Overexpression of Peroxisome Proliferator-Activated Receptor γ Co-Activator-1α Leads to Muscle Atrophy with Depletion of ATP
2006; Elsevier BV; Volume: 169; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2006.060034
ISSN1525-2191
AutoresShinji Miura, Eriko Tomitsuka, Yasutomi Kamei, Tomomi Yamazaki, Yuko Kai, Mayumi Tamura, Kiyoshi Kita, Ichizo Nishino, Osamu Ezaki,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoPeroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) is a key nuclear receptor co-activator for mitochondrial biogenesis. Here we report that overexpression of PGC-1α in skeletal muscles increased mitochondrial number and caused atrophy of skeletal muscle, especially type 2B fiber-rich muscles (gastrocnemius, quadriceps, and plantaris). Muscle atrophy became evident at 25 weeks of age, and a portion of the muscle was replaced by adipocytes. Mice showed increased energy expenditure and reduced body weight; thyroid hormone levels were normal. Mitochondria exhibited normal respiratory chain activity per mitochondrion; however, mitochondrial respiration was not inhibited by an ATP synthase inhibitor, oligomycin, clearly indicating that oxidative phosphorylation was uncoupled. Accordingly, ATP content in gastrocnemius was markedly reduced. A similar phenotype is observed in Luft's disease, a mitochondrial disorder that involves increased uncoupling of respiration and muscle atrophy. Our results indicate that overexpression of PGC-1α in skeletal muscle increases not only mitochondrial biogenesis but also uncoupling of respiration, resulting in muscle atrophy. Peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) is a key nuclear receptor co-activator for mitochondrial biogenesis. Here we report that overexpression of PGC-1α in skeletal muscles increased mitochondrial number and caused atrophy of skeletal muscle, especially type 2B fiber-rich muscles (gastrocnemius, quadriceps, and plantaris). Muscle atrophy became evident at 25 weeks of age, and a portion of the muscle was replaced by adipocytes. Mice showed increased energy expenditure and reduced body weight; thyroid hormone levels were normal. Mitochondria exhibited normal respiratory chain activity per mitochondrion; however, mitochondrial respiration was not inhibited by an ATP synthase inhibitor, oligomycin, clearly indicating that oxidative phosphorylation was uncoupled. Accordingly, ATP content in gastrocnemius was markedly reduced. A similar phenotype is observed in Luft's disease, a mitochondrial disorder that involves increased uncoupling of respiration and muscle atrophy. Our results indicate that overexpression of PGC-1α in skeletal muscle increases not only mitochondrial biogenesis but also uncoupling of respiration, resulting in muscle atrophy. Peroxisome proliferator-activated receptor (PPAR)-γ co-activator-1α (PGC-1α), which was identified as co-activator of nuclear receptors, is expressed in brown adipose tissue, skeletal muscle, heart, kidney, and brain and is markedly up-regulated in brown adipose tissue and skeletal muscle after acute exposure to cold stress.1Puigserver P Wu Z Park CW Graves R Wright M Spiegelman BM A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3101) Google Scholar Increased expression of PGC-1α as part of activated adaptive thermogenesis occurs primarily in the mitochondria of brown adipose tissue and skeletal muscle through stimulation of mitochondrial biogenesis and respiration.2Wu Z Puigserver P Andersson U Zhang C Adelmant G Mootha V Troy A Cinti S Lowell B Scarpulla RC Spiegeman BM Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1.Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3229) Google Scholar PGC-1α is also believed to be a key molecule involved in fatty acid oxidation because it was found to interact with PPARα to promote transcription of nuclear genes encoding mitochondrial fatty acid oxidation enzymes.3Vega RB Huss JM Kelly DP The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor alpha in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes.Mol Cell Biol. 2000; 20: 1868-1876Crossref PubMed Scopus (952) Google Scholar, 4Lehman JJ Barger PM Kovacs A Saffitz JE Medeiros DM Kelly DP Peroxisome proliferator-activated receptor gamma coactivator-1 promotes cardiac mitochondrial biogenesis.J Clin Invest. 2000; 106: 847-856Crossref PubMed Scopus (1026) Google Scholar In in vivo studies of muscles overexpressing PGC-1α, the skeletal muscles showed a red color characteristic of oxidative muscle and elevated levels of enzymes related to mitochondrial oxidative phosphorylation and fatty acid oxidation.5Lin J Wu H Tarr PT Zhang CY Wu Z Boss O Michael LF Puigserver P Isotani E Olson EN Lowell BB Bassel-Duby R Spiegelman BM Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres.Nature. 2002; 418: 797-801Crossref PubMed Scopus (2072) Google Scholar These same muscles showed reduced GLUT4 mRNA expression and impaired insulin tolerance.6Miura S Kai Y Ono M Ezaki O Overexpression of peroxisome proliferator-activated receptor gamma coactivator-1alpha down-regulates GLUT4 mRNA in skeletal muscles.J Biol Chem. 2003; 278: 31385-31390Crossref PubMed Scopus (120) Google Scholar In brown adipose tissue mitochondria, as observed during prolonged exposure to cold, PGC-1α promotes uncoupling of respiration through induction of uncoupling protein 1 (UCP1).1Puigserver P Wu Z Park CW Graves R Wright M Spiegelman BM A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3101) Google Scholar When respiration is uncoupled, the membrane potential energy is channeled to heat production rather than to adenosine triphosphate (ATP) production, which may cause functional abnormalities and cell death. In C2C12 myocytes, increased uncoupling of respiration driven by PGC-1α was observed.7St.-Pierre J Lin J Krauss S Tarr PT Yang R Newgard CB Spiegelman BM Bioenergetic analysis of peroxisome proliferator-activated receptor gamma coactivators 1alpha and 1beta (PGC-1alpha and PGC-1beta) in muscle cells.J Biol Chem. 2003; 278: 26597-26603Crossref PubMed Scopus (477) Google Scholar Overexpression of PGC-1α promoted mitochondrial biogenesis and resulted in dilated cardiomyopathy.4Lehman JJ Barger PM Kovacs A Saffitz JE Medeiros DM Kelly DP Peroxisome proliferator-activated receptor gamma coactivator-1 promotes cardiac mitochondrial biogenesis.J Clin Invest. 2000; 106: 847-856Crossref PubMed Scopus (1026) Google Scholar These data suggest that PGC-1α overexpression in skeletal muscles increases uncoupling of respiration, decreases ATP content, and results in muscle atrophy. However, this hypothesis has not been verified. In the present study, we examined mitochondrial respiratory function and morphological changes in the skeletal muscles of mice overexpressing PGC-1α and found that PGC-1α transgenic mice had markedly decreased ATP content in skeletal muscles and developed myopathy at 25 weeks of age, which recapitulates the phenotype of Luft's disease.8Ernster L Ikkos D Luft R Enzymic activities of human skeletal muscle mitochondria: a tool in clinical metabolic research.Nature. 1959; 184: 1851-1854Crossref PubMed Scopus (103) Google Scholar, 9Luft R Ikkos D Palmieri G Ernster L Afzelius B A case of severe hypermetabolism of nonthyroid origin with a defect in the maintenance of mitochondrial respiratory control: a correlated clinical, biochemical, and morphological study.J Clin Invest. 1962; 41: 1776-1804Crossref PubMed Scopus (616) Google Scholar, 10DiMauro S Bonilla E Lee CP Schotland DL Scarpa A Conn Jr, H Chance B Luft's disease. Further biochemical and ultrastructural studies of skeletal muscle in the second case.J Neurol Sci. 1976; 27: 217-232Abstract Full Text PDF PubMed Scopus (117) Google Scholar D-line mice expressed 10-fold higher PGC-1α mRNA and E-line mice expressed 13-fold higher PGC-1α mRNA in skeletal muscle compared to wild-type mice, as described in our previous study.6Miura S Kai Y Ono M Ezaki O Overexpression of peroxisome proliferator-activated receptor gamma coactivator-1alpha down-regulates GLUT4 mRNA in skeletal muscles.J Biol Chem. 2003; 278: 31385-31390Crossref PubMed Scopus (120) Google Scholar Male chimeras harboring the PGC-1 transgene were mated with pure C57BL/6J females (Tokyo Laboratory Animals Science, Tokyo, Japan) to obtain F1 offspring. The heterozygous F1 male offspring from this breeding were then backcrossed with purebred C57BL/6J females to obtain F2 offspring, and this process was continued until the F3 generation of mice was obtained. Heterozygous transgenic mice were used for the following studies. Mice were fed a standard laboratory chow diet (CE2; Clea, Tokyo, Japan). Mice were exposed to a 12-hour light/dark cycle and maintained at a constant temperature of 22°C. The mice were cared for in accordance with "Principles of Laboratory Animal Care" (National Institutes of Health publication no. 85-23, revised 1985: http://grants1.nih.gov/grants/olaw/references/phspol.htm) and our institutional guidelines. Oxygen consumption was measured with a metabolic chamber as described previously.11Tsuboyama-Kasaoka N Takahashi M Tanemura K Kim HJ Tange T Okuyama H Kasai M Ikemoto S Ezaki O Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice.Diabetes. 2000; 49: 1534-1542Crossref PubMed Scopus (489) Google Scholar For estimation of running wheel activity, mice were housed individually in cages (9 × 22 × 9 cm) equipped with a running wheel (20-cm diameter; Shinano Co., Tokyo, Japan). Each wheel revolution was registered by a magnetic switch connected to a counter. The number of revolutions was recorded daily for 10 days. Samples of the tibialis anterior muscle at 16 weeks of age and hindlimb at 25 weeks of age were frozen in liquid nitrogen-cooled isopentane, and transverse serial sections were stained with hematoxylin and eosin (H&E), modified Gomori trichrome, and Oil red O.12Black JT Judge D Demers L Gordon S Ragged-red fibers. A biochemical and morphological study.J Neurol Sci. 1975; 26: 479-488Abstract Full Text PDF PubMed Scopus (27) Google Scholar These sections were also analyzed by enzyme histochemistry to evaluate cytochrome c oxidase (COX)13Seligman AM Karnovsky MJ Wasserkrug HL Hanker JS Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB).J Cell Biol. 1968; 38: 1-14Crossref PubMed Scopus (733) Google Scholar and succinate dehydrogenase (SDH) activities.14Dubowitz V Brooke M A Modern Approach, Muscle Biopsy. Saunders, London1973Google Scholar For electron microscopy, extensor digitorum longus muscles from 16-week-old mice were fixed in buffered 2% isotonic glutaraldehyde (pH 7.4), postfixed in osmium tetroxide, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead nitrate and examined with an H-7000 electron microscope (Hitachi, Tokyo, Japan). Skeletal muscles (gastrocnemius and quadriceps) from both 14-week-old wild-type and PGC-1α transgenic male mice were pooled from three mice in each group. Tissues were cut with scalpels and treated with 1 ml of protease buffer containing 0.1 mol/L KCl, 50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L MgSO4, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 5 mg/ml bovine serum albumin, 1 mmol/L ATP, and 2 mg/ml proteinase K for 2 minutes on ice. Tissues were then washed with ATP buffer containing 0.1 mol/L KCl, 50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L MgSO4, 1 mmol/L EDTA, 5 mg/ml bovine serum albumin, and 1 mmol/L ATP. The washed tissues were homogenized in 10 ml of ATP buffer with a glass/Teflon homogenizer in a power-driven Potter-Elvehjem homogenizer (Kadoguchi-Keiki, Tokyo, Japan). The homogenate was brought to 15 ml and centrifuged at 600 × g for 10 minutes to remove cell debris and nuclei. The supernatant was then centrifuged at 4500 × g for 15 minutes. The pellet was washed with KCl buffer containing 0.1 mol/L KCl, 50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L MgSO4, 1 mmol/L EDTA, and 5 mg/ml bovine serum albumin and centrifuged at 7000 × g for 15 minutes. The mitochondrial pellet was resuspended in mitochondrial buffer containing 225 mmol/L mannitol and 75 mmol/L sucrose.15Pallotti F Lenaz G Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.Methods Cell Biol. 2001; 65: 1-35Crossref PubMed Google Scholar Protein concentrations were estimated by Lowry method.16Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the Folin phenol reagent.J Biol Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar Mitochondrial respiration was measured with Biological Oxygen Monitor 5300 (Yellow Springs Instrument, Yellow Springs, OH) was performed using Clark-type oxygen electrodes.17Trounce IA Kim YL Jun AS Wallace DC Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines.Methods Enzymol. 1996; 264: 484-509Crossref PubMed Google Scholar Respiration buffer containing 225 mmol/L mannitol, 75 mmol/L sucrose, 10 mmol/L Tris-HCl (pH 7.2), 5 mmol/L potassium phosphate (pH 7.2), and 10 mmol/L KCl was used. Five mmol/L succinate as substrate and 100 μg of mitochondria and 5 μg/ml rotenone were added, and respiration was started by the addition of 5 mmol/L potassium succinate. State 3 respiration was started by the addition of 200 μmol/L ADP (Sigma, St. Louis, MO). Finally, 2.5 μg/ml oligomycin (Sigma) was added as an inhibitor of ATP synthetase to block all phosphorylation-related respiration.2Wu Z Puigserver P Andersson U Zhang C Adelmant G Mootha V Troy A Cinti S Lowell B Scarpulla RC Spiegeman BM Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1.Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3229) Google Scholar SDH activity was measured by monitoring the change in absorbance 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,4-tetrazolium bromide (MTT) at 570 nm in the presence of phenazine methosulfate with the 17 mmol/L−1cm−1 extinction coefficient for MTT.18Kita K Vibat CR Meinhardt S Guest JR Gennis RB One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556.J Biol Chem. 1989; 264: 2672-2677Abstract Full Text PDF PubMed Google Scholar Succinate-ubiquinone oxidoreductase activity was measured in the presence of 2,3–2,6-dichlorophenolindophenol (DCIP) with the 21 mmol/L−1cm−1 extinction coefficient for DCIP.18Kita K Vibat CR Meinhardt S Guest JR Gennis RB One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556.J Biol Chem. 1989; 264: 2672-2677Abstract Full Text PDF PubMed Google Scholar NADH-ubiquinone reductase activity was assayed in 50 mmol/L potassium phosphate buffer (pH 7.7), 200 μmol/L NADH, 2 mmol/L KCN, and 90 μmol/L ubiquinone-1. Oxidation of NADH was monitored at 340 nm with a mmol/L−1 extinction coefficient of 6.2 for NADH.19Miyadera H Amino H Hiraishi A Taka H Murayama K Miyoshi H Sakamoto K Ishii N Hekimi S Kita K Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans.J Biol Chem. 2001; 276: 7713-7716Crossref PubMed Scopus (170) Google Scholar NADH oxidase activity was assayed in 50 mmol/L potassium phosphate buffer (pH 7.7) and 200 μmol/L NADH in the presence or absence of 100 nmol/L antimycin A and 2 mmol/L KCN. The oxidation of NADH was monitored at 340 nm with a mmol/L−1 extinction coefficient of 6.2 for NADH.20Ventura B Genova ML Bovina C Formiggini G Lenaz G Control of oxidative phosphorylation by complex I in rat liver mitochondria: implications for aging.Biochim Biophys Acta. 2002; 1553: 249-260Crossref PubMed Scopus (87) Google Scholar NADH-cytochrome c reductase, succinate-cytochrome c reductase, ubiquinol-cytochrome c reductase, and COX activities were measured as described previously.17Trounce IA Kim YL Jun AS Wallace DC Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines.Methods Enzymol. 1996; 264: 484-509Crossref PubMed Google Scholar, 21Birch-Machin MA Turnbull DM Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues.Methods Cell Biol. 2001; 65: 97-117Crossref PubMed Google Scholar Citrate synthase activity was measured as described previously.22Srere PA Citrate synthase.Methods Enzymol. 1969; 13: 3-11Crossref Scopus (2038) Google Scholar Each measurement was performed in three times. Northern blot analysis was performed as described previously.23Tsuboyama-Kasaoka N Tsunoda N Maruyama K Takahashi M Kim H Ikemoto S Ezaki O Up-regulation of uncoupling protein 3 (UCP3) mRNA by exercise training and down-regulation of UCP3 by denervation in skeletal muscles.Biochem Biophys Res Commun. 1998; 247: 498-503Crossref PubMed Scopus (116) Google Scholar To measure mitochondrial DNA (mtDNA) copy number, cDNA probes for mtDNA-COX II and nuclear genome gene-COX IV were generated as described previously.6Miura S Kai Y Ono M Ezaki O Overexpression of peroxisome proliferator-activated receptor gamma coactivator-1alpha down-regulates GLUT4 mRNA in skeletal muscles.J Biol Chem. 2003; 278: 31385-31390Crossref PubMed Scopus (120) Google Scholar For each sample, 10 μg of total DNA (containing both genomic and mitochondrial DNAs) from gastrocnemius were digested with EcoRI and NcoI, separated by electrophoresis on 0.8% agarose gels, and transferred overnight to nylon membranes.1Puigserver P Wu Z Park CW Graves R Wright M Spiegelman BM A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3101) Google Scholar Blots were then hybridized to COX II and COX IV probes radiolabeled with 32P-dCTP. The blots were washed, and DNA were quantified with an image analyzer (BAS 1800; Fuji Film, Tokyo, Japan) and expressed as the intensity of phosphostimulated luminescence. Reactions were performed in the 96-well format with SYBR Green PCR Master Mix and a 7500 real-time PCR system (Applied Biosystems, Foster City, CA) with 3 ng of total genomic DNA as template. As a preliminary experiment, we determined appropriate amounts of DNA for the assay. Copy number of COX II was normalized to those of COX IV and 36B4. 36B4 encodes acidic ribosomal phosphoprotein PO,24Laborda J 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO.Nucleic Acids Res. 1991; 19: 3998Crossref PubMed Scopus (434) Google Scholar and the 36B4 cDNA probe is widely used as a control in RNase protection experiments to study regulation of the transcription of several genes by estradiol. Mouse-specific primer pairs were: COX II forward, 5′-CCGACTAAATCAAGCAACAGTAACA-3′ and COX II reverse, 5′-AAATTTCAGAGCATTGGCCATAG-3′; COX IV forward, 5′-CTATGTGTATGGCCCCATCC-3′ and COX IV reverse, 5′-AGCGGGCTCTCACTTCTTC-3′; and 36B4 forward, 5′-GGCCCTGCACTCTCGCTTTC-3′ and 36B4 reverse, 5′-TGCCAGGACGCGCTTGT-3′. The mtDNA content is the mtDNA copy number normalized to the copy number of a gene contained in the nuclear genome. The mtDNA copy number in PGC-1α transgenic mice was expressed by percentages of those in control wild-type mice with the formula: [(mtDNA in PGC-1α transgenic mice/nuclear genome gene in PGC-1α transgenic mice)/(mtDNA in wild-type mice/nuclear genome gene in wild-type mice)] × 100%. Southern blotting and real-time PCR were used to estimate the copy number of specific genes in skeletal muscles. The mitochondrial gene used for mtDNA copy estimation was COX II, and the copy number of COX II was normalized to the copy number of one of two genes, COX IV or 36B4, contained in the nuclear genome. Gastrocnemius was homogenized with 1.0 N perchloric acid and centrifuged at 10,000 × g for 15 minutes. After neutralization of the supernatant with calcium carbonate, ATP and AMP concentrations were determined by high performance liquid chromatogram (Phenomenex Luna 5 μ NH2; mobile phase, sodium phosphate buffer; detector, 260 nm; Torrance, CA).25Watanabe A Tsuneishi E Takimoto Y Analysis of ATP and its breakdown products in beef by reversed-phase HPLC.J Food Sci. 1989; 54: 1169-1172Crossref Scopus (38) Google Scholar, 26Scott MD Baudendistel LJ Dahms TE Rapid separation of creatine, phosphocreatine and adenosine metabolites by ion-pair reversed-phase high-performance liquid chromatography in plasma and cardiac tissue.J Chromatogr. 1992; 576: 149-154Crossref PubMed Scopus (17) Google Scholar Mice were anesthetized with pentobarbital sodium, Nembutal (0.08 mg/g body weight; Abbot Laboratories, Chicago, IL), and scanned with a Lunar PIXI mus2 densitometer (Lunar Corp., Madison, WI), equipped for dual-energy X-ray absorptiometry (DEXA).27Nagy TR Clair AL Precision and accuracy of dual-energy X-ray absorptiometry for determining in vivo body composition of mice.Obes Res. 2000; 8: 392-398Crossref PubMed Scopus (245) Google Scholar All data are presented as mean ± SEM. Data from multiple groups were compared by one-way analysis of variance (StatView 5.0; Abacus Concepts, Berkeley, CA). When differences were significant, data were compared between groups by Fisher's protected least significant difference test. Data from two experimental groups were compared by unpaired Student's t-test. Statistical significance was defined as P < 0.05. In our previous study, skeletal muscles of PGC-1α transgenic mice showed the red color characteristic of oxidative muscle and increased levels of enzymes related to mitochondrial oxidative phosphorylation. In the same mice, expression of GLUT4 mRNA was reduced, and the glucose-lowering effects of insulin were impaired.6Miura S Kai Y Ono M Ezaki O Overexpression of peroxisome proliferator-activated receptor gamma coactivator-1alpha down-regulates GLUT4 mRNA in skeletal muscles.J Biol Chem. 2003; 278: 31385-31390Crossref PubMed Scopus (120) Google Scholar To determine whether PGC-1α transgenic mice show increased energy expenditure, whole-body oxygen consumption was measured in 10-week-old mice (Figure 1A). During both the dark (active) and light (sleeping) phases, PGC-1α transgenic mice showed 1.1-fold higher oxygen consumption than control mice. Because the cumulative number of wheel revolutions was 20% less in PGC-1α transgenic mice (Figure 1B), the increased oxygen consumption in PGC-1α transgenic mice was not attributable to increased voluntary exercise. Blood triiodothyronine (T3) and thyroxine (T4) concentrations were not altered in 10-week-old PGC-1α transgenic mice. Blood T3 concentrations in wild-type mice, D-line transgenic mice, and E-line transgenic mice were 1.16 ± 0.07 ng/ml, 1.14 ± 0.04 ng/ml, and 1.06 ± 0.01 ng/ml (n = 3 each group), respectively. Blood T4 concentrations in wild-type mice, D-line transgenic mice, and E-line transgenic mice were 5.89 ± 0.63 μg/dl, 5.78 ± 0.58 μg/dl, and 5.55 ± 0.29 μg/dl (n = 3 each group), respectively. We examined whether biogenesis of mitochondria and respiratory chain activity were increased in skeletal muscles from PGC-1α transgenic mice using histochemical staining of tibialis anterior muscle (type 2B-rich fiber muscle) in 16-week-old D-line transgenic mice (Figure 2). H&E staining revealed increased variability in muscle fiber size and increased interstitial cell number and connective tissues in PGC-1α transgenic mice (Figure 2E). Modified Gomori trichrome staining revealed a marked increase in the number of mitochondria in PGC-1α transgenic mice (Figure 2F). In addition, increased COX (Figure 2G) and SDH (Figure 2H) activities in transgenic muscle indicated that the mitochondrial respiratory chain was functional and active. The pathological hallmarks of mtDNA disease, such as ragged-red fibers and COX deficiency,28Larsson NG Clayton DA Molecular genetic aspects of human mitochondrial disorders.Annu Rev Genet. 1995; 29: 151-178Crossref PubMed Scopus (410) Google Scholar were not observed. To examine the subcellular structure of myocytes in PGC-1α transgenic mice in detail, extensor digitorum longus skeletal muscle in 16-week-old E-line transgenic mice was examined by electron microscopy (Figure 2J). An increase in the thickness of the Z-band that is seen in type-1 fibers and a marked increase in the number of mitochondria in the subsarcolemmal region of muscle fibers were observed. Mitochondrial size was normal, and the alignment of myofibrils was intact in transgenic mice. To estimate numbers of mitochondria in gastrocnemius from PGC-1α transgenic mice, citrate synthase activity, the most commonly used marker of mitochondrial number,17Trounce IA Kim YL Jun AS Wallace DC Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines.Methods Enzymol. 1996; 264: 484-509Crossref PubMed Google Scholar and mtDNA (COX II) copy number relative to those of nuclear genome genes (COX IV and 36B4), were measured (Table 1). The copy number of COX IV was estimated by two different methods, Southern blotting and real-time PCR. Citrate synthase activity in PGC-1α transgenic mice was threefold higher than that in wild-type mice. The ratio of COX II copy number to genomic DNA copy number in PGC-1α transgenic mice was twofold to threefold higher than that in wild-type mice, regardless of the detection method or nuclear genome reference gene. These data suggested that if we assume no significant differences in citrate synthase activity and mtDNA copy number in mitochondria between PGC-1α transgenic and wild-type mice, the number of mitochondria in gastro-cnemius from PGC-1α transgenic mice was twofold to threefold larger than that in wild-type mice.Table 1Estimated Numbers of Mitochondria in Gastrocnemius from 14-Week-Old MiceWild typePGC-1α/D linePGC-1α/E lineCitrate synthase activity (μmol/minute/mg protein, %)0.56 (100)1.75 (313)1.66 (296)COX II/COX IV (%, determined by Southern blot)100 ± 4 (6)202 ± 21 (3)‡P < 0.001 versus wild-type mice.165 ± 15 (3)†P < 0.01, andCOX II/COX IV (%, determined by real-time PCR)100 ± 5 (6)178 ± 10 (3)†P < 0.01, and214 ± 29 (3)‡P < 0.001 versus wild-type mice.COX II/36B4 (%, determined by real-time PCR)100 ± 5 (6)203 ± 8 (3)*P < 0.05,292 ± 58 (3)‡P < 0.001 versus wild-type mice.For measurement of citrate synthase activity, tissues from each group were pooled from three animals before mitochondrial isolation, and the citrate synthase activities of pooled samples were measured. For estimation of mtDNA copy number in skeletal muscle, the relative mtDNA copy number in skeletal muscle from individual mice in each group was calculated as the ratio of COX II (mitochondrial) to COX IV or 36B4 (nuclear) genes as determined by Southern blotting and/or real-time PCR. The relative mtDNA copy number was expressed as the percentage of the ratio in wild type. Numbers of mice were indicated.* P < 0.05,† P < 0.01, and‡ P < 0.001 versus wild-type mice. Open table in a new tab For measurement of citrate synthase activity, tissues from each group were pooled from three animals before mitochondrial isolation, and the citrate synthase activities of pooled samples were measured. For estimation of mtDNA copy number in skeletal muscle, the relative mtDNA copy number in skeletal muscle from individual mice in each group was calculated as the ratio of COX II (mitochondrial) to COX IV or 36B4 (nuclear) genes as determined by Southern blotting and/or real-time PCR. The relative mtDNA copy number was expressed as the percentage of the ratio in wild type. Numbers of mice were indicated. To study the function of skeletal muscle mitochondria, we compared the respiration rate (=oxygen consumption) in isolated mitochondria from gastrocnemius and quadriceps of PGC-1α transgenic mice with that in wild-type mice in the absence and presence of oligomycin, an inhibitor of the F1F0-ATP synthase.29Brand M Brown G Cooper C Mitochondria: A Practical Approach. Oxford University Press, 1995: 39-62Google Scholar First, the respiration rate of mitochondria was measured under conditions in which mitochondrial substrate and ADP were not limiting to mimic state 3 respiration. To measure uncoupled respiration, oligomycin was added to inhibit oxidative phosphorylation before measurement of respiration rates. In the absence of oligomycin, both lines of PGC-1α transgenic mice showed a twofold to threefold increase of respiration rate on a protein basis of mitochondria-rich fraction (Figure 3A). Because the increased respiration rate observed in PGC-1α transgenic mice was likely attributable to an increased mitochondrial number, we divided the respiration rate by the citrate synthase activity to express respiration rate on a per mitochondrion basis.17Trounce IA Kim YL Jun AS Wallace DC Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines.Methods Enzymol. 1996; 264: 484-509Crossref PubMed Google Scholar To normalize the respiration rate to the mitochondrial number, citrate synthase activity rather than mtDNA copy number was used, because the same method for preparation of mitochondria was used for both respiration rate and citrate synthase activity assays. When expressed per unit of citrate synthase activity, the respiration rate was similar between PGC-1α transgenic mice and wild-type mice (Figure 3B), suggesting that respiration was comparable in each mitochondrion in PGC-1α transgenic and wild-type mice. However, the mechanism of respiratory control was different; in wild-type mice, oligomycin-insensitive respiration (=uncoupling) constituted 40% of total respiration, whereas in PGC-1α transgenic mice, most respiration was oligomycin
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