Moderate exercise is an antioxidant: upregulation of antioxidant genes by training

Exercise causes oxidative stress only when exhaustive. Strenuous exercise causes oxidation of glutathione, release of cytosolic enzymes, and other signs of cell damage. However, there is increasing evidence that reactive oxygen species (ROS) not only are toxic but also play an important role in cell signaling and in the regulation of gene expression. Xanthine oxidase is involved in the generation of superoxide associated with exhaustive exercise. Allopurinol (an inhibitor of this enzyme) prevents muscle damage after exhaustive exercise, but also modifies cell signaling pathways associated with both moderate and exhaustive exercise in rats and humans. In gastrocnemius muscle from rats, exercise caused an activation of MAP kinases. This in turn activated the NF-kappaB pathway and consequently the expression of important enzymes associated with defense against ROS (superoxide dismutase) and adaptation to exercise (eNOS and iNOS). All these changes were abolished when ROS production was prevented by allopurinol. Thus ROS act as signals in exercise because decreasing their formation prevents activation of important signaling pathways that cause useful adaptations in cells. Because these signals result in an upregulation of powerful antioxidant enzymes, exercise itself can be considered an antioxidant. We have found that interfering with free radical metabolism with antioxidants may hamper useful adaptations to training.     

Insight into skeletal muscle mechanotransduction: MAPK activation is quantitatively related to tension.

The mechanism by which mechanical forces acting through skeletal muscle cells generate intracellular signaling, known as mechanotransduction, and the details of how gene expression and cell size are regulated by this signaling are poorly understood. Mitogen-activated protein kinases (MAPKs) are known to be involved in mechanically induced signaling in various cell types, including skeletal muscle where MAPK activation has been reported in response to contraction and passive stretch. Therefore, the investigation of MAPK activation in response to mechanical stress in skeletal muscle may yield important information about the mechanotransduction process. With the use of a rat plantaris in situ preparation, a wide range of peak tensions was generated through passive stretch and concentric, isometric, and eccentric contractile protocols, and the resulting phosphorylation of c-Jun NH(2)-terminal kinase (JNK), extracellular regulated kinase (ERK), and p38 MAPKs was assessed. Isoforms of JNK and ERK MAPKs were found to be phosphorylated in a tension-dependent manner, such that eccentric > isometric > concentric > passive stretch. Peak tension was found to be a better predictor of MAPK phosphorylation than time-tension integral or rate of tension development. Differences in maximal response amplitude and sensitivity between JNK and ERK MAPKs suggest different roles for these two kinase families in mechanically induced signaling. A strong linear relationship between p54 JNK phosphorylation and peak tension over a 15-fold range in tension (r(2) = 0.89, n = 32) was observed, supporting the fact that contraction-type differences can be explained in terms of tension and demonstrating that MAPK activation is a quantitative reflection of the magnitude of mechanical stress applied to muscle. Thus the measurement of MAPK activation, as an assay of skeletal muscle mechanotransduction, may help elucidate mechanically induced hypertrophy.     

IL-1beta stimulates IL-6 production in cultured skeletal muscle cells through activation of MAP kinase signaling pathway and NF-kappa B

Recent studies suggest that the skeletal muscle may be a significant site of IL-6 production in various conditions, including exercise, inflammation, hypoperfusion, denervation, and local muscle injury. The mediators and molecular mechanisms regulating muscle IL-6 production are poorly understood. We tested the hypothesis that IL-6 production in muscle cells is regulated by IL-1beta and that mitogen-activated protein (MAP) kinase signaling and NF-kappaB activation are involved in IL-1beta-induced IL-6 production. Cultured C2C12 cells, a mouse skeletal muscle cell line, were treated with different concentrations (0.1-2 ng/ml) of IL-1beta in the absence or presence of the p38 MAP kinase inhibitor SB-208350 or the p42/44 inhibitor PD-98059. Protein and gene expression of IL-6 were determined by ELISA and real-time PCR, respectively. NF-kappaB DNA binding activity was determined by electrophoretic mobility shift assay and by transfecting myocytes with a luciferase reporter plasmid containing a promoter construct with multiple repeats of NF-kappaB binding site. Treatment of myotubes with IL-1beta resulted in a dose- and time-dependent increase of IL-6 production accompanied by an approximately 25-fold increase in IL-6 mRNA levels. IL-1beta stimulated NF-kappaB DNA binding activity and gene activation. SB-208350 and PD-98059 inhibited the increase in IL-6 production induced by IL-1beta. The present results support the concept that skeletal muscle is an important site of IL-6 production. In addition, the results suggest the IL-1beta regulates muscle IL-6 production at least in part by activating the MAP kinase pathway and NF-kappaB.     

Up-regulation of mitogen activated protein kinases in mdx skeletal muscle following chronic treadmill exercise

Dystrophin, a product of the Duchenne muscular dystrophy gene, is a cytoskeletal protein of skeletal and cardiac muscle fibers. Dystrophin-deficient muscle fibers are abnormally vulnerable to mechanical stress including physical exercise, which is a powerful stimulator of mitogen-activated protein kinases (MAPKs). To examine how treadmill exercise affects MAPK family members in dystrophin-deficient skeletal muscle, we subjected both mdx mice, an animal model for Duchenne muscular dystrophy, and C57BL/10 mice to treadmill exercise and examined the phosphorylated protein levels of extracellular-signal regulated kinase (ERK1/2), p38 MAPK and c-Jun N terminal kinase 1 and 2 (JNK1 and JNK2) in the gastrocnemius muscle. Phosphorylation of ERK1/2, p38 MAPK and JNK2, but not JNK1, increased more in the muscles of exercise trained mdx mice than in muscles of trained C57BL/10 or untrained mdx mice. These results show that physical exercise aberrantly up-regulates the phosphorylated form of ERK1/2, p38 MAPK and JNK2 in dystrophin-deficient skeletal muscle and that their up-regulation might play a role in the degeneration and regeneration process of dystrophic features.     

Modulation of skeletal muscle antioxidant defense by exercise: Role of redox signaling

Contraction-induced production of reactive oxygen species has been shown to cause oxidative stress to skeletal muscle. As an adaptive response, muscle antioxidant defense systems are upregulated in response to exercise. Nuclear factor kappaB and mitogen-activated protein kinase are two major oxidative-stress-sensitive signal transduction pathways that have been shown to activate the gene expression of a number of enzymes and proteins that play important roles in maintenance of intracellular oxidant-antioxidant homeostasis. This mini-review will discuss the main mechanisms and gene targets for these signaling pathways during exercise and the biological significance of the adaptation.     

Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. Involvement Of the mitogen- and stress-activated protein kinase 1

Growing evidence suggests that activation of mitogen-activated protein kinase (MAPK) signal transduction mediates changes in muscle gene expression in response to exercise. Nevertheless, little is known about upstream or downstream regulation of MAPK in response to muscle contraction. Here we show that ex vivo muscle contraction stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38(MAPK) phosphorylation. Phosphorylation of ERK1/2 or p38(MAPK) was unaffected by protein kinase C inhibition (GF109203X), suggesting that protein kinase C is not involved in mediating contraction-induced MAPK signaling. Contraction-stimulated phosphorylation of ERK1/2 and p38(MAPK) was completely inhibited by pretreatment with PD98059 (MAPK kinase inhibitor) and SB203580 (p38(MAPK) inhibitor), respectively. Muscle contraction also activated MAPK downstream targets p90 ribosomal S6 kinase (p90(Rsk)), MAPK-activated protein kinase 2 (MAPKAP-K2), and mitogen- and stress-activated protein kinase 1 (MSK1). Use of PD98059 or SB203580 revealed that stimulation of p90(Rsk) and MAPKAP-K2 most closely reflects ERK and p38(MAPK) stimulation, respectively. Stimulation of MSK1 in contracting skeletal muscle required the activation of both ERK and p38(MAPK). These data demonstrate that muscle contraction, separate from systemic influence, activates MAPK signaling. Furthermore, we are the first to show that contractile activity stimulates MAPKAP-K2 and MSK1.     

IGF-I/PI3K/Akt and IGF-I/MAPK/ERK pathways in vivo in skeletal muscle are regulated by nutrition and contribute to somatic growth in the fine flounder

The insulin-like growth factor-I (IGF-I) is a key regulator of skeletal muscle growth in vertebrates, promoting mitogenic and anabolic effects through the activation of the MAPK/ERK and the PI3K/Akt signaling pathways. Nutrition also affects skeletal muscle growth, activating intracellular pathways and inducing protein synthesis and accretion. Thus, both hormonal and nutritional signaling regulate muscle mass. In this context, plasma IGF-I levels and the activation of both pathways in response to food were evaluated in the fine flounder using fasting and refeeding trials. The present study describes for the first time in a nonmammalian species that the MAPK/ERK and PI3K/Akt are activated by exogenous circulating IGF-I, as well as showing that the MAPK/ERK pathway activation is modulated by the nutritional status. Also, these results show that there is a time-dependent regulation of IGF-I plasma levels and its signaling pathways in muscle. Together, these results suggest that the nutritionally managed IGF-I could be regulating the activation of the MAPK/ERK and the PI3K/Akt signaling pathways differentially according to the nutritional status, triggering different effects in growth parameters and therefore contributing to somatic growth in fish. This study contributes to the understanding of the nutrient regulation of IGF-I and its signaling pathways in skeletal muscle growth in nonmammalian species, therefore providing insight concerning the events controlling somatic growth in vertebrates.     

Activation mechanisms of endothelial NF-kappaB, IKK, and MAP kinase by tert-butyl hydroperoxide

Lipid peroxidation plays a major role in vascular dysfunction and age-related cardiovascular diseases. A major product of lipid peroxidation, tert-butyl hydroperoxide (t-BHP), has been reported to modulate vascular reactivity and cellular signaling. To better understand vascular abnormality, we set out to delineate the activation mechanism of nuclear factor kappa B (NF-kappaB) by t-BHP and the regulation of MAPK in endothelial cells. The results showed that t-BHP induces NF-kappaB activation by an inhibitor of kappaB (IkappaB) phosphorylation through IkappaB kinase (IKK) activation. Our data from this t-BHP study also showed increased p38 MAP kinase and ERK activity; however, interestingly, t-BHP showed no influence on JNK. Pretreatment with the p38 MAP kinase inhibitor, SB203580 and the ERK1/2 inhibitor, PD98059, prevented t-BHP-induced increases in p65 translocation, NF-kappaB luciferase activity, and phospho-IKKalpha/beta. Data suggested that t-BHP induces NF-kappaB activation through the IKK pathway, which involves p38 MAPK and ERK activation. This study illustrates a role of t-BHP in NF-kappaB activation and MAPK related-signaling pathways. The t-BHP-induced activation of NF-kappaB and MAPK could be a major player in vascular dysfunctions, as seen in oxidative stressed responses and the vascular inflammatory process.     

Angiotensin II-induced skeletal muscle insulin resistance mediated by NF-kappaB activation via NADPH oxidase

Reduced insulin sensitivity is a key factor in the pathogenesis of type 2 diabetes and hypertension. Skeletal muscle insulin resistance is particularly important for its major role in insulin-mediated glucose disposal. Angiotensin II (ANG II) is integral in regulating blood pressure and plays a role in the pathogenesis of hypertension. In addition, we have documented that ANG II-induced skeletal muscle insulin resistance is associated with generation of reactive oxygen species (ROS). However, the linkage between ROS and insulin resistance in skeletal muscle remains unclear. To explore potential mechanisms, we employed the transgenic TG(mRen2)27 (Ren-2) hypertensive rat, which harbors the mouse renin transgene and exhibits elevated tissue ANG II levels, and skeletal muscle cell culture. Compared with Sprague-Dawley normotensive control rats, Ren-2 skeletal muscle exhibited significantly increased oxidative stress, NF-kappaB activation, and TNF-alpha expression, which were attenuated by in vivo treatment with an angiotensin type 1 receptor blocker (valsartan) or SOD/catalase mimetic (tempol). Moreover, ANG II treatment of L6 myotubes induced NF-kappaB activation and TNF-alpha production and decreased insulin-stimulated Akt activation and GLUT-4 glucose transporter translocation to plasma membranes. These effects were markedly diminished by treatment of myotubes with valsartan, the antioxidant N-acetylcysteine, NADPH oxidase-inhibiting peptide (gp91 ds-tat), or NF-kappaB inhibitor (MG-132). Similarly, NF-kappaB p65 small interfering RNA reduced NF-kappaB p65 subunit expression and nuclear translocation and TNF-alpha production but improved insulin-stimulated phosphorylation (Ser(473)) of Akt and translocation of GLUT-4. These findings suggest that NF-kappaB plays an important role in ANG II/ROS-induced skeletal muscle insulin resistance.     

The effects of acetaldehyde and acrolein on muscle catabolism in C2 myotubes

The toxic aldehydes acetaldehyde and acrolein were previously suggested to damage skeletal muscle. Several conditions in which exposure to acetaldehyde and acrolein is increased were associated with muscle wasting and dysfunction. These include alcoholic myopathy, renal failure, oxidative stress, and inflammation. A main exogenous source of both acetaldehyde and acrolein is cigarette smoking, which was previously associated with increased muscle catabolism. Recently, we have shown that exposure of skeletal myotubes to cigarette smoke stimulated muscle catabolism via increased oxidative stress, activation of p38 MAPK, and upregulation of muscle-specific E3 ubiquitin ligases. In this study, we aimed to investigate the effects of acetaldehyde and acrolein on catabolism of skeletal muscle. Skeletal myotubes differentiated from the C2 myoblast cell line were exposed to acetaldehyde or acrolein and their effects on signaling pathways related to muscle catabolism were studied. Exposure of myotubes to acetaldehyde did not promote muscle catabolism. However, exposure to acrolein caused increased generation of free radicals, activation of p38 MAPK, upregulation of the muscle-specific E3 ligases atrogin-1 and MuRF1, degradation of myosin heavy chain, and atrophy of myotubes. Inhibition of p38 MAPK by SB203580 abolished acrolein-induced muscle catabolism. Our findings demonstrate that acrolein but not acetaldehyde activates a signaling cascade resulting in muscle catabolism in skeletal myotubes. Although within the limitations of an in vitro study, these findings indicate that acrolein may promote muscle wasting in conditions of increased exposure to this aldehyde.