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.     

Alterations in mitochondrial dynamics with age‐related Sirtuin1/Sirtuin3 deficiency impair cardiomyocyte contractility

Sirtuin1 (SIRT1) and Sirtuin3 (SIRT3) protects cardiac function against ischemia/reperfusion (I/R) injury. Mitochondria are critical in response to myocardial I/R injury as disturbance of mitochondrial dynamics contributes to cardiac dysfunction. It is hypothesized that SIRT1 and SIRT3 are critical components to maintaining mitochondria homeostasis especially mitochondrial dynamics to exert cardioprotective actions under I/R stress. The results demonstrated that deficiency of SIRT1 and SIRT3 in aged (24–26 months) mice hearts led to the exacerbated cardiac dysfunction in terms of cardiac systolic dysfunction, cardiomyocytes contractile defection, and abnormal cardiomyocyte calcium flux during I/R stress. Moreover, the deletion of SIRT1 or SIRT3 in young (4–6 months) mice hearts impair cardiomyocyte contractility and shows aging‐like cardiac dysfunction upon I/R stress, indicating the crucial role of SIRT1 and SIRT3 in protecting myocardial contractility from I/R injury. The biochemical and seahorse analysis showed that the deficiency of SIRT1/SIRT3 leads to the inactivation of AMPK and alterations in mitochondrial oxidative phosphorylation (OXPHOS) that causes impaired mitochondrial respiration in response to I/R stress. Furthermore, the remodeling of the mitochondria network goes together with hypoxic stress, and mitochondria undergo the processes of fusion with the increasing elongated branches during hypoxia. The transmission electron microscope data showed that cardiac SIRT1/SIRT3 deficiency in aging alters mitochondrial morphology characterized by the impairment of mitochondria fusion under I/R stress. Thus, the age‐related deficiency of SIRT1/SIRT3 in the heart affects mitochondrial dynamics and respiration function that resulting in the impaired contractile function of cardiomyocytes in response to I/R.     

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.     

Resveratrol inhibition of inducible nitric oxide synthase in skeletal muscle involves AMPK but not SIRT1

The plant-derived polyphenol resveratrol (RSV) modulates life span and metabolism, and it is thought that these effects are largely mediated by activating the deacetylase enzyme SIRT1. However, RSV also activates the cell energy sensor AMP-activated protein kinase (AMPK). We have previously reported that AMPK activators inhibit inducible nitric oxide synthase (iNOS), a key proinflammatory mediator of insulin resistance in endotoxemia and obesity. The aim of this study was to evaluate whether RSV inhibits iNOS induction in insulin target tissues and to determine the role of SIRT1 and AMPK activation in this effect. We found that RSV (40 mg/kg ip) treatment decreased iNOS induction and NO production in skeletal muscle and white adipose tissue, but not in liver, of endotoxin (LPS)-challenged mice. This effect of the polyphenol was recapitulated in vitro, where RSV (10-80 μM) robustly inhibited iNOS protein induction and NO production in cytokine/LPS-treated L6 myocytes and 3T3-L1 adipocytes. However, no effect of RSV was observed on iNOS induction in FAO hepatocytes. Further studies using inhibitors of SIRT1 revealed that the deacetylase enzyme is not involved in RSV action on iNOS. In marked contrast, RSV activates AMPK in L6 myocytes, and blunting its activation using Compound C or RNA interference partly blocked the inhibitory effect of RSV on NO production. These results show that RSV specifically inhibits iNOS induction in muscle through a mechanism involving AMPK but not SIRT1 activation. This anti-inflammatory action of RSV likely contributes to the therapeutic effect of this plant polyphenol.     

Resveratrol,novel application by preconditioning to attenuate myocardial ischemia/reperfusion injury in mice through regulate AMPK pathway and autophagy level

Myocardial ischemia/reperfusion injury (MI/RI) is the main cause of deaths in the worldwide, leading to severe cardiac dysfunction. Resveratrol (RSV) is a polyphenol plant‐derived compound. Our study aimed to elucidate the underlying molecular mechanism of preconditioning RSV in protecting against MI/RI. Mice were ligated and re‐perfused by the left anterior descending branch with or without RSV (30 mg/kg·ip) for 7 days. Firstly, we found that RSV pretreatment significantly alleviated myocardial infarct size, improved cardiac function and decreased oxidative stress. Furthermore, RSV activated p‐AMPK and SIRT1, ameliorated inflammation including the level of TNF‐α and IL‐1β, and promoting autophagy level. Moreover, neonatal rat ventricular myocytes (NRVMs) and H9c2 cells with knockdown the expression of AMPK, SIRT1 or FOXO1 were used to uncover the underlying molecular mechanism for the cardio‐protection of RSV. In NRVMs, RSV increased cellular viability, decreased LDH release and reduced oxidative stress. Importantly, Compound C(CpC) and EX527 reversed the effect of RSV against MI/RI in vivo and in vitro and counteracted the autophagy level induced by RSV. Together, our study indicated that RSV could alleviate oxidative stress in cardiomyocytes through activating AMPK/SIRT1‐FOXO1 signallingpathway and enhanced autophagy level, thus presenting high potential protection on MI/RI.     

Two Weeks of Metformin Treatment Enhances Mitochondrial Respiration in Skeletal Muscle of AMPK Kinase Dead but Not Wild Type Mice

Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5′AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α₂ (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.     

Macrophage α1 AMP-activated Protein Kinase (α1AMPK) Antagonizes Fatty Acid-induced Inflammation through SIRT1

In this study, we aim to determine cellular mechanisms linking nutrient metabolism to the regulation of inflammation and insulin resistance. The nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 show striking similarities in nutrient sensing and regulation of metabolic pathways. We find that the expression, activity, and signaling of the major isoform α1AMPK in adipose tissue and macrophages are substantially down-regulated by inflammatory stimuli and in nutrient-rich conditions, such as exposure to lipopolysaccharide (LPS), free fatty acids (FFAs), and diet-induced obesity. Activating AMPK signaling in macrophages by 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside or constitutively active α1AMPK (CA-α1) significantly inhibits; although inhibiting α1AMPK by short hairpin RNA knock-down or dominant-negative α1AMPK (DN-α1) increases LPS- and FFA-induced tumor necrosis factor α expression. Chromatin immunoprecipitation and luciferase reporter assays show that activation of AMPK by CA-α1 in macrophages significantly inhibits LPS- or FFA-induced NF-κB signaling. More importantly, in a macrophage-adipocyte co-culture system, we find that inactivation of macrophage AMPK signaling inhibits adipocyte insulin signaling and glucose uptake. Activation of AMPK by CA-α1 increases the SIRT1 activator NAD⁺ content and SIRT1 expression in macrophages. Furthermore, α1AMPK activation mimics the effect of SIRT1 on deacetylating NF-κB, and the full capacity of AMPK to deacetylate NF-κB and inhibit its signaling requires SIRT1. In conclusion, AMPK negatively regulates lipid-induced inflammation, which acts through SIRT1, thereby contributing to the protection against obesity, inflammation, and insulin resistance. Our study defines a novel role for AMPK in bridging the signaling between nutrient metabolism and inflammation.     

Regulation of HSL serine phosphorylation in skeletal muscle and adipose tissue

Hormone-sensitive lipase (HSL) is important for the degradation of triacylglycerol in adipose and muscle tissue, but the tissue-specific regulation of this enzyme is not fully understood. We investigated the effects of adrenergic stimulation and AMPK activation in vitro and in circumstances where AMPK activity and catecholamines are physiologically elevated in humans in vivo (during physical exercise) on HSL activity and phosphorylation at Ser(563) and Ser(660), the PKA regulatory sites, and Ser(565), the AMPK regulatory site. In human experiments, skeletal muscle, subcutaneous adipose and venous blood samples were obtained before, at 15 and 90 min during, and 120 min after exercise. Skeletal muscle HSL activity was increased by approximately 80% at 15 min compared with rest and returned to resting rates at the cessation of and 120 min after exercise. Consistent with changes in plasma epinephrine, skeletal muscle HSL Ser(563) and Ser(660) phosphorylation were increased by 27% at 15 min (P < 0.05), remained elevated at 90 min, and returned to preexercise values postexercise. Skeletal muscle HSL Ser(565) phosphorylation and AMPK signaling were increased at 90 min during, and after, exercise. Phosphorylation of adipose tissue HSL paralleled changes in skeletal muscle in vivo, except HSL Ser(660) was elevated 80% in adipose compared with 35% in skeletal muscle during exercise. Studies in L6 myotubes and 3T3-L1 adipocytes revealed important tissue differences in the regulation of HSL. AMPK inhibited epinephrine-induced HSL activity in L6 myotubes and was associated with reduced HSL Ser(660) but not Ser(563) phosphorylation. HSL activity was reduced in L6 myotubes expressing constitutively active AMPK, confirming the inhibitory effects of AMPK on HSL activity. Conversely, in 3T3-L1 adipocytes, AMPK activation after epinephrine stimulation did not prevent HSL activity or glycerol release, which coincided with maintenance of HSL Ser(660) phosphorylation. Taken together, these data indicate that HSL activity is maintained in the face of AMPK activation as a result of elevated HSL Ser(660) phosphorylation in adipose tissue but not skeletal muscle.     

Whole Body Deletion of AMP-activated Protein Kinase β2 Reduces Muscle AMPK Activity and Exercise Capacity

AMP-activated protein kinase (AMPK) β subunits (β1 and β2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK β2 (β2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that β2 KO mice are viable and breed normally. β2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the β1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in β2 KO mice. During treadmill running β2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of β2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet β2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK β2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.     

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.     

Calorie restriction in overweight males ameliorates obesity-related metabolic alterations and cellular adaptations through anti-aging effects,possibly including AMPK and SIRT1 activation

Background

Calorie restriction (CR) is accepted as an experimental anti-aging paradigm. Several important signal transduction pathways including AMPK and SIRT1 are implicated in the regulation of physiological processes of CR. However, the mechanisms responsible for adaptations remain unclear in humans.

Scope of review

Four overweight male participants were enrolled and treated with 25% CR of their baseline energy requirements for 7 weeks. Characteristics, including body weight (BW), body mass index (BMI), %fat, visceral fat area (VFA), mean blood pressure (MBP) and VO₂ max, as well as metabolic parameters, such as insulin, lipid profiles and inflammatory makers and the expression of phosphorylated AMPK and SIRT1 in peripheral blood mononuclear cells (PBMNCs), were determined at baseline and then after 7 weeks. In addition, we assessed the effects of the serum collected from the participants on AMPK and SIRT1 activation and mitochondrial biogenesis in cultured human skeletal muscle cells.

Major conclusions

After CR, BW, BMI, %fat, VFA and MBP all significantly decreased, while VO₂ max increased, compared to those at baseline. The levels of fasting insulin, free fatty acid, and inflammatory makers, such as interleukin-6 and visfatin, were significantly reduced, whereas the expression of phosphorylated AMPK and SIRT1 was significantly increased in PBMNCs collected after CR, compared to those at baseline. The skeletal muscle cells that were cultured in serum collected after CR showed an increase in AMPK and SIRT1 activity as well as mitochondrial biogenesis.

General significance

CR is beneficial for obesity-related metabolic alterations and induces cellular adaptations against aging, possibly through AMPK and SIRT1 activation via circulating factors.     

Berberine modulates deacetylation of PPARγ to promote adipose tissue remodeling and thermogenesis via AMPK/SIRT1 pathway

Pharmacological stimulation of adipose tissue remodeling and thermogenesis to increase energy expenditure is expected to be a viable therapeutic strategy for obesity. Berberine has been reported to have pharmacological activity in adipose tissue to anti-obesity, while the mechanism remains unclear. Here, we observed that berberine significantly reduced the body weight and insulin resistance of high-fat diet mice by promoting the distribution of brown adipose tissue and thermogenesis. We have further demonstrated that berberine activated energy metabolic sensing pathway AMPK/SIRT1 axis to increase the level of PPARγ deacetylation, which leads to promoting adipose tissue remodeling and increasing the expression of the thermogenic protein UCP-1. These findings suggest that berberine that enhances the AMPK/SIRT1 pathway can act as a selective PPARγ activator to promote adipose tissue remodeling and thermogenesis. This study proposes a new mechanism for the regulation of berberine in adipose tissue and offers a great prospect for berberine in obesity treatment     

The Full Capacity of AICAR to Reduce Obesity-Induced Inflammation and Insulin Resistance Requires Myeloid SIRT1

Chronic Inflammation is a key link between obesity and insulin resistance. We previously showed that two nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 interact to regulate macrophage inflammation. AMPK is also a molecular target of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), which has been shown to reduce insulin resistance in various animal models. Here we aim to determine whether the therapeutic effects of AICAR against insulin resistance involve its anti-inflammatory function, which requires macrophage SIRT1. Long-term administration of low-dose AICAR significantly suppressed adipose inflammation in established diet-induced obese mice. This was associated with improved glucose homeostasis and insulin sensitivity without changes of body weight. In contrast, SIRT1 deletion in myeloid SIRT1 knockout (MSKO) mice increased infiltration of classically activated M1 macrophages and decreased alternatively activated M2 macrophages in adipose tissue. As a result, MSKO mice on high fat (HF) diets exhibited impaired insulin signaling in skeletal muscle, fat, and liver, and developed systemic insulin resistance in glucose tolerance tests, insulin tolerance tests, and hyperinsulinemic-euglycemic clamp experiments. Interestingly, the beneficial effects of AICAR on adipose inflammation and insulin sensitivity were absent in MSKO mice fed HF diets, suggesting that the full capacity of AICAR to antagonize obesity-induced inflammation and insulin resistance requires myeloid SIRT1. In summary, AICAR negatively regulates HF diet-induced inflammation, which requires myeloid SIRT1, thereby contributing to the protection against insulin resistance. Myeloid SIRT1 is a therapeutic target of the anti-inflammatory and insulin-sensitizing effects of AICAR.     

The Effect of Temperature on Oxidative Phosphorylation with Insect Flight Muscle Mitochondria

The effect of different temperatures on the biochemical activity and morphology of insect flight muscle mitochondria was examined. It was found that respiration and phosphorylation have the same thermal response at temperatures of 25°C. and below. The energy of activation for both systems is approximately 12,300 calories. Oxidation and phosphorylation can be uncoupled effectively by temperature, for at temperatures above 25°C. there is more rapid heat inactivation of phosphorylation. This is evident from reduced P/O values as well as from morphological deterioration in the mitochondrial population. The thermal response of both this sarcosomal enzyme system and the respiration in the living fly are quantitatively similar.     

Insulin down-regulates the expression of ubiquitin E3 ligases partially by inhibiting the activity and expression of AMP-activated protein kinase in L6 myotubes

While insulin is an anabolic hormone, AMP-activated protein kinase (AMPK) is not only a key energy regulator, but it can also control substrate metabolism directly by inducing skeletal muscle protein degradation. The hypothesis of the present study was that insulin inhibits AMPK and thus down-regulates the expression of the ubiquitin E3 ligases, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in skeletal muscle cells. Differentiated L6 myotubes were treated with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and/or compound C to stimulate and/or block AMPK respectively. These treatments were also conducted in the presence or absence of insulin and the cells were analysed by western blot and quantitative real-time PCR. In addition, nuleotide levels were determined using HPLC. The activation of AMPK with AICAR enhanced the mRNA levels of MAFbx and MuRF1. Insulin reduced the phosphorylation and activity AMPK, which was accompanied by reduced MAFbx and MuRF1 mRNA levels. Using a protein kinase B (PKB/Akt) inhibitor, we found that insulin regulates AMPK through the activation of Akt. Furthermore, insulin down-regulated AMPK α2 mRNA. We conclude that insulin inhibits AMPK through Akt phosphorylation in L6 myotubes, which may serve as a possible signalling pathway for the down-regulation of protein degradation. In addition, decreased expression of AMPK α2 may partially participate in inhibiting the activity of AMPK.     

Capacity of oxidative phosphorylation in human skeletal muscle: New perspectives of mitochondrial physiology

Maximal ADP-stimulated mitochondrial respiration depends on convergent electron flow through Complexes I + II to the Q-junction of the electron transport system (ETS). In most studies of respiratory control in mitochondrial preparations, however, respiration is limited artificially by supplying substrates for electron input through either Complex I or II. High-resolution respirometry with minimal amounts of tissue biopsy (1–3 mg wet weight of permeabilized muscle fibres per assay) provides a routine approach for multiple substrate-uncoupler-inhibitor titrations. Under physiological conditions, maximal respiratory capacity is obtained with glutamate + malate + succinate, reconstituting the operation of the tricarboxylic acid cycle and preventing depletion of key metabolites from the mitochondrial matrix. In human skeletal muscle, conventional assays with pyruvate + malate or glutamate + malate yield submaximal oxygen fluxes at 0.50–0.75 of capacity of oxidative phosphorylation (OXPHOS). Best estimates of muscular OXPHOS capacity at 37 °C (pmol O₂ s⁻¹ mg⁻¹ wet weight) with isolated mitochondria or permeabilized fibres, suggest a range of 100–150 and up to 180 in healthy humans with normal body mass index and top endurance athletes, but reduction to 60–120 in overweight healthy adults with predominantly sedentary life style. The apparent ETS excess capacity (uncoupled respiration) over ADP-stimulated OXPHOS capacity is high in skeletal muscle of active and sedentary humans, but absent in mouse skeletal muscle. Such differences of mitochondrial quality in skeletal muscle are unexpected and cannot be explained at present. A comparative database of mitochondrial physiology may provide the key for understanding the functional implications of mitochondrial diversity from mouse to man, and evaluation of altered mitochondrial respiratory control patterns in health and disease.     

Muscle-Specific SIRT1 Gain-of-Function Increases Slow-Twitch Fibers and Ameliorates Pathophysiology in a Mouse Model of Duchenne Muscular Dystrophy

SIRT1 is a metabolic sensor and regulator in various mammalian tissues and functions to counteract metabolic and age-related diseases. Here we generated and analyzed mice that express SIRT1 at high levels specifically in skeletal muscle. We show that SIRT1 transgenic muscle exhibits a fiber shift from fast-to-slow twitch, increased levels of PGC-1α, markers of oxidative metabolism and mitochondrial biogenesis, and decreased expression of the atrophy gene program. To examine whether increased activity of SIRT1 protects from muscular dystrophy, a muscle degenerative disease, we crossed SIRT1 muscle transgenic mice to mdx mice, a genetic model of Duchenne muscular dystrophy. SIRT1 overexpression in muscle reverses the phenotype of mdx mice, as determined by histology, creatine kinase release into the blood, and endurance in treadmill exercise. In addition, SIRT1 overexpression also results in increased levels of utrophin, a functional analogue of dystrophin, as well as increased expression of PGC-1α targets and neuromuscular junction genes. Based on these findings, we suggest that pharmacological interventions that activate SIRT1 in skeletal muscle might offer a new approach for treating muscle diseases.     

Gestational Diabetes Is Characterized by Reduced Mitochondrial Protein Expression and Altered Calcium Signaling Proteins in Skeletal Muscle

The rising prevalence of gestational diabetes mellitus (GDM) affects up to 18% of pregnant women with immediate and long-term metabolic consequences for both mother and infant. Abnormal glucose uptake and lipid oxidation are hallmark features of GDM prompting us to use an exploratory proteomics approach to investigate the cellular mechanisms underlying differences in skeletal muscle metabolism between obese pregnant women with GDM (OGDM) and obese pregnant women with normal glucose tolerance (ONGT). Functional validation was performed in a second cohort of obese OGDM and ONGT pregnant women. Quantitative proteomic analysis in rectus abdominus skeletal muscle tissue collected at delivery revealed reduced protein content of mitochondrial complex I (C-I) subunits (NDUFS3, NDUFV2) and altered content of proteins involved in calcium homeostasis/signaling (calcineurin A, α1-syntrophin, annexin A4) in OGDM (n = 6) vs. ONGT (n = 6). Follow-up analyses showed reduced enzymatic activity of mitochondrial complexes C-I, C-III, and C-IV (−60–75%) in the OGDM (n = 8) compared with ONGT (n = 10) subjects, though no differences were observed for mitochondrial complex protein content. Upstream regulators of mitochondrial biogenesis and oxidative phosphorylation were not different between groups. However, AMPK phosphorylation was dramatically reduced by 75% in the OGDM women. These data suggest that GDM is associated with reduced skeletal muscle oxidative phosphorylation and disordered calcium homeostasis. These relationships deserve further attention as they may represent novel risk factors for development of GDM and may have implications on the effectiveness of physical activity interventions on both treatment strategies for GDM and for prevention of type 2 diabetes postpartum.