Arginine promotes skeletal muscle fiber type transformation from fast-twitch to slow-twitch via Sirt1/AMPK pathway

This study investigated the effect of arginine on skeletal muscle fiber type transformation in mice and in C2C12 myotubes. Our data showed that dietary supplementation of arginine in mice significantly up-regulated the slow myosin heavy chain (MyHC), troponin I-SS, sirtuin1 (Sirt1) and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) protein expressions, as well as significantly down-regulated the fast MyHC protein expression. In C2C12 myotubes, arginine significantly increased the protein level of slow MyHC and the number of slow MyHC-positive cells, as well as significantly decreased the protein level of fast MyHC and the number of fast MyHC-positive cells. We also showed that arginine increased the activities of succinic dehydrogenase and malate dehydrogenase and decreased the activity of lactate dehydrogenase in mice and in C2C12 myotubes. Here we found that AMP-activated protein kinase (AMPK) was activated by arginine in mice and in C2C12 myotubes. However, inhibition of AMPK activity by compound C significantly attenuated the effects of arginine on slow MyHC and fast MyHC expressions in C2C12 myotubes. Finally, we showed that inhibition of Sirt1 expression by EX527 attenuated arginine-induced increase in the protein levels of phospho-AMPK and slow MyHC, the mRNA level of nitric oxide synthase (NOS) and the contents of NOS and NO, as well as decrease in fast MyHC protein level. Together, our findings indicated that arginine promotes skeletal muscle fiber type switching from fast-twitch to slow-twitch via Sirt1/AMPK pathway.     

Resveratrol regulates muscle fiber type conversion via miR-22-3p and AMPK/SIRT1/PGC-1α pathway

This study investigated the effects of resveratrol and miR-22-3p on muscle fiber type conversion in mouse C2C12 myotubes. Here we showed that resveratrol significantly increased the protein level of slow myosin heavy chain (MyHC) and the activities of succinic dehydrogenase and malate dehydrogenase, as well as markedly decreased the protein level of fast MyHC and the activity of lactate dehydrogenase. Immunofluorescence staining showed that resveratrol remarkably upregulated the number of slow MyHC-positive myotubes and downregulated the number of fast MyHC-positive myotubes, suggesting that resveratrol promoted muscle fiber type conversion from fast-twitch to slow-twitch in C2C12 myotubes. We also showed that miR-22-3p had an opposite function on muscle fiber type conversion and resveratrol was able to repress the expression of miR-22-3p. Furthermore, AMP-activated protein kinase (AMPK) inhibitor Compound C and miR-22-3p mimics could attenuate and eliminate muscle fiber type conversion from fast-twitch to slow-twitch cause by resveratrol, respectively. Together, we provided the first evidence that resveratrol promotes muscle fiber type conversion from fast-twitch to slow-twitch via miR-22-3p and AMPK/SIRT1/PGC-1α pathway in C2C12 myotubes.     

Diversity of myosin heavy chain expression in satellite cells from mouse soleus and EDL muscles.

Postnatal myoblasts, the satellite cells, originating from slow and fast skeletal muscle fibres differentiate and fuse into myotubes expressing different phenotype of myosin heavy chain (MyHC) isoforms. Little is known, however, of factors which establish and maintain this phenotypic diversity. We used immunofluorescent labelling and Western blotting to examine the expression of slow and fast MyHC isoforms in myotubes formed in vitro from satellite cells isolated from mouse fast twitch extensor digitorum longus (EDL) and slow twitch soleus muscles. Satellite cells were cultured in serum-rich growth medium promoting myoblast proliferation until cross-striated and self-contracting myotubes were formed. We report that in both cultures myotubes expressed slow as well as fast MyHC isoforms, but the level of slow MyHC was higher in soleus culture than in EDL culture. Hence, the pattern of expression of slow and fast MyHC was characteristic of the muscle fibre type from which these cells derive. These results support the concept of phenotypic diversity among satellite cells in mature skeletal muscles and suggest that this diversity is generated in vitro irrespectively of serum mitogens.     

Lycopene increases the proportion of slow-twitch muscle fiber by AMPK signaling to improve muscle anti-fatigue ability

Lycopene has a wide range of biological functions, especially its antioxidant capacity. However, effects of lycopene on muscle fatigue resistant and muscle fiber type conversion are unknown. In this study, we found that lycopene significantly prolonged the swimming time to exhaustion in mice. We also showed that lycopene increased the proportion of slow-twitch muscle fiber by promoting muscle fiber type conversion from fast-twitch to slow-twitch in mice and in C2C12 myotubes. The AMP-activated protein kinase (AMPK) signaling was activated by lycopene. AMPK upstream and downstream regulators including nuclear respiratory factor 1, calcium calmodulin-dependent protein kinase kinase-β, sirtuin 1 and peroxisome proliferator activated receptor-γ coactivator-1ɑ were also increased by lycopene. AMPK inhibitor compound C markedly attenuated the lycopene-induced skeletal muscle fiber type conversion in C2C12 myotubes. Taken together, we provided the first evidence that lycopene increases the proportion of slow-twitch muscle fiber through AMPK signaling pathway to improve fatigue resistant of skeletal muscle.     

Rescue of dystrophic skeletal muscle by PGC-1α involves a fast to slow fiber type shift in the mdx mouse

Increased utrophin expression is known to reduce pathology in dystrophin-deficient skeletal muscles. Transgenic over-expression of PGC-1α has been shown to increase levels of utrophin mRNA and improve the histology of mdx muscles. Other reports have shown that PGC-1α signaling can lead to increased oxidative capacity and a fast to slow fiber type shift. Given that it has been shown that slow fibers produce and maintain more utrophin than fast skeletal muscle fibers, we hypothesized that over-expression of PGC-1α in post-natal mdx mice would increase utrophin levels via a fiber type shift, resulting in more slow, oxidative fibers that are also more resistant to contraction-induced damage. To test this hypothesis, neonatal mdx mice were injected with recombinant adeno-associated virus (AAV) driving expression of PGC-1α. PGC-1α over-expression resulted in increased utrophin and type I myosin heavy chain expression as well as elevated mitochondrial protein expression. Muscles were shown to be more resistant to contraction-induced damage and more fatigue resistant. Sirt-1 was increased while p38 activation and NRF-1 were reduced in PGC-1α over-expressing muscle when compared to control. We also evaluated if the use a pharmacological PGC-1α pathway activator, resveratrol, could drive the same physiological changes. Resveratrol administration (100 mg/kg/day) resulted in improved fatigue resistance, but did not achieve significant increases in utrophin expression. These data suggest that the PGC-1α pathway is a potential target for therapeutic intervention in dystrophic skeletal muscle.     

Chronic intake of proanthocyanidins and docosahexaenoic acid improves skeletal muscle oxidative capacity in diet-obese rats

Obesity has become a worldwide epidemic. The cafeteria diet (CD) induces obesity and oxidative-stress-associated insulin resistance. Polyunsaturated fatty acids and polyphenols are dietary compounds that are intensively studied as products that can reduce the health complications related to obesity. We evaluate the effects of 21 days of supplementation with grape seed proanthocyanidins extract (GSPE), docosahexaenoic-rich oil (DHA-OR) or both compounds (GSPE+DHA-OR) on skeletal muscle metabolism in diet-obese rats. The supplementation with different treatments did not reduce body weight, although all groups used more fat as fuel, particularly when both products were coadministered; muscle β-oxidation was activated, the mitochondrial functionality and oxidative capacity were higher, and fatty acid uptake gene expressions were up-regulated. In addition to these outcomes shared by all treatments, GSPE reduced insulin resistance and improved muscle status. Both treatments increased 5’-AMP-activated protein kinase (AMPK) phosphorylation, which was consistent with higher plasma adiponectin levels. Moreover, AMPK activation by DHA-OR was also correlated with an up-regulation of peroxisome proliferator-activated receptor alpha (Pparα). GSPE+DHA-OR, in addition to activating AMPK and enhancing fatty acid oxidation, increased the muscle gene expression of uncoupling protein 2 (Ucp2). In conclusion, GSPE+DHA-OR induced modifications that improved muscle status and could counterbalance the deleterious effects of obesity, and such modifications are mediated, at least in part, through the AMPK signaling pathway.     

Cold-induced PGC-1alpha expression modulates muscle glucose uptake through an insulin receptor/Akt-independent, AMPK-dependent pathway

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) participates in control of expression of genes involved in adaptive thermogenesis, muscle fiber type differentiation, and fuel homeostasis. The objective of the present study was to evaluate the participation of cold-induced PGC-1alpha expression in muscle fiber type-specific activity of proteins that belong to the insulin-signaling pathway. Rats were exposed to 4 degrees C for 4 days and acutely treated with insulin in the presence or absence of an antisense oligonucleotide to PGC-1alpha. Cold exposure promoted a significant increase of PGC-1alpha and uncoupling protein-3 protein expression in type I and type II fibers of gastrocnemius muscle. In addition, cold exposure led to higher glucose uptake during a hyperinsulinemic clamp, which was accompanied by higher expression and membrane localization of GLUT4 in both muscle fiber types. Cold exposure promoted significantly lower insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and Ser473 phosphorylation of acute transforming retrovirus thymoma (Akt) and an insulin-independent increase of Thr172 phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Inhibition of PGC-1alpha expression in cold-exposed rats by antisense oligonucleotide treatment diminished glucose clearance rates during a hyperinsulinemic clamp and reduced expression and membrane localization of GLUT4. Reduction of PGC-1alpha expression resulted in no modification of insulin-induced tyrosine phosphorylation of the IR and Ser473 phosphorylation of Akt. Finally, reduction of PGC-1alpha resulted in lower Thr172 phosphorylation of AMPK. Thus cold-induced hyperexpression of PGC-1alpha participates in control of skeletal muscle glucose uptake through a mechanism that controls GLUT4 expression and subcellular localization independent of the IR and Akt activities but dependent on AMPK.     

Angiotensin II receptor blocker telmisartan enhances running endurance of skeletal muscle through activation of the PPAR‐δ/AMPK pathway

Clinical trials have shown that angiotensin II receptor blockers reduce the new onset of diabetes in hypertensives; however, the underlying mechanisms remain unknown. We investigated the effects of telmisartan on peroxisome proliferator activated receptor γ (PPAR‐δ) and the adenosine monophosphate (AMP)‐activated protein kinase (AMPK) pathway in cultured myotubes, as well as on the running endurance of wild‐type and PPAR‐δ‐deficient mice. Administration of telmisartan up‐regulated levels of PPAR‐δ and phospho‐AMPKα in cultured myotubes. However, PPAR‐δ gene deficiency completely abolished the telmisartan effect on phospho‐AMPKαin vitro. Chronic administration of telmisartan remarkably prevented weight gain, enhanced running endurance and post‐exercise oxygen consumption, and increased slow‐twitch skeletal muscle fibres in wild‐type mice, but these effects were absent in PPAR‐δ‐deficient mice. The mechanism is involved in PPAR‐δ‐mediated stimulation of the AMPK pathway. Compared to the control mice, phospho‐AMPKα level in skeletal muscle was up‐regulated in mice treated with telmisartan. In contrast, phospho‐AMPKα expression in skeletal muscle was unchanged in PPAR‐δ‐deficient mice treated with telmisartan. These findings highlight the ability of telmisartan to improve skeletal muscle function, and they implicate PPAR‐δ as a potential therapeutic target for the prevention of type 2 diabetes.     

Sensitivity of cultured and skinned chick myotube to calcium, strontium, and barium ions examined by recording isometric contractions.

Sensitivity of cultured chick myotubes to alkaline earth metal ions was investigated by recording contractile isometric tension through a semiconductor transducer. The myotubes were obtained by culturing myoblasts of chick embryo breast muscles, and skinned chemically before physiological experiments. Contractions developed in response to Ca2+ in a bathing medium higher than 3 x 10(-7) M and reached maximum at 1 x 10(-5) M. Sr2+ was less effective than Ca2+; the threshold concentration was 1 x 10(-5) M and the tension reached maximum at 1 x 10(-3) M. Ba2+ was the least effective among the three alkaline earth metal ions; only one fifth of the Ca(2+)-induced maximum tension was attained at 1 x 10(-3) M. The sensitivity was similar to that of the mature pectoral muscle fiber, a fast twitch muscle fiber, rather than that of the anterior latissimus dorsi, a slow tonic muscle fiber. The sensitivity was shown to be dependent on its troponin C by replacing it with troponin C from the mature pectoral or cardiac muscle. This indicates that TnC of a fast-muscle type is expressed in the cultured chick myotube as in the mature pectoral muscle. The contractile apparatus was thus shown to be well developed in the cultured myotube with characteristics similar to the mature fast twitch muscle fiber.