are expressed as absolute weight and as the ratio muscle weight (g)/total body weight. Specifically, we studied wild type mice on chow (n = 9) and high fat diets (n = 8) and UCP3 KO mice on chow (n = 11) and high fat diets (n = 9). Total body lipid content was assessed using alcoholic potassium hydroxide digestion with saponification of all fats, neutralization, and then enzymatic determination of glycerol as described previously (44.Salmon D.M. Flat J.P. Int. J. Obes. 1985; 9: 443-449PubMed Google Scholar). Triglyceride content is expressed as g/carcass and as percentage of total body weight. Food intake was measured in 10-week-old UCP3 KO female (n = 7), UCP3 KO male (n = 5), control female (n = 8), and control male (n = 8) mice for 3 weeks after a 1-week period of adaptation. Animals were housed individually and had free access to water. Food was weighed weekly, and the differences were assumed to represent grams of food eaten per week. Data are presented as g/day. To correct for body size, results are also expressed as g/day/body weight2/3. The cages were inspected for food spillage, and none was noted. Rectal temperature was assessed using a rectal probe (Yellow Spring Instruments Co.) in control (C) and UCP3 KO mice under the following situations: (a) fed state (C, n = 9; KO, n = 9) and (b) following 1, 4, and 24 h of cold exposure (C,n = 5; KO, n = 5). Thirty-two mice (5–9 months old) were matched for age and randomly divided into four groups as follows: 1) wild type-sedentary; 2) wild type-exercised; 3) UCP3 KO-sedentary; 4) UCP3 KO-exercised. Animals were given water and fed ad libitumand were maintained on a 12-h light/dark cycle. On the day before the exercise test, mice (wild type (WT) = 9, KO = 9) were accommodated to the treadmill apparatus and run twice for 2 h with a 30-min rest period between each running protocol. Within each 2-h running trial, the mice ran to physical exhaustion according to the following protocol: mice were accommodated to the treadmill apparatus by running at 10 meters/min for 10 min at 0% grade. Afterward, the treadmill speed was increased by 5 meters/min every 30 min so that the animals were running at a final speed of 30 meters/min for the final half of the 2-h protocol. The percentage grade of the treadmill was increased at 60 (3%), 90 (6%), and 105 min (9%). The animals continued to run at 30 meters/min up the 9% grade until they became physically exhausted (inability to avoid a shock device located in the back of the running stall) or the test was terminated at 2 h. The time (seconds) to exhaustion was recorded, and exhaustion was verified by the absence of a righting reflex (inability of the animal to right itself when placed on its back). Because UCP3 activity might be bioenergetically necessary to recover from vigorous exercise challenge, the mice rested for 30 min, and the protocol was repeated to evaluate their exercise capacity following recovery. The following day, the same 2 h running protocol to exhaustion was employed, and assessment of skeletal muscle carbohydrate and lipid metabolism was made immediately post-exercise. Food was withdrawn from both sedentary and exercised animals 3 h before the exercise test. Immediately after exercise, animals were anesthetized (ketamine/zylazine, 100 mg/10 mg/kg), and soleus and extensor digitorum longus muscles were excised, cleaned of adipose and connective tissue, and transferred to a 24-well tissue culture plate in a shaking bath at 29 °C. One soleus and one extensor digitorum longus were used to measure fatty acid oxidation, and contralateral muscles were used to determine glucose oxidation. Muscles were incubated as described previously (45.Muoio D.M. Dohm G.L. Fiedorek F.T.J. Tapscott E.B. Coleman R.A. Diabetes. 1997; 46: 1360-1363Crossref PubMed Google Scholar) in 1.0 ml of modified Krebs-Henseleit buffer continuously gassed with 95% O2, 5% CO2. After 15 min at 29 °C, muscles were placed in fresh modified Krebs-Henseleit buffer containing 1.0 mmol/liter sodium oleate, 1.0 mmol/liter carnitine, and 1% bovine serum albumin and pre-incubated for an additional 20 min at 37 °C. Muscles were then transferred to identical media but with either [U-14C]glucose (1.0 mCi/ml) or [1-14C]oleate (1.0 mCi/ml) and incubated 90 min at 37 °C. Afterward, the CO2 produced by muscle was driven from the media by adding 100 μl of 70% perchloric acid to each well. [14C]CO2 was trapped onto NaOH- saturated Whatman No. 3 filter paper, and oxidation rates were quantified as described previously (45.Muoio D.M. Dohm G.L. Fiedorek F.T.J. Tapscott E.B. Coleman R.A. Diabetes. 1997; 46: 1360-1363Crossref PubMed Google Scholar). Blood glucose
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