Sirtuin 1 (SIRT1) deacetylase activity is not required for mitochondrial biogenesis or peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) deacetylation following endurance exercise

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1α and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1α expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1α expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1α after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1α. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1α or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1α activity after exercise.     

Role of AMPKalpha2 in basal, training-, and AICAR-induced GLUT4, hexokinase II, and mitochondrial protein expression in mouse muscle

We investigated the role of AMPKalpha2in basal, exercise training-, and AICAR-induced protein expression of GLUT4, hexokinase II (HKII), mitochondrial markers, and AMPK subunits. This was conducted in red (RG) and white gastrocnemius (WG) muscle from wild-type (WT) and alpha2-knockout (KO) mice after 28 days of activity wheel running or daily AICAR injection. Additional experiments were conducted to measure acute activation of AMPK by exercise and AICAR. At basal, mitochondrial markers were reduced by approximately 20% in alpha2-KO muscles compared with WT. In both muscle types, AMPKalpha2 activity was increased in response to both stimuli, whereas AMPKalpha1 activity was increased only in response to exercise. Furthermore, AMPK signaling was estimated to be 60-70% lower in alpha2-KO compared with WT muscles. In WG, AICAR treatment increased HKII, GLUT4, cytochrome c, COX-1, and CS, and the alpha2-KO abolished the AICAR-induced increases, whereas no AICAR responses were observed in RG. Exercise training increased GLUT4, HKII, COX-1, CS, and HAD protein in WG, but the alpha2-KO did not affect training-induced increases. Furthermore, AMPKalpha1, -alpha2, -beta1, -beta2, and -gamma3 subunits were reduced in RG, but not in WG, by 30-60% in response to exercise training. In conclusion, the alpha2-KO was associated with an approximately 20% reduction in mitochondrial markers in both muscle types and abolished AICAR-induced increases in protein expression in WG. However, the alpha2-KO did not reduce training-induced increases in HKII, GLUT4, COX-1, HAD, or CS protein in WG, suggesting that AMPKalpha2 may not be essential for metabolic adaptations of skeletal muscles to exercise training.     

Resveratrol regulates muscle fiber type conversion via miR-22-3p and AMPK/SIRT1/PGC-1α pathway

This study investigated the effects of resveratrol and miR-22-3p on muscle fiber type conversion in mouse C2C12 myotubes. Here we showed that resveratrol significantly increased the protein level of slow myosin heavy chain (MyHC) and the activities of succinic dehydrogenase and malate dehydrogenase, as well as markedly decreased the protein level of fast MyHC and the activity of lactate dehydrogenase. Immunofluorescence staining showed that resveratrol remarkably upregulated the number of slow MyHC-positive myotubes and downregulated the number of fast MyHC-positive myotubes, suggesting that resveratrol promoted muscle fiber type conversion from fast-twitch to slow-twitch in C2C12 myotubes. We also showed that miR-22-3p had an opposite function on muscle fiber type conversion and resveratrol was able to repress the expression of miR-22-3p. Furthermore, AMP-activated protein kinase (AMPK) inhibitor Compound C and miR-22-3p mimics could attenuate and eliminate muscle fiber type conversion from fast-twitch to slow-twitch cause by resveratrol, respectively. Together, we provided the first evidence that resveratrol promotes muscle fiber type conversion from fast-twitch to slow-twitch via miR-22-3p and AMPK/SIRT1/PGC-1α pathway in C2C12 myotubes.     

Adaptation of rat skeletal muscle to creatine depletion: AMP deaminase and AMP deamination.

AMP deaminase catalyzes deamination of the AMP formed in contracting muscles to inosine 5'-monophosphate (IMP). Slow-twitch muscle has only approximately 30% as high a level of AMP deaminase activity as fast-twitch muscle in the rat, and rates of IMP formation during intense contractile activity are much lower in slow-twitch muscle. We found that feeding the creatine analogue beta-guanidinopropionic acid (beta-GPA) to rats, which results in creatine depletion, causes a large decrease in muscle AMP deaminase. This adaptation was used to evaluate the role of AMP deaminase activity level in accounting for differences in IMP production in slow-twitch and fast-twitch muscles. beta-GPA feeding for 3 wk lowered AMP deaminase activity in fast-twitch epitrochlearis muscle to a level similar to that found in the normal slow-twitch soleus muscle but had no effect on the magnitude of the increase in IMP in response to intense contractile activity. Despite a similar decrease in ATP in the normal soleus and the epitrochlearis from beta-GPA-fed rats, the increase in IMP was only approximately 30% as great in the soleus in response to intense contractile activity. These results demonstrate that the accumulation of less IMP in slow- compared with fast-twitch skeletal muscle during contractile activity is not due to the lower level of AMP deaminase in slow-twitch muscle.     

Flutamide restores cardiac function after trauma-hemorrhage via an estrogen-dependent pathway through upregulation of PGC-1

Although previous studies have shown that flutamide improves cardiovascular function after trauma-hemorrhage, the mechanisms responsible for the salutary effect remain unknown. We hypothesized that flutamide mediates its beneficial effects via an estrogen-dependent pathway through upregulation of peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC-1). PGC-1, a key regulator of cardiac mitochondrial ATP production, induces mitochondrial DNA (mtDNA)-encoded genes such as cytochrome-c oxidase (COX) subunit I, II, and III (COX I, COX II, and COX III), which regulates mitochondrial oxidative phosphorylation. To test this hypothesis, male rats underwent trauma-hemorrhage (mean arterial pressure of 35-40 mmHg for approximately 90 min) followed by resuscitation. At the onset of resuscitation, rats received vehicle, flutamide (25 mg/kg body wt), flutamide in combination with estrogen receptor (ER) antagonist ICI-182,780 (3 mg/kg body wt), or ICI-182,780 alone. Flutamide administration after trauma-hemorrhage restored the depressed cardiac function and increased cardiac testosterone, estrogen levels, and aromatase activity. These increases were accompanied by normalized cardiac ER-alpha and ER-beta protein levels, PGC-1, and COX I mRNA expression, mitochondrial COX activity, and ATP contents. However, cardiac dihydrotestosterone, 5alpha-reductase II, androgen receptor protein levels, and mtDNA-encoded genes COX II and COX III were unaffected by flutamide treatment. The flutamide-mediated restoration of cardiac function, the increases in aromatase activity and estrogen levels, ER-alpha, ER-beta, PGC-1, COX I, COX activity, and ATP contents were, however, abolished when ER antagonist ICI-182,780 was administrated along with flutamide. These findings suggest that the salutary effect of flutamide on cardiac function after trauma-hemorrhage is mediated via an estrogen-dependent pathway through upregulation of PGC-1.     

Examination of the calcium-modulated protein S100 alpha and its target proteins in adult and developing skeletal muscle.

In this study radioimmunoassay, immunohistochemistry, Northern blot analysis, and a gel overlay technique have been used to examine the level, subcellular distribution, and potential target proteins of the S100 family of calcium-modulated proteins in adult and developing rat skeletal muscles. Adult rat muscles contained high levels of S100 proteins but the particular form present was dependent on the muscle type: cardiac muscle contained exclusively S100 alpha, slow-twitch skeletal muscle fibers contained predominantly S100 alpha, vascular smooth muscle contained both S100 alpha and S100 beta, and fast-twitch skeletal muscle fibers contained low but detectable levels of S100 alpha and S100 beta. While the distribution of S100 mRNAs paralled the protein distribution in all muscles there was no direct correlation between the mRNA and protein levels in different muscle types, suggesting that S100 protein expression is differentially regulated in different muscle types. Immunohistochemical analysis of the cellular distribution of S100 proteins in adult skeletal muscles revealed that S100 alpha staining was associated with muscle cells, while S100 beta staining was associated with nonmuscle cells. Radioimmunoassays of developing rat skeletal muscles demonstrated that all developing muscles contained low levels of S100 alpha at postnatal day 1 and that as development proceeded the S100 alpha levels increased. In contrast to adult muscle S100 alpha expression was confined to fast-twitch fibers in developing skeletal muscle until postnatal day 21. At postnatal day 1, developing contractile elements were S100 alpha positive, but no staining periodicity was detectable. At postnatal day 21, S100 alpha exhibited the same subcellular localization as seen in the adult: colocalization with the A-band and/or longitudinal sarcoplasmic reticulum. Comparison of the S100 alpha-binding protein profiles in fast- and slow-twitch fibers of various species revealed few, if any, species- or fiber type-specific S100 binding proteins. Isolated sarcoplasmic reticulum fractions and myofibrils contained multiple S100 alpha-binding proteins. The colocalization of S100 alpha and S100 alpha-binding proteins with the contractile apparatus and sarcoplasmic reticulum suggest that S100 alpha may regulate excitation and/or contraction in slow-twitch fibers.     

Endurance training increases skeletal muscle LKB1 and PGC-1alpha protein abundance: effects of time and intensity

Recent research suggests that LKB1 is the major AMP-activated protein kinase kinase (AMPKK). Peroxisome-proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) is a master coordinator of mitochondrial biogenesis. Previously we reported that skeletal muscle LKB1 protein increases with endurance training. The purpose of this study was to determine whether training-induced increases in skeletal muscle LKB1 and PGC-1alpha protein exhibit a time course and intensity-dependent response similar to that of citrate synthase. Male Sprague-Dawley rats completed endurance- and interval-training protocols. For endurance training, rats trained for 4, 11, 25, or 53 days. Interval-training rats trained identically to endurance-trained rats, except that after 25 days interval training was combined with endurance training. Time course data were collected from endurance-trained red quadriceps (RQ) after each time point. Interval training data were collected from soleus, RQ, and white quadriceps (WQ) muscle after 53 days only. Mouse protein 25 (MO25) and PGC-1alpha protein increased significantly after 4 days. Increased citrate synthase activity, increased LKB1 protein, and decreased AMPKK activity were found after 11 days. Maximal increases occurred after 4 days for hexokinase II, 25 days for MO25, and 53 days for citrate synthase, LKB1, and PGC-1alpha. In WQ, but not RQ or soleus, interval training had an additive effect to endurance training and induced significant increases in all proteins measured. These results demonstrate that LKB1 and PGC-1alpha protein abundances increase with endurance and interval training similarly to citrate synthase. The increase in LKB1 and PGC-1alpha with endurance and interval training may function to maintain the training-induced increases in mitochondrial mass.     

Statin-Induced Increases in Atrophy Gene Expression Occur Independently of Changes in PGC1α Protein and Mitochondrial Content

One serious side effect of statin drugs is skeletal muscle myopathy. Although the mechanism(s) responsible for statin myopathy remains to be fully determined, an increase in muscle atrophy gene expression and changes in mitochondrial content and/or function have been proposed to play a role. In this study, we examined the relationship between statin-induced expression of muscle atrophy genes, regulators of mitochondrial biogenesis, and markers of mitochondrial content in slow- (ST) and fast-twitch (FT) rat skeletal muscles. Male Sprague Dawley rats were treated with simvastatin (60 or 80 mg·kg^-1·day^-1) or vehicle control via oral gavage for 14 days. In the absence of overt muscle damage, simvastatin treatment induced an increase in atrogin-1, MuRF1 and myostatin mRNA expression; however, these were not associated with changes in peroxisome proliferator gamma co-activator 1 alpha (PGC-1α) protein or markers of mitochondrial content. Simvastatin did, however, increase neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and AMPK α-subunit protein expression, and tended to increase total NOS activity, in FT but not ST muscles. Furthermore, simvastatin induced a decrease in β-hydroxyacyl CoA dehydrogenase (β-HAD) activity only in FT muscles. These findings suggest that the statin-induced activation of muscle atrophy genes occurs independent of changes in PGC-1α protein and mitochondrial content. Moreover, muscle-specific increases in NOS expression and possibly NO production, and decreases in fatty acid oxidation, could contribute to the previously reported development of overt statin-induced muscle damage in FT muscles.