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.     

Transcutaneous Carbon Dioxide Induces Mitochondrial Apoptosis and Suppresses Metastasis of Oral Squamous Cell Carcinoma In Vivo

Squamous cell carcinoma (SCC) is the main histological type of oral cancer. Its growth rate and incidence of metastasis to regional lymph nodes is influenced by various factors, including hypoxic conditions. We have previously reported that transcutaneous CO₂ induces mitochondrial apoptosis and decreases lung metastasis by reoxygenating sarcoma cells. However, previous studies have not determined the sequential mechanism by which transcutaneous CO₂ suppresses growth of epithelial tumors, including SCCs. Moreover, there is no report that transcutaneous CO₂ suppresses lymphogenous metastasis using human cell lines xenografts. In this study, we examined the effects of transcutaneous CO₂ on cancer apoptosis and lymphogenous metastasis using human SCC xenografts. Our results showed that transcutaneous CO₂ affects expressions of PGC-1α and TFAM and protein levels of cleavage products of caspase-3, caspase-9 and PARP, which relatives mitochondrial apoptosis. They also showed that transcutaneous CO₂ significantly inhibits SCC tumor growth and affects expressions of HIF-1α, VEGF, MMP-2 and MMP-9, which play essential roles in tumor angiogenesis, invasion and metastasis. In conclusion, transcutaneous CO₂ suppressed tumor growth, increased mitochondrial apoptosis and decreased the number of lymph node metastasis in human SCC by decreasing intra-tumoral hypoxia and suppressing metastatic potential with no observable effect in vivo. Our findings indicate that transcutaneous CO₂ could be a novel therapeutic tool for treating human SCC.     

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.     

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.     

Activation of ITLN-1 attenuates oxidative stress injury via activating SIRT1/PGC1-α signaling in neuroblastoma cells

Cerebral injury is closely associated with enhanced oxidative stress. A newly discovered secretory adipocytokine, intelectin-1 (ITLN-1), has been shown to have beneficial effects in neuroprotection in epidemiological studies. However, the specific molecular mechanism of ITLN-1 in protecting against cerebral oxidative stress needs further investigation. In this study, we hypothesize that ITLN-1 plays a protective role against oxidative stress injury through the SIRT1/PGC1-α signaling pathway in neuromatocytes. We used hydrogen peroxide (H₂O₂) as a oxidative stress model to simulate oxidative stress injury. Then, small interfering RNAs (siRNAs) was used to knock down SIRT1 in N2a cells with or without ITLN overexpression, followed by H₂O₂-induced injury. We observed that H₂O₂ injury significantly decreased the levels of ITLN-1, SIRT1, and PGC-1α. However, ITLN overexpression reversed H₂O₂-induced decline in cell viability and rise in apoptosis and intracellular ROS levels in N2a cells, while ITLN siRNA worsened the neurocyte injury. Furthermore, SIRT1 knockdown reversed the positive effect of ITLN overexpression on oxidative stress injury in N2a cells. Taken together, these findings suggest that ITLN-1 exerts neuroprotective effects against oxidative stress injury primarily through the SIRT1/PGC-1α axis.     

Nutrient excess and altered mitochondrial proteome and function contribute to neurodegeneration in diabetes

Diabetic neuropathy is a major complication of diabetes that results in the progressive deterioration of the sensory nervous system. Mitochondrial dysfunction has been proposed to play an important role in the pathogenesis of the neurodegeneration observed in diabetic neuropathy. Our recent work has shown that mitochondrial dysfunction occurs in dorsal root ganglia (DRG) sensory neurons in streptozotocin (STZ) induced diabetic rodents. In neurons, the nutrient excess associated with prolonged diabetes may trigger a switching off of AMP kinase (AMPK) and/or silent information regulator T1 (SIRT1) signaling leading to impaired peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α) expression/activity and diminished mitochondrial activity. This review briefly summarizes the alterations of mitochondrial function and proteome in sensory neurons of STZ-diabetic rodents. We also discuss the possible involvement of AMPK/SIRT/PGC-1α pathway in other diabetic models and different tissues affected by diabetes.     

Chronic AMPK stimulation attenuates adaptive signaling in dystrophic skeletal muscle

In the present study, we evaluated how a pharmacologically induced phenotype shift in dystrophic skeletal muscle would affect subsequent intracellular signaling in response to a complementary, adaptive physiological stimulus. mdx mice were treated with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; 500 mg·kg(-1)·day(-1)) for 30 days, and then one-half of the animals were subjected to a bout of treadmill running to induce acute AMPK and p38 MAPK signaling. The mRNA levels of phenotypic modifiers, including peroxisome proliferator-activated receptor-δ (PPARδ), PPARγ coactivator-1α (PGC-1α), receptor interacting protein 140 (RIP 140), and silent information regulator two ortholog 1 (SIRT1) were assessed in skeletal muscle, as well as the expression of the protein arginine methyltransferase genes PRMT1 and CARM1. We found unique AMPK and p38 phosphorylation and expression signatures between dystrophic and healthy muscle. In dystrophic skeletal muscle, treadmill running induced PPARδ, PGC-1α, and SIRT1 mRNAs, three molecules that promote the slow, oxidative myogenic program. In the mdx animals that received the chronic AICAR treatment, running-elicited AMPK and p38 phosphorylation was attenuated compared with vehicle-treated mice. Similarly, acute stress-evoked expression of PPARδ, PGC-1α, and SIRT1 was also blunted by chronic pharmacological AMPK stimulation. Skeletal muscle PRMT1 and CARM1 protein contents were higher in mdx mice compared with wild-type littermates. The acute running-evoked induction of PRMT1 and CARM1 mRNAs was also attenuated by the AICAR treatment. Our data demonstrate that prior pharmacological conditioning is a salient determinant in how dystrophic muscle adapts to subsequent complementary, acute physiological stress stimuli. These results provide insight into possible therapeutic applications of synthetic agonists in neuromuscular diseases, such as during chronic administration to Duchenne muscular dystrophy patients.     

The effect of SIRT1 protein knock down on PGC-1α acetylation during skeletal muscle contraction

[Purpose]

The purpose of this study was to investigate the effect of Sirtuin 1 (SIRT1) and General control nonderepressible 5 (GCN5) knock down on peroxisome proliferator- activated receptor gamma coactivator 1-alpha (PGC-1α) deacetylation during electrical stimulated skeletal muscle contraction.

[Methods]

Skeletal muscle primary cell were isolated from C57BL/6 mice gastrocnemius and transfected lentiviral SIRT1 and GCN5 shRNA. Knock downed muscle cell were stimulated by electrical stimulation (1Hz, 3min) and collected for PGC-1α deceatylation assays. Immunoprecipitation performed for PGC-1α deacetylation, acetyl-lysine level was measured.

[Results]

Our resulted showed SIRT1 knock down not influenced to PGC-1α deacetylation during electrical stimulation induced muscle contraction while GCN5 knock down decreased PGC-1α deacetylation significantly (p<0.05).

[Conclusion]

This study can be concluded that GCN5 is a critical factor for muscle contraction induced PGC-1α deacetylation.     

Kinsenoside-mediated lipolysis through an AMPK-dependent pathway in C3H10T1/2 adipocytes: Roles of AMPK and PPARα in the lipolytic effect of kinsenoside

BackgroundCurrently, more than one-third of the global population is overweight or obese, which is a risk factor for major causes of death including cardiovascular disease, numerous cancers, and diabetes. Kinsenoside, a major active component of Anoectochilus formosanus exhibits antihyperglycemic, antihyperliposis, and hepatoprotective effects and can be used to prevent and manage obesity.PurposeThis study examined the catabolic effects of kinsenoside on lipolysis in adipocytes transformed from C3H10T1/2 cells.Study design/methodsThe lipolytic effect of kinsenoside in C3H10T1/2 adipocytes was evaluated by oil-red O staining and glycerol production. The underlying mechanisms were assessed by Western blots, chromatin immunoprecipitation (IP), Co-IP, EMSA and siRNAs verification.ResultsWe demonstrated that kinsenoside increased both adipose triglyceride lipase (ATGL)-mediated lipolysis, which was upregulated by AMP-activated protein kinase (AMPK) activation, and the hydrolysis of triglycerides to glycerol and fatty acids that require transportation into mitochondria for further β-oxidation. We also demonstrated that kinsenoside increased the phosphorylation of peroxisome proliferator-activated receptor alpha (PPARα) and CRE-binding protein (CREB), and the protein levels of silent information regulator T1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and carnitine palmitoyltransferase I (CPT1) through an AMPK-dependent mechanism. SIRT1 deacetylated PGC-1α, facilitating AMPK-mediated PGC-1α phosphorylation and increasing the interaction of PPARα with its coactivator, PGC-1α. This interaction elevated the expression of CPT1, a shuttle for the mitochondrial transport of fatty acids, in kinsenoside-treated cells. In addition, AMPK-phosphorylation-mediated CREB activation caused kinsenoside-mediated PGC-1α upregulation.ConclusionAMPK activation not only elevated ATGL expression for lipolysis but also induced CPT1 expression for further mitochondrial translocation of fatty acids. The results suggested that the mechanism underlying the catabolic effects of kinsenoside on lipolysis and increased CPT1 induction was mediated through an AMPK-dependent pathway.     

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.     

Skeletal muscle-specific expression of PGC-1α-b, an exercise-responsive isoform, increases exercise capacity and peak oxygen uptake

Background

Maximal oxygen uptake (VO_2max) predicts mortality and is associated with endurance performance. Trained subjects have a high VO_2max due to a high cardiac output and high metabolic capacity of skeletal muscles. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, a fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training increases PGC-1α in skeletal muscle, PGC-1α-mediated changes may contribute to the improvement of exercise capacity and VO_2max. There are three isoforms of PGC-1α mRNA. PGC-1α-b protein, whose amino terminus is different from PGC-1α-a protein, is a predominant PGC-1α isoform in response to exercise. We investigated whether alterations of skeletal muscle metabolism by overexpression of PGC-1α-b in skeletal muscle, but not heart, would increase VO_2max and exercise capacity.

Methodology/Principal Findings

Transgenic mice showed overexpression of PGC-1α-b protein in skeletal muscle but not in heart. Overexpression of PGC-1α-b promoted mitochondrial biogenesis 4-fold, increased the expression of fatty acid transporters, enhanced angiogenesis in skeletal muscle 1.4 to 2.7-fold, and promoted exercise capacity (expressed by maximum speed) by 35% and peak oxygen uptake by 20%. Across a broad range of either the absolute exercise intensity, or the same relative exercise intensities, lipid oxidation was always higher in the transgenic mice than wild-type littermates, suggesting that lipid is the predominant fuel source for exercise in the transgenic mice. However, muscle glycogen usage during exercise was absent in the transgenic mice.

Conclusions/Significance

Increased mitochondrial biogenesis, capillaries, and fatty acid transporters in skeletal muscles may contribute to improved exercise capacity via an increase in fatty acid utilization. Increases in PGC-1α-b protein or function might be a useful strategy for sedentary subjects to perform exercise efficiently, which would lead to prevention of life-style related diseases and increased lifespan.     

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.     

Effect of Dietary Macronutrient Composition on AMPK and SIRT1 Expression and Activity in Human Skeletal Muscle

Adenosine monophosphate-activated protein kinase (AMPK), silent mating type information regulation 2 homologue 1 (SIRT 1), and peroxisome proliferator-activated receptor γ co-activator α (PGC1α) constitute an energy sensing cellular network that controls mitochondrial biogenesis. Caloric restriction activates both AMPK and SIRT-1 to increase ATP production from fat oxidation. We characterized AMPK and SIRT 1 expression and activity in human skeletal muscle in response to dietary fat or carbohydrate intake on the background of either overfeeding or caloric restriction. AMPK phosphorylation and acetylation of PGC1α (as a measure of SIRT activity) were determined. Euglycemic-hyperinsulinemic clamp and muscle biopsies were performed in human subjects participating in 2 separate studies. In study 1, 21 lean healthy individuals were overfed for 5 days, while in study 2, 18 obese otherwise healthy individuals consumed a calorie-restricted diet for 5 days. Under both conditions - overfeeding and caloric restriction - high fat/low carbohydrate (HF/LC) diet significantly increased phosphorylation of AMPK and deacetylation of PGC1α in skeletal muscle without affecting total amounts of AMPK, PGC1α, or SIRT 1. In contrast, low fat/high carbohydrate (LF/HC) hypocaloric diet reduced phosphorylation of AMPK and deacetylation of PGC1α. Our data indicate that a relative deficiency in carbohydrate intake or, albeit less likely, a relative excess of fat intake even in the absence of caloric deprivation is sufficient to activate the AMPK-SIRT 1-PGC1α energy-sensing cellular network in human skeletal muscle.     

Dual role of Zn in maintaining structural integrity and suppressing deacetylase activity of SIRT1

Zn²⁺ directly participates in catalysis of histone deacetylase (HDAC) Classes I, II, IV enzymes while its role in HDAC Class III activity is not well established. Herein we investigated the effects of Zn²⁺ on the deacetylase activity of sirtuin 1 (silent mating type information regulation 2 homolog 1, SIRT1). We found that the inherent Zn²⁺ at the zinc-finger motif of SIRT1 is essential for the structural integrity and the deacetylase activity of SIRT1, whereas the exogenous Zn²⁺ strongly inhibits the deacetylase activity with an IC₅₀ of 0.82 μM for Zn(Gly)₂. SIRT1 activity suppressed by the exogenous Zn²⁺ can be fully recovered by the metal chelator EDTA but not by the activator resveratrol. We also identified Zn²⁺ as a noncompetitive inhibitor for the substrates of NAD⁺ and the acetyl peptide P53-AMC. The 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence titration experiments and site-directed mutagenesis study suggested that the exogenous Zn²⁺ binds to SIRT1 but not at the zinc-finger motif. These results indicate that Zn²⁺ plays a dual role in SIRT1 activity. Inherent Zn²⁺ at the zinc-finger motif is structurally related and essential for SIRT1 activity. On the other hand, Zn²⁺ may also bind to another site different from the zinc-finger motif or the binding sites for the substrates or resveratrol and act as a potent inhibitor of SIRT1.