It has been reported that feeding mice resveratrol activates AMPK and SIRT1 in skeletal muscle leading to deacetylation and activation of PGC-1α, increased mitochondrial biogenesis, and improved running endurance. This study was done to further evaluate the effects of resveratrol, SIRT1, and PGC-1α deacetylation on mitochondrial biogenesis in muscle. Feeding rats or mice a diet containing 4 g resveratrol/kg diet had no effect on mitochondrial protein levels in muscle. High concentrations of resveratrol lowered ATP concentration and activated AMPK in C2C12 myotubes, resulting in an increase in mitochondrial proteins. Knockdown of SIRT1, or suppression of SIRT1 activity with a dominant-negative (DN) SIRT1 construct, increased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C2C12 cells. Expression of a DN SIRT1 in rat triceps muscle also induced an increase in mitochondrial proteins. Overexpression of SIRT1 decreased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C2C12 myotubes. Overexpression of SIRT1 also resulted in a decrease in mitochondrial proteins in rat triceps muscle. We conclude that, contrary to some previous reports, the mechanism by which SIRT1 regulates mitochondrial biogenesis is by inhibiting PGC-1α coactivator activity, resulting in a decrease in mitochondria. We also conclude that feeding rodents resveratrol has no effect on mitochondrial biogenesis in muscle.
The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) has been identified as an inducible regulator of mitochondrial function. Skeletal muscle PGC-1α expression is induced post-exercise. Therefore, we sought to determine its role in the regulation of muscle fuel metabolism. Studies were performed using conditional, muscle-specific, PGC-1α gain-of-function and constitutive, generalized, loss-of-function mice. Forced expression of PGC-1α increased muscle glucose uptake concomitant with augmentation of glycogen stores, a metabolic response similar to post-exercise recovery. Induction of muscle PGC-1α expression prevented muscle glycogen depletion during exercise. Conversely, PGC-1α-deficient animals exhibited reduced rates of muscle glycogen repletion post-exercise. PGC-1α was shown to increase muscle glycogen stores via several mechanisms including stimulation of glucose import, suppression of glycolytic flux, and by down-regulation of the expression of glycogen phosphorylase and its activating kinase, phosphorylase kinase α. These findings identify PGC-1α as a critical regulator of skeletal muscle fuel stores. The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) has been identified as an inducible regulator of mitochondrial function. Skeletal muscle PGC-1α expression is induced post-exercise. Therefore, we sought to determine its role in the regulation of muscle fuel metabolism. Studies were performed using conditional, muscle-specific, PGC-1α gain-of-function and constitutive, generalized, loss-of-function mice. Forced expression of PGC-1α increased muscle glucose uptake concomitant with augmentation of glycogen stores, a metabolic response similar to post-exercise recovery. Induction of muscle PGC-1α expression prevented muscle glycogen depletion during exercise. Conversely, PGC-1α-deficient animals exhibited reduced rates of muscle glycogen repletion post-exercise. PGC-1α was shown to increase muscle glycogen stores via several mechanisms including stimulation of glucose import, suppression of glycolytic flux, and by down-regulation of the expression of glycogen phosphorylase and its activating kinase, phosphorylase kinase α. These findings identify PGC-1α as a critical regulator of skeletal muscle fuel stores. Glucose and fatty acids are the chief fuel sources for skeletal muscle. During prolonged bouts of low intensity exercise, muscle energy needs are met through utilization of both substrates with mitochondrial fatty acid oxidation serving a "glucose sparing" function (1Hawley J.A. Clin. Exp. Pharmacol. Physiol. 2002; 29: 218-222Crossref PubMed Scopus (202) Google Scholar, 2Holloszy J.O. Kohrt W.M. Hansen P.A. Front. 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Whereas the role of PGC-1α in the control of muscle fatty acid oxidation and mitochondrial respiratory capacity is well established, its contribution to the regulation of skeletal muscle glucose metabolism has not been well defined. Several recent lines of evidence suggest that PGC-1α exerts control on muscle glucose metabolism. First, PGC-1α serves as a key regulator of hepatic gluconeogenesis, an important source of substrate for muscle (32Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Nature. 2003; 423: 550-555Crossref PubMed Scopus (1165) Google Scholar, 33Rhee J. Inoue Y. Yoon J.C. Puigserver P. Fam M. Gonzalez F.J. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4012-4017Crossref PubMed Scopus (469) Google Scholar, 34Rodgers J.T. Lerin C. Haas W. Gygi S.P. Spiegelman B.M. Puigserver P. Nature. 2005; 434: 113-118Crossref PubMed Scopus (2548) Google Scholar). Second, PGC-1α has been shown to activate transcription of the GLUT4 gene in myogenic cells in culture (35Michael L.F. Wu Z. Cheatham R.B. Puigserver P. Adelmant G. Lehman J.J. Kelly D.P. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3820-3825Crossref PubMed Scopus (536) Google Scholar). Third, PGC-1α activates expression of the gene encoding pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of glucose oxidation (22Wende A.R. Huss J.M. Schaeffer P.J. Giguère V. Kelly D.P. Mol. Cell. Biol. 2005; 25: 10684-10694Crossref PubMed Scopus (282) Google Scholar). In the current study, we sought to clarify the role of PGC-1α in the regulation of muscle fuel metabolism in vivo. To this end, we developed an inducible, muscle-specific PGC-1α transgenic system to be used in comparison with normal and PGC-1α-deficient mice (31Leone T.C. Lehman J.J. Finck B.N. Schaeffer P.J. Wende A.R. Boudina S. Courtois M. Wozniak D.F. Sambandam N. Bernal-Mizrachi C. Chen Z. Holloszy J.O. Medeiros D.M. Schmidt R.E. Saffitz J.E. Abel E.D. Semenkovich C.F. Kelly D.P. PLoS Biol. 2005; 3: 672-687Crossref Scopus (772) Google Scholar). We found that muscle-specific activation of PGC-1α increases muscle glucose uptake and glycogen levels at baseline and prevented depletion of glycogen stores following exercise. This glycogen "sparing" effect involved several mechanisms including increased capacity for fatty acid oxidation, induction of glucose transporter (GLUT1 and GLUT4) expression leading to augmentation of muscle glucose import, inhibition of glycolysis, and suppression of pathways involved in glycogen degradation. Animal Studies—All animal experiments and euthanasia protocols were conducted in accordance with the National Institutes of Health guidelines for humane treatment of animals and were reviewed and approved by the Animal Studies Committee of Washington University. Mice doubly transgenic for PGC-1α under control of the tetracycline response element promoter (TRE-PGC-1α) (30Russell L.K. Mansfield C.M. Lehman J.J. Kovacs A. Courtois M. Saffitz J.E. Medeiros D.M. Valencik M.L. McDonald J.A. Kelly D.P. Circ. Res. 2004; 94: 525-533Crossref PubMed Scopus (310) Google Scholar), and for the tetracycline transactivator under control of the muscle-specific creatine kinase promoter (MCK-tTA) (36Ghersa P. Gobert R.P. Sattonnet-Roche P. Richards C.A. Pich E.M. van Huijsduijnen R.H. Gene Ther. 1998; 5: 1213-1220Crossref PubMed Scopus (38) Google Scholar) were generated. Breeding pairs and offspring were maintained on chow containing doxycycline (200 mg/kg; Research Diet Inc., Brunswick, NJ). To activate the TRE-PGC-1α transgene, doxy-chow was removed and replaced with standard chow (Lab Diet 5052; Purina Mills Inc., St. Louis, MO). Unless otherwise stated, transgene induction was initiated at ∼5-6 weeks of age and experiments were performed 3-4 weeks later. For all studies, age- and sex-matched littermates were used. The generation and initial characterization of PGC-1α-deficient (PGC-1α-/-) mice has been described (31Leone T.C. Lehman J.J. Finck B.N. Schaeffer P.J. Wende A.R. Boudina S. Courtois M. Wozniak D.F. Sambandam N. Bernal-Mizrachi C. Chen Z. Holloszy J.O. Medeiros D.M. Schmidt R.E. Saffitz J.E. Abel E.D. Semenkovich C.F. Kelly D.P. PLoS Biol. 2005; 3: 672-687Crossref Scopus (772) Google Scholar). Approximately 2-3-month-old PGC-1α-/- mice were used for all studies with age- and sex-matched wild-type controls unless specified otherwise. Low Intensity Exercise—To assess endurance exercise capacity and glycogen depletion, male and female mice were run to exhaustion on an Exer4-OxyMax motorized treadmill (Columbus Instruments, Columbus, OH). Briefly, mice were acclimated to the treadmill for 2 days prior to the experimental protocol by running for 9 min at 10 m/min followed by 1 min at 20 m/min. On the day of the experiment, mice were run for 1 h at 10 m/min followed by an increase in speed of 2 m/min each additional 15 min until failure. Mice were defined as exhausted if they remained on the shock grid for five continuous seconds. High Intensity Exercise—Mice were acclimated to the treadmill as above. On the day of the experiment, mice were run alternating 1 min of running with 2 min of rest. The running intervals started at 10 m/min and increased 5 m/min each interval until a speed of 50 m/min was reached. Thereafter, speed was further increased 5 m/min every 6th interval until failure. Exhaustion was defined as above. For either exercise protocol, tail blood was taken prior to exercise and immediately following failure for glucose (HemoCue AB,Ángelholm, Sweden) and lactate (Lactate Pro Arkray, Kyoto, Japan) measurements, as per the manufacturers' instructions. To prevent blood loss, wounds were cauterized following sample collection. For glycogen replenishment studies, mice were gavaged with a glucose solution (0.56 m, 1 mg of glucose/g of mouse) immediately following completion of the exercise protocol and euthanized by CO2 inhalation at varying time points after cessation of exercise. The vastus muscles were harvested and immediately clamp-frozen in liquid nitrogen for later glycogen content, RNA, and protein analyses. Histology and Microscopy—Gastrocnemius was analyzed histologically. For light microscopy, frozen tissue was used for succinate dehydrogenase staining. Formalin-fixed samples were embedded in paraffin, sectioned, and stained with hematoxylin/eosin (H & E) or periodic acid Schiff. For electron microscopy, tissue was fixed in 2% glutaraldehyde and 1% paraformaldehyde, post-fixed in 1% osmium tetroxide, embedded in plastic epoxy resin (poly bed 812). Sections on copper grids were then stained with uranyl acetate and lead citrate for visualization. RNA and Protein Expression Analyses—Total cellular RNA was isolated from gastrocnemius using the RNAzol method (Tel-Test, Friendswood, TX). Northern blot hybridizations with random-primed 32P-labeled cDNA probes were performed using QuikHyb (Stratagene). Real-time quantitative reverse transcription-PCR (RT-PCR) was performed as previously described (37Huss J.M. Pinéda Torra I. Staels B. Giguère V. Kelly D.P. Mol. Cell. Biol. 2004; 24: 9079-9091Crossref PubMed Scopus (382) Google Scholar) and results were normalized to the expression of 36B4. Mouse-specific primers used for RNA analysis may be found in supplemental materials Table S1. Protein extracts were resolved by SDS-PAGE (Criterion, Bio-Rad) and transferred to nitrocellulose membranes (Whatman). Western blotting was performed using antibodies against GLUT4 (Santa Cruz, Santa Cruz, CA, and a gift from Michael Mueckler) (38Ren J.M. Marshall B.A. Gulve E.A. Gao J. Johnson D.W. Holloszy J.O. Mueckler M. J. Biol. Chem. 1993; 268: 16113-16115Abstract Full Text PDF PubMed Google Scholar), Actin (Research Diagnostics, Concord, MA), hexokinase (Santa Cruz), GPh (Fitzgerald Industries, Concord, MA), pGS (sites 3b to 5) (EMB Biosciences, San Diego, CA), GS (gift from John Lawrence (39Lawrence Jr., J.C. Hiken J.F. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. 1983; 258: 10710-10719Abstract Full Text PDF PubMed Google Scholar)), GLUT1 (gift from Michael Mueckler (40Tanner J.W. Leingang K.A. Mueckler M.M. Glenn K.C. Biochem. J. 1992; 282: 99-106Crossref PubMed Scopus (33) Google Scholar)), PDK4 (gift from Robert Harris (41Wu P. Sato J. Zhao Y. Jaskiewicz J. Popov K.M. Harris R.A. Biochem. J. 1998; 329: 197-201Crossref PubMed Scopus (262) Google Scholar)), PhKα (gift from Gerald Carlson (42Wilkinson D.A. Marion T.N. Tillman D.M. Norcum M.T. Hainfeld J.F. Seyer J.M. Carlson G.M. J. Mol. Biol. 1994; 235: 974-982Crossref PubMed Scopus (47) Google Scholar)), and pGPh (gift from Matthew Brady (43Gasa R. Jensen P.B. Berman H.K. Brady M.J. DePaoli-Roach A.A. Newgard C.B. J. Biol. Chem. 2000; 275: 26396-26403Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar)). Detection was performed by measuring horseradish peroxidase activity by chemiluminescent signal using ECL (Amersham Biosciences). Protein bands were analyzed by densitometry using ImageJ software (rsb.info.nih.gov/ij). Isolated Mitochondrial Respiration—Mitochondria were isolated from whole hindlimb using a trypsin digestion procedure as previously described (44Saks V.A. Kuznetsov A.V. Kupriyanov V.V. Miceli M.V. Jacobus W.E. J. Biol. Chem. 1985; 260: 7757-7764Abstract Full Text PDF PubMed Google Scholar). Briefly, tissue was minced, washed, and suspended in isolation medium (300 mm sucrose, 10 mm NaHepes, 0.2 mm EDTA, 1 mg/ml bovine serum albumin, pH 7.4). Following digestion, samples were gently homogenized with a glass Teflon homogenizer. Following centrifugation and two washes, isolated mitochondria were incubated with palmitate-containing media and respiration was determined as previously described (31Leone T.C. Lehman J.J. Finck B.N. Schaeffer P.J. Wende A.R. Boudina S. Courtois M. Wozniak D.F. Sambandam N. Bernal-Mizrachi C. Chen Z. Holloszy J.O. Medeiros D.M. Schmidt R.E. Saffitz J.E. Abel E.D. Semenkovich C.F. Kelly D.P. PLoS Biol. 2005; 3: 672-687Crossref Scopus (772) Google Scholar). Oxygen consumption was measured at 25 °C using an optical probe (Oxygen FOXY Probe; Ocean Optics, Dunedin, FL). Following basal state measurements, maximal (ADP-stimulated) state 3 respiration was determined by adding ADP to a 1 mm final concentration. Thereafter, rates of uncoupled respiration was determined indirectly following addition of oligomycin (1 μg/ml). The solubility of oxygen at 25 °C was taken as 246.87 mmol of O2 ml-1. Respiration rates are expressed as nanomole of O2 min-1 mg protein-1 as determined by the Pierce Micro BCA protein assay. Citrate Synthase Activity Assay—Citrate synthase activity was determined in tibialis anterior muscle as described (45Chi M.M. Hintz C.S. Coyle E.F. Martin W.H. II I Ivy J.L. Nemeth P.M. Holloszy J.O. Lowry O.H. Am. J. Physiol. 1983; 244: C276-C287Crossref PubMed Google Scholar). Measurement of Glucose Transport Activity—Animals were anesthetized with an intraperitoneal injection of sodium phenobarbital (5 mg/100 g body weight). Epitrochlearis muscle (and EDL, data not shown) was isolated following an overnight fast. Muscles were immediately washed with Krebs-Henseleit bicarbonate (KHB) and incubated for 20 min in 1.0 ml of KHB buffer containing 1 μm 2-deoxy-d-[1,2-3H]glucose and 0.1% bovine serum albumin and the rate of accumulation of 2-deoxyglucose (2-DG) in intracellular water was determined as described (46Hansen P.A. Gulve E.A. Holloszy J.O. J. Appl. Physiol. 1994; 76: 979-985Crossref PubMed Scopus (213) Google Scholar). Values were corrected to tissue weight. Glucose 6-Phosphate Assay—The concentration of Glu-6-P was determined as described (47Lowry O.H. Passonneau J.V. A Flexible System of Enzymatic Analysis. Academic Press, New York1972Google Scholar) by assaying gastrocnemius tissue extract with Glu-6-P dehydrogenase (G8289, Sigma) and 0.2 mm NADP in 50 mm Tris (pH 7.4) and 3 mm MgCl2. Resulting changes in absorption at 340 nm were compared with a standard of 0-33 nmol of Glu-6-P and corrected to protein concentration. Isolated Muscle Glycolysis Studies—Glycolysis was assayed in epitrochlearis muscle isolated from mice following an overnight fast using a modified protocol from isolated working hearts (48Sambandam N. Morabito D. Wagg C. Finck B.N. Kelly D.P. Lopaschuk G.D. Am. J. Physiol. 2006; 290: H87-H95Crossref PubMed Scopus (102) Google Scholar). Briefly, isolated muscle was immediately placed in KHB buffer with 8 mm glucose and 32 mm mannitol for 1 h at 37 °C for recovery. Muscle was then transferred to fresh KHB as above, supplemented with 125 μm oleate for substrate selection and preincubated for 30 min at 37 °C. Finally, muscle was transferred to fresh KHB as above, spiked with 1 μCi/ml [5-3H]glucose (15 Ci/mmol), and incubated for 1 h at 37 °C. The release of 3H2O from glucose into incubation buffer was measured by evaporative 3H2O transfer from the incubation buffer to water in a sealed scintillation vial. Tritium was measured by scintillation counting and disintegrations/min were normalized to control values corrected for tissue weight. Glycogen Measurements—Muscle extract from either vastus or gastrocnemius (data not shown) was analyzed as previously described (49Passonneau J.V. Lauderdale V.R. Anal. Biochem. 1974; 60: 405-412Crossref PubMed Scopus (625) Google Scholar). Briefly, snap frozen tissue was powdered under liquid nitrogen and homogenized in a 0.3 m perchloric acid solution. This extract was assayed with and without amyloglucosidase digestion (A7420, Sigma) in 50 mm Na acetate (pH 5.5) and 0.02% bovine serum albumin. Resulting changes in absorption at 340 nm were compared with a standard of 0 to 80 μmol of glucose. Results are presented as glucose released from glycogen corrected to tissue weight. Statistical Analysis—Statistical comparisons were made using unpaired t test or analysis of variance coupled to Tukey's post hoc test when appropriate. All data are presented as mean ± S.E., with a statistically significant difference defined as a value of p < 0.05. Short-term, Muscle-specific Expression of PGC-1α Activates Mitochondrial Biogenesis and Oxidative Gene Regulatory Programs—Double transgenic mice (PGC-1α-TRE(+) mice) were generated by breeding MCK-tTA ("tet-off" transactivator) and TRE-PGC-1α (transresponder) lines so that the short-term effects of muscle-specific overexpression of PGC-1α could be investigated. Single transgenic transresponder (PGC-1α-TRE(-)) littermates receiving doxycycline were used as controls. PGC-1α-TRE(+) mice were maintained on doxycycline-containing chow until 5-6 weeks of age, after which doxycycline was removed. The transgene was not expressed in the presence of doxycycline (Fig. 1A). Transgene expression was detectable in muscle after a 2-week doxy "wash-out" period and continued to increase until 3-4 weeks after doxycycline removal (Fig. 1A). Examination of various tissues revealed that transgene expression was specific to skeletal muscle (Fig. 1B and data not shown). As described previously for constitutive skeletal muscle PGC-1α transgenic mice (28Lin J. Wu H. Tarr P.T. Zhang C.Y. Wu Z. Boss O. Michael L.F. Puigserver P. Isotanni E. Olson E.N. Lowell B.B. Bassel-Duby R. Spiegelman B.M. Nature. 2002; 418: 797-801Crossref PubMed Scopus (2033) Google Scholar), forced expression of PGC-1α increased red coloration of the muscle within 3 weeks of the induction of transgene expression (Fig. 1C). Histological examination revealed enhanced succinate dehydrogenase staining in gastrocnemius, consistent with an increase in proportion of oxidative fibers in this muscle (Fig. 2A). To further examine the cellular and structural changes accompanying PGC-1α overexpression in skeletal muscle, electron microscopic analysis was performed. Forced expression of PGC-1α resulted in a dramatic increase in the cellular volume density of mitochondria (Fig. 2A). Importantly, muscle from PGC-1α-TRE(+) animals exhibited lipid droplets associated with mitochondria and granular staining resembling glycogen β-particles suggesting an increase in cellular fuel stores (Fig. 2A). Respiratory function studies were performed on mitochondria isolated from whole hindlimb of PGC-1α-TRE(+) and PGC-1α-TRE(-) mice. Using palmitoylcarnitine as substrate, state 3 (ADP-stimulated) respiration rates were significantly higher in mitochondria isolated from the PGC-1α-TRE(+) mice (Fig. 2B). In the presence of oligomycin, respiration was largely abolished in both PGC-1α-TRE(+) and PGC-1α-TRE(-) mitochondria, suggesting that the majority of the PGC-1α-stimulated respiration was coupled to ATP production. In contrast to the results obtained with palmitoylcarnitine, respiration rates with pyruvate were not different between the groups (data not shown). The activity of the citric acid cycle enzyme, citrate synthase (Fig. 2B), and the expression of nuclear and mitochondrial genes encoding enzymes involved in mitochondrial fatty acid β-oxidation, electron transport, and oxidative phosphorylation were coordinately increased in the muscle of the PGC-1α-TRE(+) mice (Fig. 2C). Expression of the cellular fatty acid transporter, CD36, was also induced in the muscle of PGC-1α-TRE(+) mice (Fig. 2C). Taken together, these results demonstrate that PGC-1α augments the capacity for muscle mitochondrial fatty acid oxidation. PGC-1α Drives Increased Muscle Glucose Uptake and Storage—We next sought to evaluate the effects of PGC-1α overexpression on muscle glucose metabolism. Rates of 2-DG uptake were determined ex vivo in epitrochlearis muscles isolated from PGC-1α-TRE(+) and PGC-1α-TRE(-) mice. Mean 2-DG uptake rates in PGC-1α-TRE(+) muscle were significantly greater compared with control muscles (Fig. 3A). Consistent with the glucose uptake data, expression of GLUT4, GLUT1, and hexokinase was induced in muscle of the PGC-1α-TRE(+) mice (Fig. 3B). To determine the fate of the glucose transported into PGC-1α-TRE(+) muscle, glycolysis rates were measured in isolated skeletal muscle by following the release of 3H2O from [5-3H]glucose. Surprisingly, PGC-1α overexpression led to a marked reduction (over 50%) in glycolytic flux (Fig. 3A). The decreased glycolytic rate was associated with a modest but significant reduction in the expression of the gene encoding phosphofructokinase, which catalyzes a tightly regulated, rate-limiting step in the glycolytic pathway (Fig. 3B). Recently we demonstrated that PGC-1α exerts repression on glucose oxidation by increasing expre
Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator of nuclear genes that encode a range of mitochondrial proteins including cytochrome c, various other respiratory chain subunits, and delta-aminolevulinate synthase. Activation of NRF-1 in fibroblasts has been shown to induce increases in cytochrome c expression and mitochondrial respiratory capacity. To further evaluate the role of NRF-1 in the regulation of mitochondrial biogenesis and respiratory capacity, we generated transgenic mice overexpressing NRF-1 in skeletal muscle. Cytochrome c expression was increased approximately twofold and delta-aminolevulinate synthase was increased approximately 50% in NRF-1 transgenic muscle. The levels of some mitochondrial proteins were increased 50-60%, while others were unchanged. Muscle respiratory capacity was not increased in the NRF-1 transgenic mice. A finding that provides new insight regarding the role of NRF-1 was that expression of MEF2A and GLUT4 was increased in NRF-1 transgenic muscle. The increase in GLUT4 was associated with a proportional increase in insulin-stimulated glucose transport. These results show that an isolated increase in NRF-1 is not sufficient to bring about a coordinated increase in expression of all of the proteins necessary for assembly of functional mitochondria. They also provide the new information that NRF-1 overexpression results in increased expression of GLUT4.
It has been reported that supplementation with the antioxidant vitamins C and E prevents the adaptive increases in mitochondrial biogenesis and GLUT4 expression induced by endurance exercise. We reevaluated the effects of these antioxidants on the adaptive responses of rat skeletal muscle to swimming in a short-term study consisting of 9 days of vitamins C and E with exercise during the last 3 days and a longer-term study consisting of 8 wk of antioxidant vitamins with exercise during the last 3 wk. The rats in the antioxidant groups were given 750 mg·kg body wt −1 ·day −1 vitamin C and 150 mg·kg body wt −1 ·day −1 vitamin E. In rats euthanized immediately after exercise, plasma TBARs were elevated in the control rats but not in the antioxidant-supplemented rats, providing evidence for an antioxidant effect. In rats euthanized 18 h after exercise there were large increases in insulin responsiveness of glucose transport in epitrochlearis muscles mediated by an approximately twofold increase in GLUT4 expression in both the short- and long-term treatment groups. The protein levels of a number of mitochondrial marker enzymes were also increased about twofold. Superoxide dismutases (SOD) 1 and 2 were increased about twofold in triceps muscle after 3 days of exercise, but only SOD2 was increased after 3 wk of exercise. There were no differences in the magnitudes of any of these adaptive responses between the control and antioxidant groups. These results show that very large doses of antioxidant vitamins do not prevent the exercise-induced adaptive responses of muscle mitochondria, GLUT4, and insulin action to exercise and have no effect on the level of these proteins in sedentary rats.
Background It has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis. Methodology/Principal Findings Rats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance. Conclusions/Significance The results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance.
There are reports that the β-adrenergic agonist clenbuterol induces a large increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in skeletal muscle. This has led to the hypothesis that the increases in PGC-1α and mitochondrial biogenesis induced in muscle by endurance exercise are mediated by catecholamines. In the present study, we evaluated this possibility and found that injecting rats with clenbuterol or norepinephrine induced large increases in PGC-1α and mitochondrial proteins in brown adipose tissue but had no effect on PGC-1α expression or mitochondrial biogenesis in skeletal muscle. In brown adipocytes, the increase in PGC-1α expression induced by β-adrenergic stimulation is mediated by activation of p38 mitogen-activated protein kinase (p38 MAPK), which phosphorylates and activates the cAMP response element binding protein (CREB) family member activating transcription factor 2 (ATF2), which binds to a cyclic AMP response element (CRE) in the PGC-1α promoter and mediates the increase in PGC-1α transcription. Phospho-CREB does not have this effect. Our results show that the reason for the lack of effect of β-adrenergic stimulation on PGC-1α expression in muscle is that catecholamines do not activate p38 or increase ATF2 phosphorylation in muscle.
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