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.     

Effects of Resveratrol and SIRT1 on PGC-1α Activity and Mitochondrial Biogenesis: A Reevaluation

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.     

NAMPT regulates mitochondria biogenesis via NAD metabolism and calcium binding proteins during skeletal muscle contraction

[Purpose]

The purpose of this study was to investigate the effect that muscle contraction induced NAD metabolism via NAMPT has on mitochondrial biogenesis.

[Methods]

Primary skeletal muscle cells were isolated from the gastrocnemius in C57BL/6 mice. The muscle cells were stimulated by electrical current at 1Hz for 3 minutes in conditions of normal or NAD metabolism related inhibitor treatment. NAD/NADH level, Sirt1 and mitochondria biogenesis related signal factor’s changes were examined in normal or NAD metabolism related inhibitor treated cells.

[Results]

Electrical stimulation (ES) induced muscle contractions significantly increased NAD/NADH levels, NAMPT inhibitor FK-866 inhibited ES-induced NAD formation, which caused SIRT1 expression and PGC-1α deacetylation to decrease. Moreover, NAMPT inhibition decreased mitochondrial biogenesis related mRNA, COX-1 and Tfam levels. Along with AMPK inhibitor, compound C decreases SIRT1 expression, PGC-1α deacetylation and muscle contraction induced mitochondrial biogenesis related mRNA increment. These results indicated that the AMPK-NAMPT signal is a key player for muscle contraction induced SIRT1 expression and PGC-1α deacetylation, which influences mitochondrial biogenesis. Inhibition of the AMPK upregulator, Camkkβ, STO-609 decreased AMPK phosphorylation and SIRT1 expression but did not decrease PGC-1α deacetylation. However, CAMKII inhibition via AIP decreased PGC-1α deacetylation.

[Conclusion]

In conclusion, the results indicate that NAMPT plays an important role in NAD metabolism and mitochondrial biogenesis. However, mitochondrial biogenesis is also controlled by different calcium binding protein signals including Camkkβ and CAMKII. [Keyword] Muscle contraction, NAD metabolism, SIRT1, PGC-1 α, mitochondria biogenesis.     

TRPV1 activation improves exercise endurance and energy metabolism through PGC-1α upregulation in mice

Impaired aerobic exercise capacity and skeletal muscle dysfunction are associated with cardiometabolic diseases. Acute administration of capsaicin enhances exercise endurance in rodents, but the long-term effect of dietary capsaicin is unknown. The capsaicin receptor, the transient receptor potential vanilloid 1 (TRPV1) cation channel has been detected in skeletal muscle, the role of which remains unclear. Here we report the function of TRPV1 in cultured C2C12 myocytes and the effect of TRPV1 activation by dietary capsaicin on energy metabolism and exercise endurance of skeletal muscles in mice. In vitro, capsaicin increased cytosolic free calcium and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression in C2C12 myotubes through activating TRPV1. In vivo, PGC-1α in skeletal muscle was upregulated by capsaicin-induced TRPV1 activation or genetic overexpression of TRPV1 in mice. TRPV1 activation increased the expression of genes involved in fatty acid oxidation and mitochondrial respiration, promoted mitochondrial biogenesis, increased oxidative fibers, enhanced exercise endurance and prevented high-fat diet-induced metabolic disorders. Importantly, these effects of capsaicin were absent in TRPV1-deficient mice. We conclude that TRPV1 activation by dietary capsaicin improves energy metabolism and exercise endurance by upregulating PGC-1α in skeletal muscles. The present results indicate a novel therapeutic strategy for managing metabolic diseases and improving exercise endurance.     

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.     

AMP-activated protein kinase plays an important evolutionary conserved role in the regulation of glucose metabolism in fish skeletal muscle cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.     

PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle

The pathophysiology underlying mitochondrial dysfunction in insulin-resistant skeletal muscle is incompletely characterized. To further delineate this we investigated the interaction between insulin signaling, mitochondrial regulation, and function in C2C12 myotubes and in skeletal muscle. In myotubes elevated insulin and glucose disrupt insulin signaling, mitochondrial biogenesis, and mitochondrial bioenergetics. The insulin-sensitizing thiazolidinedione pioglitazone restores these perturbations in parallel with induction of the mitochondrial biogenesis regulator PGC-1alpha. Overexpression of PGC-1alpha rescues insulin signaling and mitochondrial bioenergetics, and its silencing concordantly disrupts insulin signaling and mitochondrial bioenergetics. In primary skeletal myoblasts pioglitazone also up-regulates PGC-1alpha expression and restores the insulin-resistant mitochondrial bioenergetic profile. In parallel, pioglitazone up-regulates PGC-1alpha in db/db mouse skeletal muscle. Interestingly, the small interfering RNA knockdown of the insulin receptor in C2C12 myotubes down-regulates PGC-1alpha and attenuates mitochondrial bioenergetics. Concordantly, mitochondrial bioenergetics are blunted in insulin receptor knock-out mouse-derived skeletal myoblasts. Taken together these data demonstrate that elevated glucose and insulin impairs and pioglitazone restores skeletal myotube insulin signaling, mitochondrial regulation, and bioenergetics. Pioglitazone functions in part via the induction of PGC-1alpha. Moreover, PGC-1alpha is identified as a bidirectional regulatory link integrating insulin-signaling and mitochondrial homeostasis in skeletal muscle.     

Sirtuin 3, a New Target of PGC-1α, Plays an Important Role in the Suppression of ROS and Mitochondrial Biogenesis

Background

Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1α induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.

Results

Here we show that PGC-1α strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1α led to decreased Sirt3 gene expression. PGC-1α activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (−407/−399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRα bound to the identified ERRE and PGC-1α co-localized with ERRα in the mSirt3 promoter. Knockdown of ERRα reduced the induction of Sirt3 by PGC-1α in C₂C₁₂ myotubes. Furthermore, Sirt3 was essential for PGC-1α-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1α in C₂C₁₂ myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1α on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1α on mitochondrial biogenesis in C₂C₁₂ myotubes.

Conclusion

Our results indicate that Sirt3 functions as a downstream target gene of PGC-1α and mediates the PGC-1α effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease.     

An acute bout of high-intensity interval training increases the nuclear abundance of PGC-1α and activates mitochondrial biogenesis in human skeletal muscle

Low-volume, high-intensity interval training (HIT) increases skeletal muscle mitochondrial capacity, yet little is known regarding potential mechanisms promoting this adaptive response. Our purpose was to examine molecular processes involved in mitochondrial biogenesis in human skeletal muscle in response to an acute bout of HIT. Eight healthy men performed 4 × 30-s bursts of all-out maximal intensity cycling interspersed with 4 min of rest. Muscle biopsy samples (vastus lateralis) were obtained immediately before and after exercise, and after 3 and 24 h of recovery. At rest, the majority of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis, was detected in cytosolic fractions. Exercise activated p38 MAPK and AMPK in the cytosol. Nuclear PGC-1α protein increased 3 h into recovery from exercise, a time point that coincided with increased mRNA expression of mitochondrial genes. This was followed by an increase in mitochondrial protein content and enzyme activity after 24 h of recovery. These findings support the hypothesis that an acute bout of low-volume HIT activates mitochondrial biogenesis through a mechanism involving increased nuclear abundance of PGC-1α.     

Dynamic mobilization of PGC-1α mediates mitochondrial biogenesis for the protection of RGC-5 cells by resveratrol during serum deprivation

Mitochondrial dysfunction contributing to the pathogenesis of glaucomatous neurodegeneration has stimulated considerable interest recently. In this study, we explored the role of peroxisome proliferator activated receptor-γ co-activator 1α (PGC-1α) in resveratrol-triggered mitochondrial biogenesis for preventing apoptosis in a retinal ganglion cell line RGC-5. Our results showed that serum deprivation induced cell apoptosis in a time-dependent manner. Applying resveratrol maintained the normal mitochondrial membrane potential, decreased the levels of both total and cleaved caspase-3, and inhibited the release of cytochrome c, which subsequently enhanced cell survival. Moreover, resveratrol stimulated mitochondrial biogenesis by increasing the absolute quantity of mitochondria as well as their DNA copies. Treatment with resveratrol promoted the protein expression of SIRT1, but not PGC-1α; instead, resveratrol facilitated PGC-1α translocation from the cytoplasm to the nucleus and up-regulated NRF1 and TFAM, which were blocked by nicotinamide. Collectively, we demonstrate that the SIRT1-dependent PGC-1α subcellular translocation following resveratrol application potentially attenuates serum deprivation-elicited RGC-5 cell death, thereby raising the possibility of mitigating glaucomatous retinopathy by enhancement of mitochondrial biogenesis.     

Effect of exercise intensity and AICAR on isoform-specific expressions of murine skeletal muscle PGC-1α mRNA: a role of β₂-adrenergic receptor activation

There are three isoforms of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) mRNA, which promotes mitochondrial biogenesis in skeletal muscles. Compared with PGC-1α-a mRNA, PGC-1α-b or PGC-1α-c mRNA is transcribed by a different exon 1 of the PGC-1α gene. In this study, effects of exercise intensity and 5-aminoimidazole-4-carboxamide-1β-d-ribofuranoside (AICAR) on isoform-specific expressions of PGC-1α were investigated. All isoforms were increased in proportion to exercise intensity of treadmill running (10-30 m/min for 30 min). Preinjection of β?-adrenergic receptor (AR) antagonist (ICI 118551) inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs, but not the increase in PGC-1α-a mRNA, in response to high-intensity exercise. Although high-intensity exercise activated α2-AMP-activated protein kinase (α2-AMPK) in skeletal muscles, inactivation of α2-AMPK activity did not affect high-intensity exercise-induced mRNA expression of all PGC-1α isoforms, suggesting that activation of α2-AMPK is not mandatory for an increase in PGC-1α mRNA by high-intensity exercise. A single injection in mice of AICAR, an AMPK activator, increased mRNAs of all PGC-1α isoforms. AICAR increased blood catecholamine concentrations, and preinjection of β?-AR antagonist inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs but not the increase in PGC-1α-a mRNA. Direct exposure of epitrochlearis muscle to AICAR increased PGC-1α-a but not the -b isoform. These data indicate that exercise-induced PGC-1α expression was dependent on the intensity of exercise. Exercise or AICAR injection increased PGC-1α-b and PGC-1α-c mRNAs via β?-AR activation, whereas high-intensity exercise increased PGC-1α-a expression by a multiple mechanism in which α2-AMPK is one of the signaling pathways.     

Inverse Regulation of the Cytosolic Ca2+ Buffer Parvalbumin and Mitochondrial Volume in Muscle Cells via SIRT1/PGC-1α Axis

Skeletal muscles show a high plasticity to cope with various physiological demands. Different muscle types can be distinguished by the force, endurance, contraction/relaxation kinetics (fast-twitch vs. slow-twitch muscles), oxidative/glycolytic capacity, and also with respect to Ca²⁺-signaling components. Changes in Ca²⁺ signaling and associated Ca²⁺-dependent processes are thought to underlie the high adaptive capacity of muscle fibers. Here we investigated the consequences and the involved mechanisms caused by the ectopic expression of the Ca²⁺-binding protein parvalbumin (PV) in C2C12 myotubes in vitro, and conversely, the effects caused by its absence in in fast-twitch muscles of parvalbumin null-mutant (PV−/−) mice in vivo. The absence of PV in fast-twitch muscle tibialis anterior (TA) resulted in an increase in the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and of its positive regulator, the deacetylase sirtuin 1 (SIRT1). TA muscles from PV−/− mice also have an increased mitochondrial volume. Mild ionophore treatment of control (PV-devoid) C2C12 myotubes causing a moderate elevation in [Ca²⁺]_c resulted in an increase in mitochondrial volume, together with elevated PGC-1α and SIRT1 expression levels, whilst it increased PV expression levels in myotubes stably transfected with PV. In PV-expressing myotubes the mitochondrial volume, PGC-1α and SIRT1 were significantly lower than in control C2C12 myotubes already at basal conditions and application of ionophore had no effect on either one. SIRT1 activation causes a down-regulation of PV in transfected myotubes, whilst SIRT1 inhibition has the opposite effect. We conclude that PV expression and mitochondrial volume in muscle cells are inversely regulated via a SIRT1/PGC-1α signaling axis.     

Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts

Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.