Extract of Phyllanthus emblica L. fruit stimulates basal glucose uptake and ameliorates palmitate-induced insulin resistance through AMPK activation in C2C12 myotubes.

BACKGROUND: The fruit of Phyllanthus emblica L., a traditional medicine in China and India, is used to treat diabetes mellitus. Its water extract (WEPE) has demonstrated hypoglycemic effects in diabetic rats, but its mechanisms on glucose utilization and insulin resistance in skeletal muscle remain unclear. Therefore, this study aims to investigate the effects and underlying mechanisms of WEPE on glucose utilization and insulin resistance using C2C12 myotubes. METHODS: Effects of WEPE on glucose uptake, GLUT4 translocation, and AMPK and AKT phosphorylation were investigated in C2C12 myotubes and palmitate-treated myotubes. An AMPK inhibitor and siRNA were used to explore the mechanisms of WEPE. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay, and protein expression and GLUT4 translocation were assessed via western blotting. RESULTS: In normal myotubes, WEPE significantly stimulated glucose uptake and GLUT4 translocation to the plasma membrane at concentrations of 125 and 250 µg/mL. This was accompanied by an increase in the phosphorylation of AMPK and its downstream targets. However, both compound C and AMPK siRNA blocked the WEPE-induced GLUT4 translocation and glucose uptake. Moreover, pretreatment with STO-609, a calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor, inhibited WEPE-induced AMPK phosphorylation and attenuated the WEPE-stimulated glucose uptake and GLUT4 translocation. In myotubes treated with palmitate, WEPE prevented palmitate-induced insulin resistance by enhancing insulin-mediated glucose uptake and AKT phosphorylation. It also restored the insulin-mediated translocation of GLUT4 from cytoplasm to membrane. However, these effects of WEPE on glucose uptake and GLUT4 translocation were blocked by pretreatment with compound C. CONCLUSIONS: WEPE significantly stimulated basal glucose uptake though CaMKKß/AMPK pathway and markedly ameliorated palmitate-induced insulin resistance by activating the AMPK pathway in C2C12 myotubes.

Generation of Bidimensional and Three-Dimensional Muscle Culture Systems.

Skeletal muscle is a postmitotic tissue composed of contractile myofibers that are oriented and connected to different layers of connective tissue. Nevertheless, adult muscle fibers retain the capacity to regenerate in response to damage, activating the classical muscle stem cell compartment, namely, satellite cells (SCs), which are mitotically quiescent cells until required for growth or repair and are localized between the basal lamina and sarcolemma of myofibers. The transition of SCs from the quiescent state toward activation, commitment, and differentiation involves the genetic and epigenetic adaptation to novel biological functions, entailing dynamic changes in the protein expression profile. Interestingly, some of the activities and signaling regulating proliferation, commitment, differentiation, and survival/apoptosis of satellite cells have been also partially recapitulated in vitro, taking advantage of robust markers, reliable techniques, and reproducible protocols. Over the years, different techniques of muscular cell culture have been designed including primary cultures from embryonic or postnatal muscle, myogenic cell line, and three-dimensional (3D) skeletal muscle construct. Typical two-dimensional (2D) muscle cell culture cannot fully recapitulate the complexity of living muscle tissues, restricting their usefulness for physiological studies. The development of functional 3D culture models represents a valid alternative to overcome the limitations of already available in vitro model, increasing our understanding of the roles played by the various cell types and how they interact. In this chapter, the development of bidimensional and three-dimensional cell cultures have been described, improving the technical aspect of satellite cell isolation, the best culture-based conditions for muscle cell growth and differentiation, and the procedures required to develop a three-dimensional skeletal muscle construct.

Muscle degeneration in aging Drosophila flies: the role of mechanical stress.

Muscle wasting is a universal hallmark of aging which is displayed by a wide range of organisms, although the causes and mechanisms of this phenomenon are not fully understood. We used Drosophila to characterize the phenomenon of spontaneous muscle fiber degeneration (SMFD) during aging. We found that SMFD occurs across diverse types of somatic muscles, progresses with chronological age, and positively correlates with functional muscle decline. Data from vital dyes and morphological markers imply that degenerative fibers most likely die by necrosis. Mechanistically, SMFD is driven by the damage resulting from muscle contractions, and the nervous system may play a significant role in this process. Our quantitative model of SMFD assessment can be useful in identifying and validating novel genetic factors that influence aging-related muscle wasting.

JAK inhibition with tofacitinib rapidly increases contractile force in human skeletal muscle.

Reduction in muscle contractile force associated with many clinical conditions incurs serious morbidity and increased mortality. Here, we report the first evidence that JAK inhibition impacts contractile force in normal human muscle. Muscle biopsies were taken from patients who were randomized to receive tofacitinib (n = 16) or placebo (n = 17) for 48 h. Single-fiber contractile force and molecular studies were carried out. The contractile force of individual diaphragm myofibers pooled from the tofacitinib group (n = 248 fibers) was significantly higher than those from the placebo group (n = 238 fibers), with a 15.7% greater mean maximum specific force (P = 0.0016). Tofacitinib treatment similarly increased fiber force in the serratus anterior muscle. The increased force was associated with reduced muscle protein oxidation and FoxO-ubiquitination-proteasome signaling, and increased levels of smooth muscle MYLK. Inhibition of MYLK attenuated the tofacitinib-dependent increase in fiber force. These data demonstrate that tofacitinib increases the contractile force of skeletal muscle and offers several underlying mechanisms. Inhibition of the JAK-STAT pathway is thus a potential new therapy for the muscle dysfunction that occurs in many clinical conditions.

Comparative Metabolomic Analysis of Moringa oleifera Leaves of Different Geographical Origins and Their Antioxidant Effects on C2C12 Myotubes.

Moringa oleifera is widely grown throughout the tropics and increasingly used for its therapeutic and nutraceutical properties. These properties are attributed to potent antioxidant and metabolism regulators, including glucosinolates/isothiocyanates as well as flavonoids, polyphenols, and phenolic acids. Research to date largely consists of geographically limited studies that only examine material available locally. These practices make it unclear as to whether moringa samples from one area are superior to another, which would require identifying superior variants and distributing them globally. Alternatively, the finding that globally cultivated moringa material is essentially functionally equivalent means that users can easily sample material available locally. We brought together accessions of Moringa oleifera from four continents and nine countries and grew them together in a common garden. We performed a metabolomic analysis of leaf extracts (MOLE) using an LC-MSMS ZenoTOF 7600 mass spectrometry system. The antioxidant capacity of leaf samples evaluated using the Total Antioxidant Capacity assay did not show any significant difference between extracts. MOLE samples were then tested for their antioxidant activity on C2C12 myotubes challenged with an oxidative insult. Hydrogen peroxide (H2O2) was added to the myotubes after pretreatment with different extracts. H2O2 exposure caused an increase in cell death that was diminished in all samples pretreated with moringa extracts. Our results show that Moringa oleifera leaf extract is effective in reducing the damaging effect of H2O2 in C2C12 myotubes irrespective of geographical origin. These results are encouraging because they suggest that the use of moringa for its therapeutic benefits can proceed without the need for the lengthy and complex global exchange of materials between regions.

Anti-muscle atrophy effect of fermented Tenebrio molitor larvae extract by modulating the PI3K-Akt-mTOR/FoxO3α pathway in mice treated with dexamethasone.

This study investigated the anti-sarcopenic effect of fermented Tenebrio molitor larvae (mealworms) extract (FME) in both dexamethasone (DEX)-treated C2C12 cells and mice. FME (100 µg/mL) increased the diameter of myotubes and inhibited the gene and protein expression of atrogin-1 compared to DEX- or non-fermented mealworms extract (ME)-treated C2C12 cells. Male C57BL/6N mice were divided into five groups: Normal Control (NC), DEX (10 mg/kg, intraperitoneal), and three groups of DEX+FME (100, 200, or 500 mg FME/kg/day, oral) for two weeks. FME at doses of 200 and 500 mg/kg effectively improved grip strength when compared to the DEX group. Histological analysis of the quadriceps muscle showed a larger muscle fiber size in the DEX+FME groups compared to DEX group. FME (200 and 500 mg/kg) significantly increased cross-sectional area of the muscle fiber compared to DEX group. FME (500 mg/kg) significantly decreased the ubiquitin, atrogin-1 and MuRF-1 protein levels, and increased levels of MHC and MyoG in DEX-treated mice. The puromycin labeling assay revealed that FME increased protein synthesis in DEX-induced muscle atrophy. The FME treatment demonstrated significant upregulation in phosphorylation levels, including mTOR, FoxO3α, Akt, and PI3K compared to DEX group. In conclusion, FME inhibited the increase in proteins associated with muscle atrophy, including, atrogin-1 and MuRF-1, by regulating the PI3K-Akt-FoxO3α pathway. FME improved the PI3K-Akt-mTOR signaling pathway, which was reduced by DEX. This study suggests that FME has the potential for use in sarcopenia therapy, possibly serving as a natural agent that counteracts the negative effects of DEX on muscle tissue.

KIC (ketoisocaproic acid) and leucine have divergent effects on tissue insulin signaling but not on whole-body insulin sensitivity in rats.

Plasma levels of branched-chain amino acids and their metabolites, the branched-chain ketoacids are increased in insulin resistance. Our previous studies showed that leucine and its metabolite KIC suppress insulin-stimulated glucose uptake in L6 myotubes along with the activation of the S6K1-IRS-1 pathway. Because other tissue and fiber types can be differentially regulated by KIC, we analyzed the effect of KIC gavage on whole-body insulin sensitivity and insulin signaling in vivo. We hypothesized that KIC gavage would reduce whole-body insulin sensitivity and increase S6K1-IRS-1 phosphorylation in various tissues and muscle fibers. Five-week-old male Sprague-Dawley rats were starved for 24 hours and then gavaged with 0.75ml/100g of water, leucine (22.3g/L) or KIC (30g/L) twice, ten minutes apart. They were then euthanized at different time points post-gavage (0.5-3h), and muscle, liver, and heart tissues were dissected. Other sets of gavaged animals underwent an insulin tolerance test. Phosphorylation (ph) of S6K1 (Thr389), S6 (Ser235/6) and IRS-1 (Ser612) was increased at 30 minutes post leucine gavage in skeletal muscles irrespective of fiber type. Ph-S6 (Ser235/6) was also increased in liver and heart 30 minutes after leucine gavage. KIC gavage increased ph-S6 (Ser235/6) in the liver. Neither Leucine nor KIC influenced whole-body insulin tolerance, nor ph-Akt (Ser473) in skeletal muscle and heart. BCKD-E1 α abundance was highest in the heart and liver, while ph-BCKD-E1 α (Ser293) was higher in the gastrocnemius and EDL compared to the soleus. Our data suggests that only leucine activates the S6K1-IRS-1 signaling axis in skeletal muscle, liver and heart, while KIC only does so in the liver. The effect of leucine and KIC on the S6K1-IRS-1 signaling pathway is uncoupled from whole-body insulin sensitivity. These results suggest that KIC and leucine may not induce insulin resistance, and the contributions of other tissues may regulate whole-body insulin sensitivity in response to leucine/KIC gavage.

Glucocorticoid Deflazacort Normalizes the Ultrastructure of Skeletal Muscles and the State of the Colon Microbiota in Dystrophin-Deficient Mice.

We studied the effect of enteral administration of the glucocorticoid deflazacort (DFC, 1.2 mg/kg per day, 28 days) on the state of skeletal muscles and tissue ultrastructure, as well as the composition of the colon microbiota in dystrophin-deficient mdx mice. DFC has been shown to reduce the intensity of degeneration/regeneration cycles in muscle fibers of mdx mice. This effect of DFC was accompanied by normalization of the size of sarcomeres of skeletal muscles of mdx mice, improvement of the ultrastructure of the subsarcolemmal population of mitochondria, and an increase in the number of organelles, as well as normalization of the number of contact interactions between the sarcoplasmic reticulum and mitochondria. In addition, DFC had a corrective effect on the colon microbiota of mdx mice, which manifested in an increase in the number of the Bifidobacterium genus microorganisms and a decrease in the level of E. coli with reduced enzymatic activity.

Sporadic inclusion body myositis-derived myotube culture revealed muscle cell-autonomous expression profiles.

Sporadic inclusion body myositis (sIBM) is a muscle disease in older people and is characterized by inflammatory cell invasion into intact muscle fibers and rimmed vacuoles. The pathomechanism of sIBM is not fully elucidated yet, and controversy exists as to whether sIBM is a primary autoimmune disease or a degenerative muscle disease with secondary inflammation. Previously, we established a method of collecting CD56-positive myoblasts from human skeletal muscle biopsy samples. We hypothesized that the myoblasts derived from these patients are useful to see the cell-autonomous pathomechanism of sIBM. With these resources, myoblasts were differentiated into myotubes, and the expression profiles of cell-autonomous pathology of sIBM were analyzed. Myoblasts from three sIBM cases and six controls were differentiated into myotubes. In the RNA-sequencing analysis of these "myotube" samples, 104 differentially expressed genes (DEGs) were found to be significantly upregulated by more than twofold in sIBM, and 13 DEGs were downregulated by less than twofold. For muscle biopsy samples, a comparative analysis was conducted to determine the extent to which "biopsy" and "myotube" samples differed. Fifty-three DEGs were extracted of which 32 (60%) had opposite directions of expression change (e.g., increased in biopsy vs decreased in myotube). Apolipoprotein E (apoE) and transmembrane protein 8C (TMEM8C or MYMK) were commonly upregulated in muscle biopsies and myotubes from sIBM. ApoE and myogenin protein levels were upregulated in sIBM. Given that enrichment analysis also captured changes in muscle contraction and development, the triggering of muscle atrophy signaling and abnormal muscle differentiation via MYMK or myogenin may be involved in the pathogenesis of sIBM. The presence of DEGs in sIBM suggests that the myotubes formed from sIBM-derived myoblasts revealed the existence of muscle cell-autonomous degeneration in sIBM. The catalog of DEGs will be an important resource for future studies on the pathogenesis of sIBM focusing on primary muscle degeneration.

Sucrose-induced metabolic syndrome differentially affects energy metabolism and fiber phenotype of EDL and soleus muscles during exercise in the rat.

Molecular mechanisms associated to improvement of metabolic syndrome (MetS) during exercise are not fully elucidated. MetS was induced in 250 g male Wistar rats by 30% sucrose in drinking water. Control rats receiving tap water were controls, both groups received solid standard diet. After 14 weeks, an endurance exercised group, and a sedentary were formed for 8 weeks. The soleus and extensor digitorum longus (EDL) muscles were dissected to determine contractile performance, expression of myosin heavy chain isoforms, PGC1α, AMPKα2, NFATC1, MEF2a, SIX1, EYA1, FOXO1, key metabolic enzymes activities. Exercise mildly improved MetS features. MetS didn't alter the contractile performance of the muscles. Exercise didn't altered expression of PGC1α, NFATC1, SIX1 and EYA1 on MetS EDL whereas NFATC1 increased in soleus. Only citrate synthase was affected by MetS on the EDL and this was partially reverted by exercise. Soleus α-ketoglutarate dehydrogenase activity was increased by exercise but MetS rendered the muscle resistant to this effect. MetS affects mostly the EDL muscle, and endurance exercise only partially reverts this. Soleus muscle seems more resilient to MetS. We highlight the importance of studying both muscles during MetS, and their metabolic remodeling on the development and treatment of MetS by exercise.

Inhibition of skeletal muscle differentiation by calciprotein particles in human primary myoblasts.

Sarcopenia is a common complication of chronic kidney disease (CKD) and has a detrimental effect on prognosis. Previous studies have explored the role of secondary calciprotein particles (CPP2) in determining the progression of complications and poor outcomes in patients with CKD. However, no study has demonstrated that CPP2 impairs skeletal myogenesis. Our study revealed that CPP2 exposure inhibits skeletal myogenesis by suppressing myotube formation and expression of skeletal muscle-specific myosin heavy chain and actin in human primary myoblasts. Moreover, CPP2 exposure altered the expression patterns of lineage-determinative transcription factors responsible for regulating myotube differentiation marker genes. This study first demonstrated that CPP2 interferes with myoblast differentiation and myotube formation in vitro.

Design, Synthesis, and Biological Evaluation of 1,4-Dihydropyridine-Indole as a Potential Antidiabetic Agent via GLUT4 Translocation Stimulation.

In the quest for the discovery of antidiabetic compounds, a series of 27 1,4-dihydropyridine-indole derivatives were synthesized using a diversity approach. These compounds were systematically evaluated for their antidiabetic activity, starting with an in vitro assessment for GLUT4 translocation stimulation in L6-GLUT4myc myotubes, followed by in vivo antihyperglycemic activity evaluation in a streptozotocin (STZ)-induced diabetic rat model. Among the synthesized compounds, 12, 14, 15, 16, 19, 27, and 35 demonstrated significant potential to stimulate GLUT4 translocation in skeletal muscle cells. Compound 19 exhibited the highest potency and was selected for in vivo evaluation. A notable reduction of 21.6% (p < 0.01) in blood glucose levels was observed after 5 h of treatment with compound 19 in STZ-induced diabetic rats. Furthermore, pharmacokinetic studies affirmed that compound 19 was favorable to oral exposure with suitable pharmacological parameters. Overall, compound 19 emerged as a promising lead compound for further structural modification and optimization.

Stroke-induced excess in capillarization relative to oxidative capacity in rats is muscle specific.

Stroke is not only associated with muscle weakness, but also associated with reduced muscle fatigue resistance and reduced desaturation during exercise that may be caused by a reduced oxidative capacity and/or microvasculature. Therefore, the objective of the present study was to determine the effects of stroke on muscle mass, fiber size and shape, capillarization and oxidative capacity of the rat m. extensor carpi radialis (ECR) and m. flexor carpi ulnaris (FCU) after a photothrombotic stroke in the forelimb region of the primary sensorimotor cortex. The main observation of the present study was that 4 weeks after induction of stroke there were no significant changes in muscle fiber size and shape. Although there was no significant capillary rarefaction, there was some evidence for remodeling of the capillary bed as reflected by a reduced heterogeneity of capillary spacing (p = 0.006) that may result in improved muscle oxygenation. In the ECR, but not in the FCU, this was accompanied by reduction in muscle fiber oxidative capacity as reflected by reduced optical density of sections stained for succinate dehydrogenase (p = 0.013). The reduced oxidative capacity and absence of significant capillary rarefaction resulted in a capillary to fiber ratio per unit of oxidative capacity that was higher after stroke in the ECR (p = 0.01), but not in the FCU. This suggests that at least during the early stages, stroke is not necessarily accompanied by muscle fiber atrophy, and that stroke-induced reductions in oxidative capacity resulting in relative excess of capillarization are muscle specific.

Tissue-Level Effect of Andrographis and Ashwagandha Metabolites on Metabolic and Inflammatory Gene Expression in Skeletal Muscle and Adipose Tissue: An Ex Vivo/In Vitro Investigation.

Adipose tissue and skeletal muscle dysfunction play a central role in cardiometabolic morbidity. Ashwagandha and Andrographis are purported to have anti-inflammatory and antioxidant activity, but this is based on exposure of cells to the parent compounds ignoring phytochemical absorption and metabolism. We explored the anti-inflammatory/antioxidant effects of ashwagandha and Andrographis in ex vivo human models of skeletal muscle and adipose tissue. Healthy participants supplemented with 2000 mg/day Andrographis (n = 10) or 1100 mg/day ashwagandha (n = 10) for 28 days. Sera collected pre (D0) and post (D28) supplementation were pooled by timepoint and added to adipose explant (AT) and primary human myotube (SKMC) culture media (15% v/v) for treatment. A Taqman panel of 56 genes was used to quantify these. In AT, treatment with ashwagandha sera decreased the expression of genes involved in antioxidant defence and inflammatory response (CCL5, CD36, IL6, IL10, ADIPOQ, NFEL2, UCP2, GPX3, GPX4; geometric 95% CI for fold change > 1) and altered the expression of genes involved in fatty acid metabolism. In SKMC, ashwagandha sera altered FOXO1 and SREBF1 expression. Andrographis sera decreased IL18 and SERPINEA3 expression in AT. This physiologically relevant in vitro screening characterises the effects of ashwagandha in AT to guide future clinical trials.

OTUB1 regulates ferroptosis to inhibit myoblast differentiation into myotubes by deubiquitinating P62.

As the largest organ in the human body, skeletal muscle is essential for breathing support, movement initiation, and maintenance homeostasis. It has been shown that programmed cell death (PCD), which includes autophagy, apoptosis, and necrosis, is essential for the development of skeletal muscle. A novel form of PCD called ferroptosis is still poorly understood in relation to skeletal muscle. In this study, we observed that the activation of ferroptosis significantly impeded the differentiation of C2C12 myoblasts into myotubes and concurrently suppressed the expression of OTUB1, a crucial deubiquitinating enzyme. OTUB1-silenced C2C12 mouse myoblasts were used to investigate the function of OTUB1 in ferroptosis. The results show that OTUB1 knockdown in vitro significantly increased C2C12 ferroptosis and inhibited myogenesis. Interestingly, the induction of ferroptosis resulting from OTUB1 knockdown was concomitant with the activation of autophagy. Furthermore, OTUB1 interacted with the P62 protein and stabilized its expression by deubiquitinating it, thereby inhibiting autophagy-dependent ferroptosis and promoting myogenesis. All of these findings demonstrate the critical role that OTUB1 plays in controlling ferroptosis, and we suggest that focusing on the OTUB1-P62 axis may be a useful tactic in the treatment and prevention of disorders involving the skeletal muscle.

Tangeretin Enhances Muscle Endurance and Aerobic Metabolism in Mice via Targeting AdipoR1 to Increase Oxidative Myofibers.

Slow oxidative myofibers play an important role in improving muscle endurance performance and maintaining body energy homeostasis. However, the targets and means to regulate slow oxidative myofibers proportion remain unknown. Here, we show that tangeretin (TG), a natural polymethoxylated flavone, significantly activates slow oxidative myofibers-related gene expression and increases type I myofibers proportion, resulting in improved endurance performance and aerobic metabolism in mice. Proteomics, molecular dynamics, cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) investigations revealed that TG can directly bind to adiponectin receptor 1 (AdipoR1). Using AdipoR1-knockdown C2C12 cells and muscle-specific AdipoR1-knockout mice, we found that the positive effect of TG on regulating slow oxidative myofiber related markers expression is mediated by AdipoR1 and its downstream AMPK/PGC-1α pathway. Together, our data uncover TG as a natural compound that regulates the identity of slow oxidative myofibers via targeting the AdipoR1 signaling pathway. These findings further unveil the new function of TG in increasing the proportion of slow oxidative myofibers and enhancing skeletal muscle performance.

P38α MAPK Coordinates Mitochondrial Adaptation to Caloric Surplus in Skeletal Muscle.

Excessive calorie intake leads to mitochondrial overload and triggers metabolic inflexibility and insulin resistance. In this study, we examined how attenuated p38α activity affects glucose and fat metabolism in the skeletal muscles of mice on a high-fat diet (HFD). Mice exhibiting diminished p38α activity (referred to as p38αAF) gained more weight and displayed elevated serum insulin levels, as well as a compromised response in the insulin tolerance test, compared to the control mice. Additionally, their skeletal muscle tissue manifested impaired insulin signaling, leading to resistance in insulin-mediated glucose uptake. Examination of muscle metabolites in p38αAF mice revealed lower levels of glycolytic intermediates and decreased levels of acyl-carnitine metabolites, suggesting reduced glycolysis and ß-oxidation compared to the controls. Additionally, muscles of p38αAF mice exhibited severe abnormalities in their mitochondria. Analysis of myotubes derived from p38αAF mice revealed reduced mitochondrial respiratory capacity relative to the myotubes of the control mice. Furthermore, these myotubes showed decreased expression of Acetyl CoA Carboxylase 2 (ACC2), leading to increased fatty acid oxidation and diminished inhibitory phosphorylation of pyruvate dehydrogenase (PDH), which resulted in elevated mitochondrial pyruvate oxidation. The expected consequence of reduced mitochondrial respiratory function and uncontrolled nutrient oxidation observed in p38αAF myotubes mitochondrial overload and metabolic inflexibility. This scenario explains the increased likelihood of insulin resistance development in the muscles of p38αAF mice compared to the control mice on a high-fat diet. In summary, within skeletal muscles, p38α assumes a crucial role in orchestrating the mitochondrial adaptation to caloric surplus by promoting mitochondrial biogenesis and regulating the selective oxidation of nutrients, thereby preventing mitochondrial overload, metabolic inflexibility, and insulin resistance.

Suppression of gp130 attenuated insulin-mediated signaling and glucose uptake in skeletal muscle cells.

The aim of the present study was to investigate the effects of Oncostatin M receptor (OSMR) subunit gp130 knockdown on insulin-stimulated glucose metabolism-related signaling pathways and glucose uptake in skeletal muscle cells. siRNA-mediated gp130 knockdown was conducted in C2C12 muscle cells, and insulin was added and incubated for 1 h. The cells were cultivated to analyze the mRNA levels of gp130, phosphorylation of STAT3, and glucose metabolism-regulated signaling pathways, and OSM levels in the culture medium were analyzed. The phosphorylation of STAT 3 was significantly decreased in gp130-/- cell. The insulin stimulation was significantly increased in both gp130-/- and gp130+/+ and the phosphorylation of IRS-1 Ser 1101 was significantly decreased in gp130-/-. PI3-kinase activity and Akt Thr 308 phosphorylation were significantly decreased in gp130-/-. The insulin-stimulated increase in glucose uptake rate was significantly attenuated in gp130-/-. In the culture medium, OSM levels were significantly lower in gp130+/+compared to gp130-/- cell. In conclusion, the knockdown of gp130 caused a decrease in STAT 3 phosphorylation and resulted in the attenuation of insulin-mediated glucose metabolism signaling in skeletal muscle cells. Thus, an excessive increase in extracellular OSM may induce blunted insulin action in skeletal muscle cells.

Preventing loss of sirt1 lowers mitochondrial oxidative stress and preserves C2C12 myotube diameter in an in vitro model of cancer cachexia.

Cancer cachexia is a multifactorial syndrome associated with advanced cancer that contributes to mortality. Cachexia is characterized by loss of body weight and muscle atrophy. Increased skeletal muscle mitochondrial reactive oxygen species (ROS) is a contributing factor to loss of muscle mass in cachectic patients. Mice inoculated with Lewis lung carcinoma (LLC) cells lose weight, muscle mass, and have lower muscle sirtuin-1 (sirt1) expression. Nicotinic acid (NA) is a precursor to nicotinamide dinucleotide (NAD+) which is exhausted in cachectic muscle and is a direct activator of sirt1. Mice lost body and muscle weight and exhibited reduced skeletal muscle sirt1 expression after inoculation with LLC cells. C2C12 myotubes treated with LLC-conditioned media (LCM) had lower myotube diameter. We treated C2C12 myotubes with LCM for 24 h with or without NA for 24 h. C2C12 myotubes treated with NA maintained myotube diameter, sirt1 expression, and had lower mitochondrial superoxide. We then used a sirt1-specific small molecule activator SRT1720 to increase sirt1 activity. C2C12 myotubes treated with SRT1720 maintained myotube diameter, prevented loss of sirt1 expression, and attenuated mitochondrial superoxide production. Our data provides evidence that NA may be beneficial in combating cancer cachexia by maintaining sirt1 expression and decreasing mitochondrial superoxide production.

Inhibition of the ubiquitin-proteasome system reduces the abundance of pyruvate dehydrogenase kinase 1 in cultured myotubes.

Pyruvate dehydrogenase kinase (PDK), which phosphorylates the pyruvate dehydrogenase complex, regulates glucose metabolism in skeletal muscle. PDK1, an isozyme whose expression is controlled by hypoxia-inducible factor-1α (HIF-1α), is thought to play a role in muscle adaptation to hypoxia. While transcriptional upregulation of PDK1 by HIF-1α is well characterised, mechanisms controlling proteolysis of PDK1 in skeletal muscle have not been thoroughly investigated. Proteasome inhibitor MG132 paradoxically reduced the abundance of PDK1 in human cancer cells and rat L6 myotubes, suggesting that MG132 might direct PDK1 towards autophagic degradation. The objectives of our current study were to determine (1) whether MG132 suppresses PDK1 levels in primary human myotubes, (2) whether chloroquine, an inhibitor of autophagy, prevents MG132-induced suppression of PDK1 in L6 myotubes, and (3) whether PYR-41, an inhibitor of ubiquitination, suppresses PDK1 in L6 myotubes. Using qPCR and/or immunoblotting, we found that despite markedly upregulating HIF-1α protein, MG132 did not alter the PDK1 expression in cultured primary human myotubes, while it suppressed both PDK1 mRNA and protein in L6 myotubes. The PDK1 levels in L6 myotubes were suppressed also during co-treatment with chloroquine and MG132. PYR-41 markedly increased the abundance of HIF-1α in primary human and L6 myotubes, while reducing the abundance of PDK1. In L6 myotubes treated with PYR-41, chloroquine increased the abundance of the epidermal growth factor receptor, but did not prevent the suppression of PDK1. Collectively, our results suggest that cultured myotubes degrade PDK1 via a pathway that cannot be inhibited by MG132, PYR-41, and/or chloroquine.