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Skeletal muscle atrophy is caused by disruption in the homeostatic balance of muscle degeneration and regeneration under various pathophysiological conditions. We have previously reported that iron accumulation induces skeletal muscle atrophy via a ubiquitin ligase-dependent pathway. However, the potential effect of iron accumulation on muscle regeneration remains unclear. To examine the effect of iron accumulation on myogenesis, we used a mouse model with cardiotoxin (CTX)-induced muscle regeneration in vivo and C2C12 mouse myoblast cells in vitro . In mice with iron overload, the skeletal muscles exhibited increased oxidative stress and decreased expression of satellite cell markers. Following CTX-induced muscle injury, these mice also displayed delayed muscle regeneration with a decrease in the size of regenerating myofibers, reduced expression of myoblast differentiation markers, and decreased phosphorylation of MAPK signaling pathways. In vitro , iron overload also suppressed the differentiation of C2C12 myoblast cells but the suppression could be reversed by superoxide scavenging using tempol. Excess iron inhibits myogenesis via oxidative stress, leading to an imbalance in skeletal muscle homeostasis.-Ikeda, Y., Satoh, A., Horinouchi, Y., Hamano, H., Watanabe, H., Imao, M., Imanishi, M., Zamami, Y., Takechi, K., Izawa-Ishizawa, Y., Miyamoto, L., Hirayama, T., Nagasawa, H., Ishizawa, K., Aihara, K.-I., Tsuchiya, K., Tamaki, T. Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.

Skeletal muscle wasting or sarcopenia is a critical health problem. Skeletal muscle atrophy is induced by an excess of iron, which is an essential trace metal for all living organisms. Excessive amounts of iron catalyze the formation of highly toxic hydroxyl radicals via the Fenton reaction. However, the molecular mechanism of iron-induced skeletal muscle atrophy has remained unclear. In this study, 8-weeks-old C57BL6/J mice were divided into 2 groups: vehicle-treated group and the iron-injected group (10 mg iron day(-1)mouse(-1)) during 2 weeks. Mice in the iron-injected group showed an increase in the iron content of the skeletal muscle and serum and ferritin levels in the muscle, along with reduced skeletal muscle mass. The skeletal muscle showed elevated mRNA expression of the muscle atrophy-related E3 ubiquitin ligases, atrogin-1 and muscle ring finger-1(MuRF1), on days 7 and 14 of iron treatment. Moreover, iron-treated mice showed reduced phosphorylation of Akt and forkhead box O3 (FOXO3a) in skeletal muscles. Inhibition of FOXO3a using siRNA in vitro in C2C12 myotube cells inhibited iron-induced upregulation of atrogin-1 and MuRF1 and reversed the reduction in myotube diameters. Iron-load caused oxidative stress, and an oxidative stress inhibitor abrogated iron-induced muscle atrophy by reactivating the Akt-FOXO3a pathway. Iron-induced skeletal muscle atrophy is suggested to involve the E3 ubiquitin ligase mediated by the reduction of Akt-FOXO3a signaling by oxidative stress., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Physical exercise has potent therapeutic and preventive effects against metabolic disorders. A number of studies have suggested that 5'-AMP-activated protein kinase (AMPK) plays a pivotal role in regulating carbohydrate and lipid metabolism in contracting skeletal muscles, while several genetically manipulated animal models revealed the significance of AMPK-independent pathways. To elucidate significance of AMPK and AMPK-independent signals in contracting skeletal muscles, we conducted a metabolomic analysis that compared the metabolic effects of 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) stimulation with the electrical contraction ex vivo in isolated rat epitrochlearis muscles, in which both α1- and α2-isoforms of AMPK and glucose uptake were equally activated. The metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry detected 184 peaks and successfully annotated 132 small molecules. AICAR stimulation exhibited high similarity to the electrical contraction in overall metabolites. Principal component analysis (PCA) demonstrated that the major principal component characterized common effects whereas the minor principal component distinguished the difference. PCA and a factor analysis suggested a substantial change in redox status as a result of AMPK activation. We also found a decrease in reduced glutathione levels in both AICAR-stimulated and contracting muscles. The muscle contraction-evoked influences related to the metabolism of amino acids, in particular, aspartate, alanine, or lysine, are supposed to be independent of AMPK activation. Our results substantiate the significance of AMPK activation in contracting skeletal muscles and provide novel evidence that AICAR stimulation closely mimics the metabolomic changes in the contracting skeletal muscles.

Aim of the Study: Morus alba (mulberry) leaf is a natural therapeutic agent that has been shown to have an antidiabetic effect. We explored the possibility that 5'-AMP-activated protein kinase (AMPK) is involved in metabolic enhancement by the Morus alba leaf., Materials and Methods: Isolated rat epitrochlearis muscle was incubated in a buffer containing Morus alba leaf hot water extract (MLE) and the AMPK activation and related events were examined., Results: In response to MLE treatment, the Thr(172) phosphorylation of the catalytic alpha subunit of AMPK, an essential step for full kinase activation increased in a dose- and time-dependent manner. Ser(79) phosphorylation of acetyl CoA carboxylase, an intracellular substrate of AMPK, increased similarly. Analysis of isoform-specific AMPK activity revealed that MLE activated both the alpha1 and alpha2 isoforms of the catalytic subunit. This increase in enzyme activity was associated with an increased rate of 3-O-methyl-D-glucose transport in the absence of insulin and with phosphorylation of AS160, a signaling intermediary leading to glucose transporter 4 translocation. The intracellular energy status, estimated from the ATP and phosphocreatine concentrations, was not affected by MLE., Conclusion: MLE stimulates skeletal muscle AMPK activity acutely without changing the intracellular energy status.

5'-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the alpha(1)- and alpha(2)-isoforms of AMPK, with a corresponding increase in the rate of 3-O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3-O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis.

5'Adenosine monophosphate-activated protein kinase (AMPK) has been implicated in exercise-induced stimulation of glucose metabolism in skeletal muscle. Although skeletal muscle expresses both the alpha1 and alpha2 isoforms of AMPK, the alpha2 isoform is activated predominantly in response to moderate-intensity endurance exercise in human and animal muscles. The purpose of this study was to determine whether activation of alpha2 AMPK plays a role in increasing the rate of glucose transport, promoting glucose transporter 4 (GLUT4) expression, and enhancing insulin sensitivity in skeletal muscle. To selectively activate the alpha2 isoform, we used 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), which is metabolized in muscle cells and preferentially stimulates the alpha2 isoform. Subcutaneous administration of 250 mg/kg AICAR activated the alpha2 isoform for 90 minutes, but not the alpha1 isoform in hind limb muscles of the C57/B6J mouse. The maximal activation of the alpha2 isoform was observed 30 to 60 minutes after administration of AICAR and was similar to the activation induced by a 30-minute swim in a current pool. The increase in alpha2 activity paralleled the phosphorylation of Thr(172), the essential residue for full kinase activation, and the activity of acetyl-coenzyme A carboxylase beta, a known substrate of AMPK in skeletal muscle. Subcutaneous injection of AICAR rapidly increased, by 30%, the rate of 2-deoxyglucose (2DG) transport into soleus muscle; 2DG transport increased within 30 minutes and remained elevated for 4 hours after administration of AICAR. Repeated intraperitoneal injection of AICAR, 3 times a day for 4 to 7 days, increased soleus GLUT4 protein by 30% concomitant with a significant 20% increase in insulin-stimulated 2DG transport. These data suggest that moderate endurance exercise promotes glucose transport, GLUT4 expression, and insulin sensitivity in skeletal muscle at least partially via activation of the alpha2 isoform of AMPK.

Recent studies have suggested that 5'AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H(2)O(2), AMPKalpha1 activity increased in a time- and dose-dependent manner, whereas AMPKalpha2 activity remained unchanged. The activation of AMPKalpha1 was associated with phosphorylation of AMPK Thr(172), suggesting that an upstream kinase is involved in the activation process. H(2)O(2)-induced AMPKalpha1 activation was blocked in the presence of the antioxidant N-acetyl-l-cysteine (NAC), and H(2)O(2) significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H(2)O(2) did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H(2)O(2) increased 3-O-methyl-d-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPKalpha1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPKalpha1-mediated mechanism.