Fibroblast growth factor 21 is expressed and secreted from skeletal muscle following electrical stimulation via extracellular ATP activation of the PI3K/Akt/mTOR signaling pathway.

Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, and energy metabolism. Although it is released mainly from the liver, in recent years it has been shown that it is a "myokine", synthesized in skeletal muscles after exercise and stress conditions through an Akt-dependent pathway and secreted for mediating autocrine and endocrine roles. To date, the molecular mechanism for the pathophysiological regulation of FGF21 production in skeletal muscle is not totally understood. We have previously demonstrated that muscle membrane depolarization controls gene expression through extracellular ATP (eATP) signaling, by a mechanism defined as "Excitation-Transcription coupling". eATP signaling regulates the expression and secretion of interleukin 6, a well-defined myokine, and activates the Akt/mTOR signaling pathway. This work aimed to study the effect of electrical stimulation in the regulation of both production and secretion of skeletal muscle FGF21, through eATP signaling and PI3K/Akt pathway. Our results show that electrical stimulation increases both mRNA and protein (intracellular and secreted) levels of FGF21, dependent on an extracellular ATP signaling mechanism in skeletal muscle. Using pharmacological inhibitors, we demonstrated that FGF21 production and secretion from muscle requires the activation of the P2YR/PI3K/Akt/mTOR signaling pathway. These results confirm skeletal muscle as a source of FGF21 in physiological conditions and unveil a new molecular mechanism for regulating FGF21 production in this tissue. Our results will allow to identify new molecular targets to understand the regulation of FGF21 both in physiological and pathological conditions, such as exercise, aging, insulin resistance, and Duchenne muscular dystrophy, all characterized by an alteration in both FGF21 levels and ATP signaling components. These data reinforce that eATP signaling is a relevant mechanism for myokine expression in skeletal muscle.

Mechanical Disturbance of Osteoclasts Induces ATP Release That Leads to Protein Synthesis in Skeletal Muscle through an Akt-mTOR Signaling Pathway.

Muscle and bone are tightly integrated through mechanical and biochemical signals. Osteoclasts are cells mostly related to pathological bone loss; however, they also start physiological bone remodeling. Therefore, osteoclast signals released during bone remodeling could improve both bone and skeletal muscle mass. Extracellular ATP is an autocrine/paracrine signaling molecule released by bone and muscle cells. Then, in the present work, it was hypothesized that ATP is a paracrine mediator released by osteoclasts and leads to skeletal muscle protein synthesis. RAW264.7-derived osteoclasts were co-cultured in Transwell® chambers with flexor digitorum brevis (FDB) muscle isolated from adult BalbC mice. The osteoclasts at the upper chamber were mechanically stimulated by controlled culture medium perturbation, resulting in a two-fold increase in protein synthesis in FDB muscle at the lower chamber. Osteoclasts released ATP to the extracellular medium in response to mechanical stimulation, proportional to the magnitude of the stimulus and partly dependent on the P2X7 receptor. On the other hand, exogenous ATP promoted Akt phosphorylation (S473) in isolated FDB muscle in a time- and concentration-dependent manner. ATP also induced phosphorylation of proteins downstream Akt: mTOR (S2448), p70S6K (T389) and 4E-BP1 (T37/46). Exogenous ATP increased the protein synthesis rate in FDB muscle 2.2-fold; this effect was blocked by Suramin (general P2X/P2Y antagonist), LY294002 (phosphatidylinositol 3 kinase inhibitor) and Rapamycin (mTOR inhibitor). These blockers, as well as apyrase (ATP metabolizing enzyme), also abolished the induction of FDB protein synthesis evoked by mechanical stimulation of osteoclasts in the co-culture model. Therefore, the present findings suggest that mechanically stimulated osteoclasts release ATP, leading to protein synthesis in isolated FDB muscle, by activating the P2-PI3K-Akt-mTOR pathway. These results open a new area for research and clinical interest in bone-to-muscle crosstalk in adaptive processes related to muscle use/disuse or in musculoskeletal pathologies.

High extracellular ATP levels released through pannexin-1 channels mediate inflammation and insulin resistance in skeletal muscle fibres of diet-induced obese mice.

AIMS/HYPOTHESIS: Skeletal muscle is a key target organ for insulin's actions and is the main regulator of blood glucose. In obese individuals and animal models, there is a chronic low-grade inflammatory state affecting highly metabolic organs, leading to insulin resistance. We have described that adult skeletal muscle fibres can release ATP to the extracellular medium through pannexin-1 (PANX1) channels. Besides, it is known that high extracellular ATP concentrations can act as an inflammatory signal. Here, we propose that skeletal muscle fibres from obese mice release high levels of ATP, through PANX1 channels, promoting inflammation and insulin resistance in muscle cells. METHODS: C57BL/6J mice were fed with normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. Muscle fibres were isolated from flexor digitorum brevis (FDB) muscle. PANX1-knockdown FDB fibres were obtained by in vivo electroporation of a short hairpin RNA Panx1 plasmid. We analysed extracellular ATP levels in a luciferin/luciferase assay. Gene expression was studied with quantitative real-time PCR (qPCR). Protein levels were evaluated by immunoblots, ELISA and immunofluorescence. Insulin sensitivity was analysed in a 2-NBDG (fluorescent glucose analogue) uptake assay, immunoblots and IPGTT. RESULTS: HFD-fed mice showed significant weight gain and insulin resistance compared with NCD-fed mice. IL-6, IL-1ß and TNF-α protein levels were increased in FDB muscle from obese mice. We observed high levels of extracellular ATP in muscle fibres from obese mice (197 ± 55 pmol ATP/µg RNA) compared with controls (32 ± 10 pmol ATP/µg RNA). ATP release in obese mice fibres was reduced by application of 100 µmol/l oleamide (OLE) and 5 µmol/l carbenoxolone (CBX), both PANX1 blockers. mRNA levels of genes linked to inflammation were reduced using OLE, CBX or 2 U/ml ATPase apyrase in muscle fibres from HFD-fed mice. In fibres from mice with pannexin-1 knockdown, we observed diminished extracellular ATP levels (78 ± 10 pmol ATP/µg RNA vs 252 ± 37 pmol ATP/µg RNA in control mice) and a lower expression of inflammatory markers. Moreover, a single pulse of 300 µmol/l ATP to fibres from control mice reduced insulin-mediated 2-NBDG uptake and promoted an elevation in mRNA levels of inflammatory markers. PANX-1 protein levels were increased two- to threefold in skeletal muscle from obese mice compared with control mice. Incubation with CBX increased Akt activation and 2-NBDG uptake in HFD fibres after insulin stimulation, rescuing the insulin resistance condition. Finally, in vivo treatment of HFD-fed mice with CBX (i.p. injection of 10 mg/kg each day) for 14 days, compared with PBS, reduced extracellular ATP levels in skeletal muscle fibres (51 ± 10 pmol ATP/µg RNA vs 222 ± 28 pmol ATP/µg RNA in PBS-treated mice), diminished inflammation and improved glycaemic management. CONCLUSIONS/INTERPRETATION: In this work, we propose a novel mechanism for the development of inflammation and insulin resistance in the skeletal muscle of obese mice. We found that high extracellular ATP levels, released by overexpressed PANX1 channels, lead to an inflammatory state and insulin resistance in skeletal muscle fibres of obese mice.

Cytosolic ROS production by NADPH oxidase 2 regulates muscle glucose uptake during exercise.

Reactive oxygen species (ROS) act as intracellular compartmentalized second messengers, mediating metabolic stress-adaptation. In skeletal muscle fibers, ROS have been suggested to stimulate glucose transporter 4 (GLUT4)-dependent glucose transport during artificially evoked contraction ex vivo, but whether myocellular ROS production is stimulated by in vivo exercise to control metabolism is unclear. Here, we combined exercise in humans and mice with fluorescent dyes, genetically-encoded biosensors, and NADPH oxidase 2 (NOX2) loss-of-function models to demonstrate that NOX2 is the main source of cytosolic ROS during moderate-intensity exercise in skeletal muscle. Furthermore, two NOX2 loss-of-function mouse models lacking either p47phox or Rac1 presented striking phenotypic similarities, including greatly reduced exercise-stimulated glucose uptake and GLUT4 translocation. These findings indicate that NOX2 is a major myocellular ROS source, regulating glucose transport capacity during moderate-intensity exercise.

The Emerging Roles of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 in Skeletal Muscle Redox Signaling and Metabolism.

Significance: Skeletal muscle is a crucial tissue to whole-body locomotion and metabolic health. Reactive oxygen species (ROS) have emerged as intracellular messengers participating in both physiological and pathological adaptations in skeletal muscle. A complex interplay between ROS-producing enzymes and antioxidant networks exists in different subcellular compartments of mature skeletal muscle. Recent evidence suggests that nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are a major source of contraction- and insulin-stimulated oxidants production, but they may paradoxically also contribute to muscle insulin resistance and atrophy. Recent Advances: Pharmacological and molecular biological tools, including redox-sensitive probes and transgenic mouse models, have generated novel insights into compartmentalized redox signaling and suggested that NOX2 contributes to redox control of skeletal muscle metabolism. Critical Issues: Major outstanding questions in skeletal muscle include where NOX2 activation occurs under different conditions in health and disease, how NOX2 activation is regulated, how superoxide/hydrogen peroxide generated by NOX2 reaches the cytosol, what the signaling mediators are downstream of NOX2, and the role of NOX2 for different physiological and pathophysiological processes. Future Directions: Future research should utilize and expand the current redox-signaling toolbox to clarify the NOX2-dependent mechanisms in skeletal muscle and determine whether the proposed functions of NOX2 in cells and animal models are conserved into humans.

Herpud1 impacts insulin-dependent glucose uptake in skeletal muscle cells by controlling the Ca2+-calcineurin-Akt axis.

Skeletal muscle plays a central role in insulin-controlled glucose homeostasis. The molecular mechanisms related to insulin resistance in this tissue are incompletely understood. Herpud1 is an endoplasmic reticulum membrane protein that maintains intracellular Ca2+ homeostasis under stress conditions. It has recently been reported that Herpud1-knockout mice display intolerance to a glucose load without showing altered insulin secretion. The functions of Herpud1 in skeletal muscle also remain unknown. Based on these findings, we propose that Herpud1 is necessary for insulin-dependent glucose disposal in skeletal muscle. Here we show that Herpud1 silencing decreased insulin-dependent glucose uptake, GLUT4 translocation to the plasma membrane, and Akt Ser473 phosphorylation in cultured L6 myotubes. A decrease in insulin-induced Akt Ser473 phosphorylation was observed in soleus but not in extensor digitorum longus muscle samples from Herpud1-knockout mice. Herpud1 knockdown increased the IP3R-dependent cytosolic Ca2+ response and the activity of Ca2+-dependent serine/threonine phosphatase calcineurin in L6 cells. Calcineurin decreased insulin-dependent Akt phosphorylation and glucose uptake. Moreover, calcineurin inhibition restored the insulin response in Herpud1-depleted L6 cells. Based on these findings, we conclude that Herpud1 is necessary for adequate insulin-induced glucose uptake due to its role in Ca2+/calcineurin regulation in L6 myotubes.

Interleukin-6 and neuregulin-1 as regulators of utrophin expression via the activation of NRG-1/ErbB signaling pathway in mdx cells.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease originated by mutations in the dystrophin gene. A promising therapeutic approach deals with functional substitution of dystrophin by utrophin, a structural homolog that might be able to compensate dystrophin absence in DMD muscle fibers. It has been described that both interleukin-6 (IL-6) and neuregulin-1 (NRG-1; Heregulin-HRG) induce utrophin expression in skeletal muscle. We investigated a possible functional link among IL-6, NRG-1 and utrophin, in normal (C57) and dystrophic (mdx) skeletal muscle cells. Western Blot analysis allowed us to demonstrate that IL-6 (100ng/mL) induces NRG-1 receptor phosphorylation (ErbB2/ErbB3) in both cell types, in a process that depends on intracellular Ca2+ and metalloproteinase activity; it also induces a transient increase of ERK1 and GABPα phosphorylation only in dystrophic myotubes. Semiquantitative PCR showed that IL-6 treatment increases utrophin mRNA levels just in mdx myotubes. We observed that utrophin mRNA induction was abolished by BAPTA-AM (an intracellular Ca2+ chelator), GM6001 (a general metalloproteinase inhibitor), genistein (a general protein tyrosine kinase inhibitor), PD-158780 (an ErbB receptor tyrosine kinase inhibitor) and PD-98059 (a MEK inhibitor), whereas Ly-294002 and wortmannin (PI3K inhibitors) did not affect utrophin induction evoked by IL-6 in dystrophic myotubes. Our results suggest that IL-6 induces utrophin expression in mdx myotubes through activation of a NRG-1/ErbBs signaling cascade. Soluble NRG-1 elicited by proteolytic processing of transmembrane NRG-1 might induce ErbBs phosphorylation and ERK1/2 pathway activation, leading to utrophin up-regulation.

Characterization of a multiprotein complex involved in excitation-transcription coupling of skeletal muscle.

BACKGROUND: Electrical activity regulates the expression of skeletal muscle genes by a process known as "excitation-transcription" (E-T) coupling. We have demonstrated that release of adenosine 5'-triphosphate (ATP) during depolarization activates membrane P2X/P2Y receptors, being the fundamental mediators between electrical stimulation, slow intracellular calcium transients, and gene expression. We propose that this signaling pathway would require the proper coordination between the voltage sensor (dihydropyridine receptor, DHPR), pannexin 1 channels (Panx1, ATP release conduit), nucleotide receptors, and other signaling molecules. The goal of this study was to assess protein-protein interactions within the E-T machinery and to look for novel constituents in order to characterize the signaling complex. METHODS: Newborn derived myotubes, adult fibers, or triad fractions from rat or mouse skeletal muscles were used. Co-immunoprecipitation, 2D blue native SDS/PAGE, confocal microscopy z-axis reconstruction, and proximity ligation assays were combined to assess the physical proximity of the putative complex interactors. An L6 cell line overexpressing Panx1 (L6-Panx1) was developed to study the influence of some of the complex interactors in modulation of gene expression. RESULTS: Panx1, DHPR, P2Y2 receptor (P2Y2R), and dystrophin co-immunoprecipitated in the different preparations assessed. 2D blue native SDS/PAGE showed that DHPR, Panx1, P2Y2R and caveolin-3 (Cav3) belong to the same multiprotein complex. We observed co-localization and protein-protein proximity between DHPR, Panx1, P2Y2R, and Cav3 in adult fibers and in the L6-Panx1 cell line. We found a very restricted location of Panx1 and Cav3 in a putative T-tubule zone near the sarcolemma, while DHPR was highly expressed all along the transverse (T)-tubule. By Panx1 overexpression, extracellular ATP levels were increased both at rest and after electrical stimulation. Basal mRNA levels of the early gene cfos and the oxidative metabolism markers citrate synthase and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were significantly increased by Panx1 overexpression. Interleukin 6 expression evoked by 20-Hz electrical stimulation (270 pulses, 0.3 ms each) was also significantly upregulated in L6-Panx1 cells. CONCLUSIONS: We propose the existence of a relevant multiprotein complex that coordinates events involved in E-T coupling. Unveiling the molecular actors involved in the regulation of gene expression will contribute to the understanding and treatment of skeletal muscle disorders due to wrong-expressed proteins, as well as to improve skeletal muscle performance.

Reactive oxygen species and calcium signals in skeletal muscle: A crosstalk involved in both normal signaling and disease.

Reactive Oxygen Species (ROS) have been profusely studied as agents of potential damage to living cells and they have been related to a number of pathological processes. Increasing evidence points to a more positive role of ROS in cell signaling and the detailed mechanism that regulates the precise amount of ROS needed for cell functioning without the deleterious effects of excess ROS still needs to be resolved in detail. In skeletal muscle the main source of ROS during normal functioning appears to be NADPH oxidase 2 (NOX2), which is activated by electrical stimuli (or exercise) through a cascade of events that include ATP release through pannexin1 channels. NOX2 is a protein complex that assembles in the T-tubule membrane before activation and ROS production by NOX2 appears to be important for muscle adaptation through gene expression and mitochondrial biogenesis as well as for improving glucose transport after insulin action. Excess ROS production (or diminished antioxidant defenses) plays a role in a number of pathological processes in skeletal muscle. Together with increased reactive nitrogen species, an increase in ROS appears to have a deleterious role in a model of Duchenne muscular dystrophy as well as muscle wasting in other diseases such as aging sarcopenia and cancer cachexia. In addition, ROS is involved in obesity and muscle insulin resistance, both of which are causally related to type 2 diabetes. A detailed description of the fine-tuning of ROS (including all sources of ROS) in skeletal muscle in health and disease will significantly contribute to our knowledge of both muscle adaptation and muscle related pathologies.

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.

Altered ROS production, NF-κB activation and interleukin-6 gene expression induced by electrical stimulation in dystrophic mdx skeletal muscle cells.

Duchenne muscular dystrophy is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. This pathology is associated with an increased production of reactive oxygen (ROS) and nitrogen species. The aim of this work was to study the alterations in NF-κB activation and interleukin-6 (IL-6) expression induced by membrane depolarization in dystrophic mdx myotubes. Membrane depolarization elicited by electrical stimulation increased p65 phosphorylation, NF-κB transcriptional activity and NF-κB-dependent IL-6 expression in wt myotubes, whereas in mdx myotubes it had the opposite effect. We have previously shown that depolarization-induced intracellular Ca2+ increases and ROS production are necessary for NF-κB activation and stimulation of gene expression in wt myotubes. Dystrophic myotubes showed a reduced amplitude and area under the curve of the Ca2+ transient elicited by electrical stimulation. On the other hand, electrical stimuli induced higher ROS production in mdx than wt myotubes, which were blocked by NOX2 inhibitors. Moreover, mRNA expression and protein levels of the NADPH oxidase subunits: p47phox and gp91phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50 µM H2O2 increased NF-κB activity; after administration of 100 and 200 µM H2O2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H2O2, with a significant effect of 100 µM and 200 µM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of electrical stimuli in both NF-κB activation and IL-6 expression. Finally, to ascertain whether stimulation of NF-κB and IL-6 gene expression by the inflammatory pathway is also impaired in mdx myotubes, we studied the effect of lipopolysaccharide on both NF-κB activation and IL-6 expression. Exposure to lipopolysaccharide induced a dramatic increase in both NF-κB activation and IL-6 expression in both wt and mdx myotubes, suggesting that the altered IL-6 gene expression after electrical stimulation in mdx muscle cells is due to dysregulation of Ca2+ release and ROS production, both of which impinge on NF-κB signaling. IL-6 is a key metabolic modulator that is released by the skeletal muscle to coordinate a multi-systemic response (liver, muscle, and adipocytes) during physical exercise; the alteration of this response in dystrophic muscles may contribute to an abnormal response to contraction and exercise.

The cholesterol-lowering agent methyl-ß-cyclodextrin promotes glucose uptake via GLUT4 in adult muscle fibers and reduces insulin resistance in obese mice.

Insulin stimulates glucose uptake in adult skeletal muscle by promoting the translocation of GLUT4 glucose transporters to the transverse tubule (T-tubule) membranes, which have particularly high cholesterol levels. We investigated whether T-tubule cholesterol content affects insulin-induced glucose transport. Feeding mice a high-fat diet (HFD) for 8 wk increased by 30% the T-tubule cholesterol content of triad-enriched vesicular fractions from muscle tissue compared with triads from control mice. Additionally, isolated muscle fibers (flexor digitorum brevis) from HFD-fed mice showed a 40% decrease in insulin-stimulated glucose uptake rates compared with fibers from control mice. In HFD-fed mice, four subcutaneous injections of MßCD, an agent reported to extract membrane cholesterol, improved their defective glucose tolerance test and normalized their high fasting glucose levels. The preincubation of isolated muscle fibers with relatively low concentrations of MßCD increased both basal and insulin-induced glucose uptake in fibers from controls or HFD-fed mice and decreased Akt phosphorylation without altering AMPK-mediated signaling. In fibers from HFD-fed mice, MßCD improved insulin sensitivity even after Akt or CaMK II inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, prevented the stimulatory effects of MßCD on glucose uptake. Addition of MßCD elicited ryanodine receptor-mediated calcium signals in isolated fibers, which were essential for glucose uptake. Our findings suggest that T-tubule cholesterol content exerts a critical regulatory role on insulin-stimulated GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may represent a useful strategy to counteract insulin resistance.

ATP signaling in skeletal muscle: from fiber plasticity to regulation of metabolism.

Tetanic electrical stimulation releases adenosine triphosphate (ATP) from muscle fibers through pannexin-1 channels in a frequency-dependent manner; extracellular ATP activates signals that ultimately regulate gene expression and is able to increase glucose transport through activation of P2Y receptors, phosphatidylinositol 3-kinase, Akt, and AS160. We hypothesize that this mechanism is an important link between exercise and the regulation of muscle fiber plasticity and metabolism.

Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca(2+) signals and an IL-6 autocrine loop.

Interleukin-6 (IL-6) is an important myokine that is highly expressed in skeletal muscle cells upon exercise. We assessed IL-6 expression in response to electrical stimulation (ES) or extracellular ATP as a known mediator of the excitation-transcription mechanism in skeletal muscle. We examined whether the canonical signaling cascade downstream of IL-6 (IL-6/JAK2/STAT3) also responds to muscle cell excitation, concluding that IL-6 influences its own expression through a positive loop. Either ES or exogenous ATP (100 µM) increased both IL-6 expression and p-STAT3 levels in rat myotubes, a process inhibited by 100 µM suramin and 2 U/ml apyrase. ATP also evoked IL-6 expression in both isolated skeletal fibers and extracts derived from whole FDB muscles. ATP increased IL-6 release up to 10-fold. STAT3 activation evoked by ATP was abolished by the JAK2 inhibitor HBC. Blockade of secreted IL-6 with a neutralizing antibody or preincubation with the STAT3 inhibitor VIII reduced STAT3 activation evoked by extracellular ATP by 70%. Inhibitor VIII also reduced by 70% IL-6 expression evoked by ATP, suggesting a positive IL-6 loop. In addition, ATP increased up to 60% the protein levels of SOCS3, a negative regulator of the IL-6 signaling pathway. On the other hand, intracellular calcium chelation or blockade of IP3-dependent calcium signals abolished STAT3 phosphorylation evoked by either extracellular ATP or ES. These results suggest that expression of IL-6 in stimulated skeletal muscle cells is mediated by extracellular ATP and nucleotide receptors, involving IP3-dependent calcium signals as an early step that triggers a positive IL-6 autocrine loop.

Insulin elicits a ROS-activated and an IP3-dependent Ca²âº release, which both impinge on GLUT4 translocation.

Insulin signaling includes generation of low levels of H2O2; however, its origin and contribution to insulin-stimulated glucose transport are unknown. We tested the impact of H2O2 on insulin-dependent glucose transport and GLUT4 translocation in skeletal muscle cells. H2O2 increased the translocation of GLUT4 with an exofacial Myc-epitope tag between the first and second transmembrane domains (GLUT4myc), an effect additive to that of insulin. The anti-oxidants N-acetyl L-cysteine and Trolox, the p47(phox)-NOX2 NADPH oxidase inhibitory peptide gp91-ds-tat or p47(phox) knockdown each reduced insulin-dependent GLUT4myc translocation. Importantly, gp91-ds-tat suppressed insulin-dependent H2O2 production. A ryanodine receptor (RyR) channel agonist stimulated GLUT4myc translocation and insulin stimulated RyR1-mediated Ca(2+) release by promoting RyR1 S-glutathionylation. This pathway acts in parallel to insulin-mediated stimulation of inositol-1,4,5-trisphosphate (IP3)-activated Ca(2+) channels, in response to activation of phosphatidylinositol 3-kinase and its downstream target phospholipase C, resulting in Ca(2+) transfer to the mitochondria. An inhibitor of IP3 receptors, Xestospongin B, reduced both insulin-dependent IP3 production and GLUT4myc translocation. We propose that, in addition to the canonical α,ß phosphatidylinositol 3-kinase to Akt pathway, insulin engages both RyR-mediated Ca(2+) release and IP3-receptor-mediated mitochondrial Ca(2+) uptake, and that these signals jointly stimulate glucose uptake.

Electrical stimuli are anti-apoptotic in skeletal muscle via extracellular ATP. Alteration of this signal in Mdx mice is a likely cause of dystrophy.

ATP signaling has been shown to regulate gene expression in skeletal muscle and to be altered in models of muscular dystrophy. We have previously shown that in normal muscle fibers, ATP released through Pannexin1 (Panx1) channels after electrical stimulation plays a role in activating some signaling pathways related to gene expression. We searched for a possible role of ATP signaling in the dystrophy phenotype. We used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. We demonstrated that low frequency electrical stimulation has an anti-apoptotic effect in normal muscle fibers repressing the expression of Bax, Bim and PUMA. Addition of exogenous ATP to the medium has a similar effect. In dystrophic fibers, the basal levels of extracellular ATP were higher compared to normal fibers, but unlike control fibers, they do not present any ATP release after low frequency electrical stimulation, suggesting an uncoupling between electrical stimulation and ATP release in this condition. Elevated levels of Panx1 and decreased levels of Cav1.1 (dihydropyridine receptors) were found in triads fractions prepared from mdx muscles. Moreover, decreased immunoprecipitation of Cav1.1 and Panx1, suggest uncoupling of the signaling machinery. Importantly, in dystrophic fibers, exogenous ATP was pro-apoptotic, inducing the transcription of Bax, Bim and PUMA and increasing the levels of activated Bax and cytosolic cytochrome c. These evidence points to an involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies.

Insulin-dependent H2O2 production is higher in muscle fibers of mice fed with a high-fat diet.

Insulin resistance is defined as a reduced ability of insulin to stimulate glucose utilization. C57BL/6 mice fed with a high-fat diet (HFD) are a model of insulin resistance. In skeletal muscle, hydrogen peroxide (H2O2) produced by NADPH oxidase 2 (NOX2) is involved in signaling pathways triggered by insulin. We evaluated oxidative status in skeletal muscle fibers from insulin-resistant and control mice by determining H2O2 generation (HyPer probe), reduced-to-oxidized glutathione ratio and NOX2 expression. After eight weeks of HFD, insulin-dependent glucose uptake was impaired in skeletal muscle fibers when compared with control muscle fibers. Insulin-resistant mice showed increased insulin-stimulated H2O2 release and decreased reduced-to-oxidized glutathione ratio (GSH/GSSG). In addition, p47phox and gp91phox (NOX2 subunits) mRNA levels were also high (~3-fold in HFD mice compared to controls), while protein levels were 6.8- and 1.6-fold higher, respectively. Using apocynin (NOX2 inhibitor) during the HFD feeding period, the oxidative intracellular environment was diminished and skeletal muscle insulin-dependent glucose uptake restored. Our results indicate that insulin-resistant mice have increased H2O2 release upon insulin stimulation when compared with control animals, which appears to be mediated by an increase in NOX2 expression.

Testosterone signals through mTOR and androgen receptor to induce muscle hypertrophy.

PURPOSE: The anabolic hormone testosterone induces muscle hypertrophy, but the intracellular mechanisms involved are poorly known. We addressed the question whether signal transduction pathways other than the androgen receptor (AR) are necessary to elicit hypertrophy in skeletal muscle myotubes. METHODS: Cultured rat skeletal muscle myotubes were preincubated with inhibitors for ERK1/2 (PD98059), PI3K/Akt (LY294002 and Akt inhibitor VIII) or mTOR/S6K1 (rapamycin), and then stimulated with 100 nM testosterone. The expression of α-actin and the phosphorylation levels of ERK1/2, Akt and S6K1 (a downstream target for mTOR) were measured by Western blot. mRNA levels were evaluated by real time RT-PCR. Myotube size and sarcomerization were determined by confocal microscopy. Inhibition of AR was assessed by bicalutamide. RESULTS: Testosterone-induced myotube hypertrophy was assessed as increased myotube cross-sectional area (CSA) and increased α-actin mRNA and α-actin protein levels, with no changes in mRNA expression of atrogenes (MAFbx and MuRF-1). Morphological development of myotube sarcomeres was evident in testosterone-stimulated myotubes. Known hypertrophy signaling pathways were studied at short times: ERK1/2 and Akt showed an increase in phosphorylation status after testosterone stimulus at 5 and 15 min, respectively. S6K1 was phosphorylated at 60 min. This response was abolished by PI3K/Akt and mTOR inhibition but not by ERK1/2 inhibition. Similarly, the CSA increase at 12 h was abolished by inhibitors of the PI3K/Akt pathway as well as by AR inhibition. CONCLUSIONS: These results suggest a crosstalk between pathways involving fast intracellular signaling and the AR to explain testosterone-induced skeletal muscle hypertrophy.

Cav1.1 controls frequency-dependent events regulating adult skeletal muscle plasticity.

An important pending question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype as slow or fast twitch muscle fibers. We have previously shown that voltage-gated L-type calcium channel (Cav1.1) acts as a voltage sensor for activation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-dependent Ca(2+) signals that regulates gene expression. ATP released by muscle cells after electrical stimulation through pannexin-1 channels plays a key role in this process. We show now that stimulation frequency determines both ATP release and Ins(1,4,5)P3 production in adult skeletal muscle and that Cav1.1 and pannexin-1 colocalize in the transverse tubules. Both ATP release and increased Ins(1,4,5)P3 was seen in flexor digitorum brevis fibers stimulated with 270 pulses at 20 Hz, but not at 90 Hz. 20 Hz stimulation induced transcriptional changes related to fast-to-slow muscle fiber phenotype transition that required ATP release. Addition of 30 µM ATP to fibers induced the same transcriptional changes observed after 20 Hz stimulation. Myotubes lacking the Cav1.1-α1 subunit released almost no ATP after electrical stimulation, showing that Cav1.1 has a central role in this process. In adult muscle fibers, ATP release and the transcriptional changes produced by 20 Hz stimulation were blocked by both the Cav1.1 antagonist nifedipine (25 µM) and by the Cav1.1 agonist (-)S-BayK 8644 (10 µM). We propose a new role for Cav1.1, independent of its calcium channel activity, in the activation of signaling pathways allowing muscle fibers to decipher the frequency of electrical stimulation and to activate specific transcriptional programs that define their phenotype.

Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration.

The endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions essential for cellular homeostasis, development, and stress responsiveness. In response to cellular stress, a well-established signaling cascade, the unfolded protein response (UPR), is activated. This intricate mechanism is an important means of re-establishing cellular homeostasis and alleviating the inciting stress. Now, emerging evidence has demonstrated that the UPR influences cellular metabolism through diverse mechanisms, including calcium and lipid transfer, raising the prospect of involvement of these processes in the pathogenesis of disease, including neurodegeneration, cancer, diabetes mellitus and cardiovascular disease. Here, we review the distinct functions of the ER and UPR from a metabolic point of view, highlighting their association with prevalent pathologies.