Interleukin-1ß triggers muscle-derived extracellular superoxide dismutase expression and protects muscles from doxorubicin-induced atrophy.

Doxorubicin, a conventional chemotherapeutic agent prescribed for cancer, causes skeletal muscle atrophy and adversely affects mobility and strength. Given that doxorubicin-induced muscle atrophy is attributable primarily to oxidative stress, its effects could be mitigated by antioxidant-focused therapies; however, these protective therapeutic targets remain ambiguous. The aim of this study was to demonstrate that doxorubicin triggers severe muscle atrophy via upregulation of oxidative stress (4-hydroxynonenal and malondialdehyde) and atrogenes (atrogin-1/MAFbx and muscle RING finger-1) in association with decreased expression of the antioxidant enzyme extracellular superoxide dismutase (EcSOD), in cultured C2C12 myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein elevated EcSOD levels on the cellular membrane of cultured myotubes, consequently inhibiting doxorubicin-induced oxidative stress and myotube atrophy. Furthermore, doxorubicin treatment reduced interleukin-1ß (IL-1ß) mRNA expression in cultured myotubes and skeletal muscle, whereas transient IL-1ß treatment increased EcSOD protein expression on the myotube membrane. Notably, transient IL-1ß treatment of cultured myotubes and local administration in mouse skeletal muscle attenuated doxorubicin-induced muscle atrophy, which was associated with increased EcSOD expression. Collectively, these findings reveal that the regulation of skeletal muscle EcSOD via maintenance of IL-1ß signalling is a potential therapeutic approach to counteract the muscle atrophy mediated by doxorubicin and oxidative stress. KEY POINTS: Doxorubicin, a commonly prescribed chemotherapeutic agent for patients with cancer, induces severe muscle atrophy owing to increased expression of oxidative stress; however, protective therapeutic targets are poorly understood. Doxorubicin induced muscle atrophy owing to increased expression of oxidative stress and atrogenes in association with decreased protein expression of extracellular superoxide dismutase (EcSOD) in cultured C2C12 myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein increased EcSOD levels on the cellular membrane of cultured myotubes, resulting in inhibition of doxorubicin-induced oxidative stress and myotube atrophy. Doxorubicin treatment decreased interleukin-1ß (IL-1ß) expression in cultured myotubes and skeletal muscle, whereas transient IL-1ß treatment in vivo and in vitro increased EcSOD protein expression and attenuated doxorubicin-induced muscle atrophy. These findings reveal that regulation of skeletal muscle EcSOD via maintenance of IL-1ß signalling is a possible therapeutic approach for muscle atrophy mediated by doxorubicin and oxidative stress.

Muscle p62 stimulates the expression of antioxidant proteins alleviating cancer cachexia.

Oxidative stress plays an important role in skeletal muscle atrophy during cancer cachexia, and more glycolytic muscles are preferentially affected. Sequestosome1/SQSTM1 (i.e., p62), particularly when phosphorylated at Ser 349 (Ser 351 in mice), competitively binds to the Kelch-like ECH-associated protein 1 (Keap1) activating Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 then stimulates the transcription of antioxidant/electrophile-responsive elements in target genes. However, a potential role for p62 in the protection of muscle wasting in cachexia remains to be determined. Here, using the well-established cachexia-inducing model of Lewis Lung Carcinoma (LLC) in mice we demonstrate higher expression of antioxidant proteins (i.e., NQO1, HO-1, GSTM1, CuZnSOD, MnSOD, and EcSOD) in the more oxidative and cachexia resistant soleus muscle than in the more glycolytic and cachexia prone extensor digitorum longus muscle. This was accompanied by higher p62 (total and phosphorylated) and nuclear Nrf2 levels in the soleus, which were paralleled by higher expression of proteins known to either phosphorylate or promote p62 phosphorylation (i.e., NBR1, CK1, PKCδ, and TAK1). Muscle-specific p62 gain-of-function (i.e., in p62 mTg mice) activated Nrf2 nuclear translocation and increased the expression of multiple antioxidant proteins (i.e., CuZnSOD, MnSOD, EcSOD, NQO1, and GSTM1) in glycolytic muscles. Interestingly, skeletal muscle Nrf2 haplodeficiency blunted the increases of most of these proteins (i.e., CuZnSOD, EcSOD, and NQO1) suggesting that muscle p62 stimulates antioxidant protein expression also via additional, yet to be determined mechanisms. Of note, p62 gain-of-function mitigated glycolytic muscle wasting in LLC-affected mice. Collectively, our findings identify skeletal muscle p62 as a potential therapeutic target for cancer cachexia.

Regular exercise stimulates endothelium autophagy via IL-1 signaling in ApoE deficient mice.

Regular exercise maintains arterial endothelial cell homeostasis and protects the arteries from vascular disease, such as peripheral artery disease and atherosclerosis. Autophagy, which is a cellular process that degrades misfolded or aggregate proteins and damaged organelles, plays an important role in maintaining organ and cellular homeostasis. However, it is unknown whether regular exercise stimulates autophagy in aorta endothelial cells of mice prone to atherosclerosis independently of their circulating lipid profile. Here, we observed that 16 weeks of voluntary exercise reduced high-fat diet-induced atherosclerotic plaque formation in the aortic root of ApoE deficient mice, and that this protection occurred without changes in circulating triglycerides, total cholesterol, and lipoproteins. Immunofluorescence analysis indicated that voluntary exercise increased levels of the autophagy protein LC3 in aortic endothelial cells. Interestingly, human umbilical vein endothelial cells (HUVECs) exposed to serum from voluntarily exercised mice displayed significantly increased LC3-I and LC3-II protein levels. Analysis of circulating cytokines demonstrated that voluntary exercise caused changes directly relevant to IL-1 signaling (ie, decreased interleukin-1 receptor antagonist [IL-1ra] while also increasing IL-1α). HUVECs exposed to IL-1α and IL-1ß recombinant protein significantly increased LC3 mRNA expression, LC3-I and LC3-II protein levels, and autophagy flux. Collectively, these results suggest that regular exercise protects arteries from ApoE deficient mice against atherosclerosis at least in part by stimulating endothelial cell autophagy via enhanced IL-1 signaling.

Skeletal muscle-specific Keap1 disruption modulates fatty acid utilization and enhances exercise capacity in female mice.

Skeletal muscle health is important for the prevention of various age-related diseases. The loss of skeletal muscle mass, which is known as sarcopenia, underlies physical disability, poor quality of life and chronic diseases in elderly people. The transcription factor NRF2 plays important roles in the regulation of the cellular defense against oxidative stress, as well as the metabolism and mitochondrial activity. To determine the contribution of skeletal muscle NRF2 to exercise capacity, we conducted skeletal muscle-specific inhibition of KEAP1, which is a negative regulator of NRF2, and examined the cell-autonomous and non-cell-autonomous effects of NRF2 pathway activation in skeletal muscles. We found that NRF2 activation in skeletal muscles increased slow oxidative muscle fiber type and improved exercise endurance capacity in female mice. We also observed that female mice with NRF2 pathway activation in their skeletal muscles exhibited enhanced exercise-induced mobilization and ß-oxidation of fatty acids. These results indicate that NRF2 activation in skeletal muscles promotes communication with adipose tissues via humoral and/or neuronal signaling and facilitates the utilization of fatty acids as an energy source, resulting in increased mitochondrial activity and efficient energy production during exercise, which leads to improved exercise endurance.

Muscle-derived SDF-1α/CXCL12 modulates endothelial cell proliferation but not exercise training-induced angiogenesis.

Chemokines are critical mediators of angiogenesis in several physiological and pathological conditions; however, a potential role for muscle-derived chemokines in exercise-stimulated angiogenesis in skeletal muscle remains poorly understood. Here, we postulated that the chemokine stromal cell-derived factor-1 (SDF-1α/C-X-C motif chemokine ligand 12: CXCL12), shown to promote neovascularization in several organs, contributes to angiogenesis in skeletal muscle. We found that CXCL12 is abundantly expressed in capillary-rich oxidative soleus and exercise-trained plantaris muscles. CXCL12 mRNA and protein were also abundantly expressed in muscle-specific peroxisome proliferator-activated receptor γ coactivator 1α transgenic mice, which have a high proportion of oxidative muscle fibers and capillaries when compared with wild-type littermates. We then generated CXCL12 muscle-specific knockout mice but observed normal baseline capillary density and normal angiogenesis in these mice when they were exercise trained. To get further insight into a potential CXCL12 role in a myofiber-endothelial cell crosstalk, we first mechanically stretched C2C12 myotubes, a model known to induce stretch-related chemokine release, and observed increased CXCL12 mRNA and protein. Human umbilical vein endothelial cells (HUVECs) exposed to conditioned medium from cyclically stretched C2C12 myotubes displayed increased proliferation, which was dependent on CXCL12-mediated signaling through the CXCR4 receptor. However, HUVEC migration and tube formation were unaltered under these conditions. Collectively, our findings indicate that increased muscle contractile activity enhances CXCL12 production and release from muscle, potentially contributing to endothelial cell proliferation. However, redundant signals from other angiogenic factors are likely sufficient to sustain normal endothelial cell migration and tube formation activity, thereby preserving baseline capillary density and exercise training-mediated angiogenesis in muscles lacking CXCL12.

p62/SQSTM1 and Nrf2 are essential for exercise-mediated enhancement of antioxidant protein expression in oxidative muscle.

Increased muscle contractile activity, as observed with regular exercise, prevents oxidative stress-induced muscle wasting, at least partially, by improving the antioxidant defense system. Phosphorylated p62/sequestosome1 competitively binds to the Kelch-like ECH-associated protein 1, activating nuclear factor erythroid 2-related factor 2 (Nrf2), which stimulates transcription of antioxidant/electrophile responsive elements. However, it remains to be determined if this process is activated by regular exercise in skeletal muscle. Here, we demonstrate that muscle contractile activity increases antioxidants, Nrf2 translocation into nuclei, and Nrf2 DNA-binding activity in association with increased p62 phosphorylation (Ser351) in mouse oxidative skeletal muscle. Skeletal muscle-specific loss of Nrf2 [i.e., Nrf2 muscle-specific knockout (mKO) mice] abolished the expression of the Nrf2 target antioxidant gene NAD(P)H-quinone oxidoreductase 1 (NQO1) in both glycolytic and oxidative muscles but reduced exercise-mediated increases of antioxidants (i.e., copper/zinc superoxide dismutase (SOD) and extracellular SOD only in oxidative muscle. Interestingly, skeletal muscle-specific loss of p62 (i.e., p62 mKO mice) also abolished the expression of NQO1 and reduced exercise-mediated increases of the same antioxidants in soleus muscle. Collectively, these findings indicate that p62 and Nrf2 cooperatively regulate the exercise-mediated increase of antioxidants in oxidative muscle.-Yamada, M., Iwata, M., Warabi, E., Oishi, H., Lira, V. A., Okutsu, M. p62/SQSTM1 and Nrf2 are essential for exercise-mediated enhancement of antioxidant protein expression in oxidative muscle.

Maternal exercise training attenuates endotoxin-induced sepsis in mice offspring.

Regular exercise during pregnancy can prevent offspring from several diseases, such as cardiovascular diseases, obesity, and type II diabetes during adulthood. However, little information is available about whether maternal exercises during pregnancy protect the offspring from infectious diseases, such as sepsis and multiple organ dysfunction syndrome (MODS). This study aimed to investigate whether maternal exercise training protects the offspring from endotoxin-induced septic shock in mice. Female C57BL/6 mice performed voluntary wheel exercises during pregnancy. All dams and offspring were fed normal chow with sedentary activity during lactation and after weaning. At 10-week-old, mice were intraperitoneally injected a lethal (30 mg/kg) or nonlethal (15 mg/kg) dose of lipopolysaccharide (LPS), following which the survival of mice that were administered a lethal dose was monitored for 60 h. Plasma, lung, and liver samples were collected 18 h after the injection to evaluate the cytokine concentration or mRNA expression from those administered a nonlethal dose. Although maternal exercise training could not prevent lethality during an LPS-induced septic shock, it significantly inhibited the LPS-induced loss of body weight in female offspring. Regular maternal exercise significantly inhibited the mRNA expression of the LPS-induced inflammatory cytokines, such as interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ), in the plasma and liver. Thus, maternal exercise inhibited the LPS-induced inflammatory response in female offspring, suggesting that regular exercise during pregnancy could be a potential candidate of the onset of sepsis and MODS in offspring.

Muscle-derived extracellular superoxide dismutase inhibits endothelial activation and protects against multiple organ dysfunction syndrome in mice.

Multiple organ dysfunction syndrome (MODS) is a detrimental clinical complication in critically ill patients with high mortality. Emerging evidence suggests that oxidative stress and endothelial activation (induced expression of adhesion molecules) of vital organ vasculatures are key, early steps in the pathogenesis. We aimed to ascertain the role and mechanism(s) of enhanced extracellular superoxide dismutase (EcSOD) expression in skeletal muscle in protection against MODS induced by endotoxemia. We showed that EcSOD overexpressed in skeletal muscle-specific transgenic mice (TG) redistributes to other peripheral organs through the circulation and enriches at the endothelium of the vasculatures. TG mice are resistant to endotoxemia (induced by lipopolysaccharide [LPS] injection) in developing MODS with significantly reduced mortality and organ damages compared with the wild type littermates (WT). Heterogenic parabiosis between TG and WT mice conferred a significant protection to WT mice, whereas mice with R213G knock-in mutation, a human single nucleotide polymorphism leading to reduced binding EcSOD in peripheral organs, exacerbated the organ damages. Mechanistically, EcSOD inhibits vascular cell adhesion molecule 1 expression and inflammatory leukocyte adhesion to the vascular wall of vital organs, blocking an early step of the pathology in organ damage under endotoxemia. Therefore, enhanced expression of EcSOD in skeletal muscle profoundly protects against MODS by inhibiting endothelial activation and inflammatory cell adhesion, which could be a promising therapy for MODS.

ADAM12: a genetic modifier of preclinical peripheral arterial disease.

In prior studies from multiple groups, outcomes following experimental peripheral arterial disease (PAD) differed considerably across inbred mouse strains. Similarly, in humans with PAD, disease outcomes differ, even when there are similarities in risk factors, disease anatomy, arteriosclerotic burden, and hemodynamic measures. Previously, we identified a locus on mouse chromosome 7, limb salvage-associated quantitative trait locus 1 (LSq-1), which was sufficient to modify outcomes following experimental PAD. We compared expression of genes within LSq-1 in Balb/c mice, which normally show poor outcomes following experimental PAD, with that in C57Bl/6 mice, which normally show favorable outcomes, and found that a disintegrin and metalloproteinase gene 12 (ADAM12) had the most differential expression. Augmentation of ADAM12 expression in vivo improved outcomes following experimental PAD in Balb/c mice, whereas knockdown of ADAM12 made outcomes worse in C57Bl/6 mice. In vitro, ADAM12 expression modulates endothelial cell proliferation, survival, and angiogenesis in ischemia, and this appeared to be dependent on tyrosine kinase with Ig-like and EGF-like domain 2 (Tie2) activation. ADAM12 is sufficient to modify PAD severity in mice, and this likely occurs through regulation of Tie2.

Enhanced skeletal muscle expression of extracellular superoxide dismutase mitigates streptozotocin-induced diabetic cardiomyopathy by reducing oxidative stress and aberrant cell signaling.

BACKGROUND: Exercise training enhances extracellular superoxide dismutase (EcSOD) expression in skeletal muscle and elicits positive health outcomes in individuals with diabetes mellitus. The goal of this study was to determine if enhanced skeletal muscle expression of EcSOD is sufficient to mitigate streptozotocin-induced diabetic cardiomyopathy. METHODS AND RESULTS: Exercise training promotes EcSOD expression in skeletal muscle and provides protection against diabetic cardiomyopathy; however, it is not known if enhanced expression of EcSOD in skeletal muscle plays a functional role in this protection. Here, we show that skeletal muscle-specific EcSOD transgenic mice are protected from cardiac hypertrophy, fibrosis, and dysfunction under the condition of type 1 diabetes mellitus induced by streptozotocin injection. We also show that both exercise training and muscle-specific transgenic expression of EcSOD result in elevated EcSOD protein in the blood and heart without increased transcription in the heart, suggesting that enhanced expression of EcSOD from skeletal muscle redistributes to the heart. Importantly, cardiac tissue in transgenic mice displayed significantly reduced oxidative stress, aberrant cell signaling, and inflammatory cytokine expression compared with wild-type mice under the same diabetic condition. CONCLUSIONS: Enhanced expression of EcSOD in skeletal muscle is sufficient to mitigate streptozotocin-induced diabetic cardiomyopathy through attenuation of oxidative stress, aberrant cell signaling, and inflammation, suggesting a cross-organ mechanism by which exercise training improves cardiac function in diabetes mellitus.

Cortisol is not the primary mediator for augmented CXCR4 expression on natural killer cells after acute exercise.

CXC-chemokine receptor 4 (CXCR4) and its ligand, stromal-derived factor 1α (SDF-1α; also known as CXCL12), are crucial for the redistribution of immune cells after acute exercise. We investigated the relationships between acute exercise and CXCR4 expression on natural killer (NK) cells. Peripheral blood mononuclear cells (PBMCs) were cultured with cortisol and analyzed for CXCR4 expression on CD3(-)/CD56(+) NK cells and NK cell migration activity. To determine the effect of exercise, we isolated PBMCs from subjects before and after a 90-min exercise at 70% peak O2 uptake (V̇o2peak) and determined the changes in CXCR4 expression on NK cells after exercise. We cultured PBMCs with plasma obtained before and after exercise and with the glucocorticoid antagonist RU-486 to determine NK cell migration activity and the effects of cortisol on CXCR4 expression in vitro. Cortisol treatment increased CXCR4 expression (P < 0.05) and migration activity (P < 0.05) of NK cells. Exercise did not affect CXCR4 expression on NK cells, whereas incubating them with postexercise plasma significantly increased CXCR4 expression (P < 0.05) and migration activity (P < 0.05). RU-486 blocked cortisol-induced CXCR4 upregulation on NK cells, but only partially blocked (7%) CXCR4 upregulation when PMBCs were incubated with postexercise plasma. Thus acute exercise increases CXCR4 expression on NK cells and their migration activity and may contribute to NK cell redistribution after acute exercise; however, cortisol did not appear to be the primary mediator of augmented CXCR4 expression.

Extracellular superoxide dismutase ameliorates skeletal muscle abnormalities, cachexia, and exercise intolerance in mice with congestive heart failure.

BACKGROUND: Congestive heart failure (CHF) is a leading cause of morbidity and mortality, and oxidative stress has been implicated in the pathogenesis of cachexia (muscle wasting) and the hallmark symptom, exercise intolerance. We have previously shown that a nitric oxide-dependent antioxidant defense renders oxidative skeletal muscle resistant to catabolic wasting. Here, we aimed to identify and determine the functional role of nitric oxide-inducible antioxidant enzyme(s) in protection against cardiac cachexia and exercise intolerance in CHF. METHODS AND RESULTS: We demonstrated that systemic administration of endogenous nitric oxide donor S-nitrosoglutathione in mice blocked the reduction of extracellular superoxide dismutase (EcSOD) protein expression, as well as the induction of MAFbx/Atrogin-1 mRNA expression and muscle atrophy induced by glucocorticoid. We further showed that endogenous EcSOD, expressed primarily by type IId/x and IIa myofibers and enriched at endothelial cells, is induced by exercise training. Muscle-specific overexpression of EcSOD by somatic gene transfer or transgenesis (muscle creatine kinase [MCK]-EcSOD) in mice significantly attenuated muscle atrophy. Importantly, when crossbred into a mouse genetic model of CHF (α-myosin heavy chain-calsequestrin), MCK-EcSOD transgenic mice had significant attenuation of cachexia with preserved whole body muscle strength and endurance capacity in the absence of reduced HF. Enhanced EcSOD expression significantly ameliorated CHF-induced oxidative stress, MAFbx/Atrogin-1 mRNA expression, loss of mitochondria, and vascular rarefaction in skeletal muscle. CONCLUSIONS: EcSOD plays an important antioxidant defense function in skeletal muscle against cardiac cachexia and exercise intolerance in CHF.

Exercise prevents maternal high-fat diet-induced hypermethylation of the Pgc-1α gene and age-dependent metabolic dysfunction in the offspring.

Abnormal conditions during early development adversely affect later health. We investigated whether maternal exercise could protect offspring from adverse effects of a maternal high-fat diet (HFD) with a focus on the metabolic outcomes and epigenetic regulation of the metabolic master regulator, peroxisome proliferator-activated receptor γ coactivator-1α (Pgc-1α). Female C57BL/6 mice were exposed to normal chow, an HFD, or an HFD with voluntary wheel exercise for 6 weeks before and throughout pregnancy. Methylation of the Pgc-1α promoter at CpG site -260 and expression of Pgc-1α mRNA were assessed in skeletal muscle from neonatal and 12-month-old offspring, and glucose and insulin tolerance tests were performed in the female offspring at 6, 9, and 12 months. Hypermethylation of the Pgc-1α promoter caused by a maternal HFD was detected at birth and was maintained until 12 months of age with a trend of reduced expression of Pgc-1α mRNA (P = 0.065) and its target genes. Maternal exercise prevented maternal HFD-induced Pgc-1α hypermethylation and enhanced Pgc-1α and its target gene expression, concurrent with amelioration of age-associated metabolic dysfunction at 9 months of age in the offspring. Therefore, maternal exercise is a powerful lifestyle intervention for preventing maternal HFD-induced epigenetic and metabolic dysregulation in the offspring.

Corticosterone accelerates atherosclerosis in the apolipoprotein E-deficient mouse.

Chronic stress is an important risk factor for atherosclerosis, which is a chief process in the development of cardiovascular disease. Increased circulating levels of corticosterone have been documented in several animal models of chronic stress. However, it remains to be established whether corticosterone is sufficient to exacerbate atherosclerosis. To test this hypothesis, apolipoprotein E (ApoE)-deficient mice were fed a high-fat diet for 13 weeks with exposure to either corticosterone or vehicle in the drinking water (CORT and Con). Corticosterone treatment significantly increased atherosclerotic plaque area at the aortic root. Such exacerbation of atherosclerosis was accompanied by significantly lower levels of circulating white blood cells and serum interleukin-1ß (IL-1ß), and significantly elevated serum concentrations of total cholesterol, low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL) and small dense low-density lipoprotein (sd-LDL) in CORT mice when compared to Con mice. These findings demonstrate that corticosterone is sufficient to exacerbate atherosclerosis in vivo despite its anti-inflammatory properties and that this marked pro-atherogenic phenotype is primarily associated with increased dyslipidaemia.

Autophagy is required for exercise training-induced skeletal muscle adaptation and improvement of physical performance.

Pathological and physiological stimuli, including acute exercise, activate autophagy; however, it is unknown whether exercise training alters basal levels of autophagy and whether autophagy is required for skeletal muscle adaptation to training. We observed greater autophagy flux (i.e., a combination of increased LC3-II/LC3-I ratio and LC3-II levels and reduced p62 protein content indicating a higher rate of initiation and resolution of autophagic events), autophagy protein expression (i.e., Atg6/Beclin1, Atg7, and Atg8/LC3) and mitophagy protein Bnip3 expression in tonic, oxidative muscle compared to muscles of either mixed fiber types or of predominant glycolytic fibers in mice. Long-term voluntary running (4 wk) resulted in increased basal autophagy flux and expression of autophagy proteins and Bnip3 in parallel to mitochondrial biogenesis in plantaris muscle with mixed fiber types. Conversely, exercise training promoted autophagy protein expression with no significant increases of autophagy flux and mitochondrial biogenesis in the oxidative soleus muscle. We also observed increased basal autophagy flux and Bnip3 content without increases in autophagy protein expression in the plantaris muscle of sedentary muscle-specific Pgc-1α transgenic mice, a genetic model of augmented mitochondrial biogenesis. These findings reveal that endurance exercise training-induced increases in basal autophagy, including mitophagy, only take place if an enhanced oxidative phenotype is achieved. However, autophagy protein expression is mainly dictated by contractile activity independently of enhancements in oxidative phenotype. Exercise-trained mice heterozygous for the critical autophagy protein Atg6 showed attenuated increases of basal autophagy flux, mitochondrial content, and angiogenesis in skeletal muscle, along with impaired improvement of endurance capacity. These results demonstrate that increased basal autophagy is required for endurance exercise training-induced skeletal muscle adaptation and improvement of physical performance.

Disconnecting mitochondrial content from respiratory chain capacity in PGC-1-deficient skeletal muscle.

The transcriptional coactivators PGC-1α and PGC-1ß are widely thought to be required for mitochondrial biogenesis and fiber typing in skeletal muscle. Here, we show that mice lacking both PGC-1s in myocytes do indeed have profoundly deficient mitochondrial respiration but, surprisingly, have preserved mitochondrial content, isolated muscle contraction capacity, fiber-type composition, in-cage ambulation, and voluntary running capacity. Most of these findings are recapitulated in cell culture and, thus, are cell autonomous. Functional electron microscopy reveals normal cristae density with decreased cytochrome oxidase activity. These data lead to the following surprising conclusions: (1) PGC-1s are in fact dispensable for baseline muscle function, mitochondrial content, and fiber typing, (2) endurance fatigue at low workloads is not limited by muscle mitochondrial capacity, and (3) mitochondrial content and cristae density can be dissociated from respiratory capacity.

Baf60c drives glycolytic metabolism in the muscle and improves systemic glucose homeostasis through Deptor-mediated Akt activation.

A shift from oxidative to glycolytic metabolism has been associated with skeletal muscle insulin resistance in type 2 diabetes. However, whether this metabolic switch is deleterious or adaptive remains under debate, in part because of a limited understanding of the regulatory network that directs the metabolic and contractile specification of fast-twitch glycolytic muscle. Here we show that Baf60c (also called Smarcd3), a transcriptional cofactor enriched in fast-twitch muscle, promotes a switch from oxidative to glycolytic myofiber type through DEP domain-containing mTOR-interacting protein (Deptor)-mediated Akt activation. Muscle-specific transgenic expression of Baf60c activates a program of molecular, metabolic and contractile changes characteristic of glycolytic muscle. In addition, Baf60c is required for maintaining glycolytic capacity in adult skeletal muscle in vivo. Baf60c expression is significantly lower in skeletal muscle from obese mice compared to that from lean mice. Activation of the glycolytic muscle program by transgenic expression of Baf60c protects mice from diet-induced insulin resistance and glucose intolerance. Further mechanistic studies revealed that Deptor is induced by the Baf60c-Six4 transcriptional complex and mediates activation of Akt and glycolytic metabolism by Baf60c in a cell-autonomous manner. This work defines a fundamental mechanism underlying the specification of fast-twitch glycolytic muscle and illustrates that the oxidative-to-glycolytic metabolic shift in skeletal muscle is potentially adaptive and beneficial in the diabetic state.

A PGC-1α isoform induced by resistance training regulates skeletal muscle hypertrophy.

PGC-1α is a transcriptional coactivator induced by exercise that gives muscle many of the best known adaptations to endurance-type exercise but has no effects on muscle strength or hypertrophy. We have identified a form of PGC-1α (PGC-1α4) that results from alternative promoter usage and splicing of the primary transcript. PGC-1α4 is highly expressed in exercised muscle but does not regulate most known PGC-1α targets such as the mitochondrial OXPHOS genes. Rather, it specifically induces IGF1 and represses myostatin, and expression of PGC-1α4 in vitro and in vivo induces robust skeletal muscle hypertrophy. Importantly, mice with skeletal muscle-specific transgenic expression of PGC-1α4 show increased muscle mass and strength and dramatic resistance to the muscle wasting of cancer cachexia. Expression of PGC-1α4 is preferentially induced in mouse and human muscle during resistance exercise. These studies identify a PGC-1α protein that regulates and coordinates factors involved in skeletal muscle hypertrophy.

Kruppel-like factor 15 regulates skeletal muscle lipid flux and exercise adaptation.

The ability of skeletal muscle to enhance lipid utilization during exercise is a form of metabolic plasticity essential for survival. Conversely, metabolic inflexibility in muscle can cause organ dysfunction and disease. Although the transcription factor Kruppel-like factor 15 (KLF15) is an important regulator of glucose and amino acid metabolism, its endogenous role in lipid homeostasis and muscle physiology is unknown. Here we demonstrate that KLF15 is essential for skeletal muscle lipid utilization and physiologic performance. KLF15 directly regulates a broad transcriptional program spanning all major segments of the lipid-flux pathway in muscle. Consequently, Klf15-deficient mice have abnormal lipid and energy flux, excessive reliance on carbohydrate fuels, exaggerated muscle fatigue, and impaired endurance exercise capacity. Elucidation of this heretofore unrecognized role for KLF15 now implicates this factor as a central component of the transcriptional circuitry that coordinates physiologic flux of all three basic cellular nutrients: glucose, amino acids, and lipids.

Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy.

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.