l-Arginine Alleviates LPS-Induced Oxidative Stress and Apoptosis via Activating SIRT1-AKT-Nrf2 and SIRT1-FOXO3a Signaling Pathways in C2C12 Myotube Cells

l-arginine (l-Arg) has been reported to possess a wide range of functions, including anti-inflammatory, anti-oxidative, and anti-apoptosis. However, the role of l-Arg in LPS-induced muscle injury and its potential protective mechanism has not been well elucidated. This study aimed to investigate the effects of l-Arg on the LPS-induced oxidative stress and apoptosis in differentiated C2C12 myotube cells. Our results demonstrated that myotube cells treated with 0.2 mg/mL LPS significantly decreased cell viability. l-Arg treatment significantly suppressed LPS induced ROS accumulation and cell apoptosis. Furthermore, l-Arg improved antioxidant-related enzymes’ activities; increased antioxidant ability via Akt-Nrf2 signaling pathway; maintained the mitochondrial membrane potential (MMP); and enhanced FOXO3a expression, leading to a decrease in the mitochondrial-associated apoptotic proteins. In addition, l-Arg exposure dramatically increased the mRNA and protein expressions of SIRT1. The cytoprotective effect of l-Arg was restricted by the SIRT1 inhibitor EX527, which led to an increase in ROS level, apoptosis rate, and decreased cell MMP. The results also demonstrated that EX527 treatment significantly eliminated the effect of l-Arg on LPS-induced oxidative damage and mitochondria-mediated cell apoptosis. Our findings revealed that l-Arg could be used as a potential nutraceutical in reducing muscle injury via regulating SIRT1-Akt-Nrf2 and SIRT1-FOXO3a-mitochondria apoptosis signaling pathways.

L-Arginine Alleviates LPS-induced Oxidative Stress and Apoptosis via Activating SIRT1-AKT-Nrf2 and SIRT1-FOXO3a Signaling Pathways in C2C12 Myotube Cells

BackgroundAs a highly plasticized tissue, muscle could exert a spontaneous immune behavior in response to external pathogen stimulation. L-arginine (L-Arg) has been reported to possess a wide range of functions, including anti-inflammatory, anti-oxidative, and anti-apoptosis. However, the role of L-Arg in LPS-induced muscle injury and its potential protective mechanism has not been well elucidated. This study aimed to investigate the effects of L-Arg on the LPS-induced oxidative stress and apoptosis in vitro models of well-differentiated C2C12 myotube cells. ResultsIn the present study, we first demonstrated that myotube cells treated with 0.2 mg/mL lipopolysaccharide (LPS) significantly decreased cell viability. Then, different concentrations of L-Arg (0, 0.5, 2.5, 5 mM)-pretreated myotube cells were exposed to LPS. The results showed that L-Arg treatment significantly suppressed reactive oxygen species (ROS) accumulation and cell apoptosis. Furthermore, L-Arg improved antioxidant-related enzymes’ activities; increased antioxidant ability via Akt-Nrf2 signaling pathway; maintained the mitochondrial membrane potential (MMP) and enhanced forkhead box protein 3a (FOXO3a) expression, lead to a decrease in the mitochondrial-associated apoptotic proteins. In addition, L-Arg exposure dramatically increased the mRNA and protein expressions of Sirtuin1 (SIRT1). The cytoprotective effect of L-Arg was restricted by the SIRT1 inhibitor EX527, which led to an increase in ROS level, apoptosis rate, and decreased cell MMP. The results also demonstrated that EX527 treatment significantly eliminated the effect of L-Arg on LPS-induced oxidative damage and mitochondria-mediated cell apoptosis. ConclusionsOur findings revealed that L-Arg could be used as a potential nutraceutical in reducing muscle injury via regulating SIRT1-Akt-Nrf2 and SIRT1-FOXO3a-mitochondria apoptosis signaling pathways.

Exosomal microRNA-let-7b-5p Derived From Pancreatic Cancer Cells Possibly Promotes Insulin Resistance in C2C12 Myotube Cells by Targeting SLC6A15

Background Cancers trigger systemic metabolic disorder usually associated glucose intolerance, which as initial apparent phenomenon. One of the features of pancreatic cancer (PC) metabolic reprogramming is the crosstalk between PC and peripheral tissues (skeletal muscle and adipose tissues), emphasized by insulin resistance (IR). In our previous study (Sci Rep. 2017;7(1):5384), we reported that mice pancreatic cancer derived exosomes could induce skeletal muscle cells(C2C12) IR and exosomal microRNAs (miRNAs) may exert an important effect. This work was carried out to investigate whether there exist a direct functional relationship between PC exosomal miRNAs and C2C12 cell genes, in the pathological process of IR.Methods The expression profiles of exosomal miRNAs were evaluated using the Agilent Mouse miRNA V21.0 chip (GSE95741). The differentially expressed genes were screened through Agilent SurePrint G3 Mouse GE V2.0 chip (GSE174058). TargetScan and miRbase databases were used for target genes prediction and the prediction results were verified by dual-luciferase reporter gene assay.Results The biochips (GSE95741 and GSE174058) revealed that exosomes derived from mouse pancreatic cancer cells had higher levels of miRNA-let-7b-5p (Log FC 8.6); SLC6A15 gene expression was down-regulated in C2C12 cells (Log FC -4.7). Related mice and human studies has showed that SLC6A15 is associated with IR of metabolic disorder. In this work, luciferase assays confirmed that a direct interaction between miRNA-let-7b-5p and the SLC6A15 3΄-untranslated region (3΄-UTR) was established, as predicted by the TargetScan and miRbase.Conclusions Our data suggest that exosomal miRNA-let-7b-5p may promote IR in C2C12 myotube cells targeting SLC6A15.

Higher glucose availability augments the metabolic responses of the C2C12 myotubes to exercise-like electrical pulse stimulation

The application of exercise-like electrical pulse simulation (EL-EPS) has become a widely used exercise mimetic in vitro. EL-EPS produces similar physiological responses as in vivo exercise, while less is known about the detailed metabolic effects. Routinely the C2C12 myotubes are cultured in high glucose medium (4.5 g/l), which may alter EL-EPS responses. In this study, we evaluate the metabolic effects of EL-EPS under the high and low glucose (1.0 g/l) conditions to understand how substrate availability affects the myotube response to EL-EPS.The C2C12 myotube, media and cell-free media metabolites were analyzed using untargeted nuclear magnetic resonance (NMR)-based metabolomics. Further, translational and metabolic changes and possible exerkine effects were analyzed. EL-EPS enhanced substrate utilization as well as production and secretion of lactate, acetate, 3-hydroxybutyrate and branched chain fatty acids (BCFAs). The increase in BCFAs correlated with branched chain amino acids (BCAAs) and BCFAs were strongly decreased when myotubes were cultured without BCAAs suggesting the action of acyl-CoA thioesterases on BCAA catabolites. Notably, not all EL-EPS responses were augmented by high glucose because EL-EPS increased phosphorylated c-Jun N-terminal kinase and interleukin-6 secretion independent of glucose availability. Administration of acetate and EL-EPS conditioned media on HepG2 hepatocytes had no adverse effects on lipolysis or triacylglycerol content.Our results demonstrate that unlike in cell-free media, the C2C12 myotube and media metabolites were affected by EL-EPS, particularly under high glucose condition suggesting that media composition should be considered in future EL-EPS studies. Further, acetate and BCFAs were identified as putative exerkines warranting more research.

Activation of IGF-1 pathway and suppression of atrophy related genes are involved in Epimedium extract (icariin) promoted C2C12 myotube hypertrophy

The regenerative effect of Epimedium and its major bioactive flavonoid icariin (ICA) have been documented in traditional medicine, but their effect on sarcopenia has not been evaluated. The aim of this study was to investigate the effects of Epimedium extract (EE) on skeletal muscle as represented by differentiated C2C12 cells. Here we demonstrated that EE and ICA stimulated C2C12 myotube hypertrophy by activating several, including IGF-1 signal pathways. C2C12 myotube hypertrophy was demonstrated by enlarged myotube and increased myosin heavy chains (MyHCs). In similar to IGF-1, EE/ICA activated key components of the IGF-1 signal pathway, including IGF-1 receptor. Pre-treatment with IGF-1 signal pathway specific inhibitors such as picropodophyllin, LY294002, and rapamycin attenuated EE induced myotube hypertrophy and MyHC isoform overexpression. In a different way, EE induced MHyC-S overexpression can be blocked by AMPK, but not by mTOR inhibitor. On the level of transcription, EE suppressed myostatin and MRF4 expression, but did not suppress atrogenes MAFbx and MuRF1 like IGF-1 did. Differential regulation of MyHC isoform and atrogenes is probably due to inequivalent AKT and AMPK phosphorylation induced by EE and IGF-1. These findings suggest that EE/ICA stimulates pathways partially overlapping with IGF-1 signaling pathway to promote myotube hypertrophy.

Expression of sialyltransferases from the ST3Gal, ST6Gal and ST6GalNAc families in mouse skeletal muscle and mouse C2C12 myotube cell cultures

I. Background. In skeletal muscles the sialic acids have a great significance for their functional maintenance and proper structural organization. Our work for the first time described the expressions of ST3Gal, ST6Gal and ST6GalNAc sialyltransferases specific for glycoproteins in mouse skeletal muscles and murine C2C12 myotube cell cultures.II. Methods and Results. Lectin histochemistry, cytochemistry and lectin blot were used to demonstrate the membrane localization and the electrophoretic profiles of α-2,3- and α-2,6-sialylated glycoproteins. The expression levels of sialyltransferases were analyzed by real time RT-PCR and western blot. The enzymes ST6Gal2 and ST6GalNAc1 were not expressed in skeletal muscle tissue and C2C12 myotubes. In both experimental groups mRNAs of the ST3Gal family prevailed over the mRNA expressions of the ST6Gal and ST6GalNAc families. The profiles of STR expressions showed differences between the two experimental groups, illustrated by the absence of expressions of the mRNA for the ST3Gal6 and ST3GalNAc3 enzymes in the C2C12 cell samples and by the different shares of the enzymes ST3Gal3 and ST3Gal4 in both experimental groups. The different patterns of enzyme expressions in both experimental groups corresponded with differences between their α-2,3- and α-2,6-sialylated glycoprotein profiles.III. Conclusions. These results could be a useful addendum to the knowledge concerning the glycosylation of the skeletal muscle tissue. In addition, this report would be helpful and informative for any researches in future where the C2C12 myotube cell cultures will take a place as an experimental model.

Palmitic Acid Impairs Myogenesis and Alters Temporal Expression of miR-133a and miR-206 in C2C12 Myoblasts

Palmitic acid (PA), a saturated fatty acid enriched in high-fat diet, has been implicated in the development of sarcopenic obesity. Herein, we chose two non-cytotoxic concentrations to better understand how excess PA could impact myotube formation or diameter without inducing cell death. Forty-eight hours of 100 µM PA induced a reduction of myotube diameter and increased the number of type I fibers, which was associated with increased miR-206 expression. Next, C2C12 myotube growth in the presence of PA was evaluated. Compared to control cells, 150 µM PA reduces myoblast proliferation and the expression of MyoD and miR-206 and miR-133a expression, leading to a reduced number and diameter of myotubes. PA (100 µM), despite not affecting proliferation, impairs myotube formation by reducing the expression of Myf5 and miR-206 and decreasing protein synthesis. Interestingly, 100 and 150 µM PA-treated myotubes had a higher number of type II fibers than control cells. In conclusion, PA affects negatively myotube diameter, fusion, and metabolism, which may be related to myomiRs. By providing new insights into the mechanisms by which PA affects negatively skeletal muscle, our data may help in the discovery of new targets to treat sarcopenic obesity.

Myotube hypertrophy is associated with cancer-like metabolic reprogramming and limited by PHGDH

Muscle fiber size and oxidative metabolism are inversely related, suggesting that a glycolytic metabolism may offer a growth advantage in muscle fibers. However, the mechanisms underlying this advantage remains unknown. Nearly 100 years ago, Warburg reported that cancer cells take up more glucose to produce glycolytic intermediates for anabolic reactions such as amino acid-protein synthesis. The aim of this study was to test whether glycolysis contributes to anabolic signalling responses and hypertrophy in post-mitotic muscle cells. Skeletal muscle hypertrophy was induced in vitro by treating mouse C2C12 myotubes with IGF-1. 14C glucose was added to differentiation medium and radioactivity in isolated protein was measured. We exposed differentiated C2C12 and primary mouse myotubes, to 2-deoxyglucose (2DG) and PHGDH siRNA upon which we assessed myotube diameter and signaling pathways involved in the regulation of muscle fiber size. Here, we present evidence that, hypertrophying C2C12 myotubes undergo a cancer-like metabolic reprogramming. First, IGF-1-induced C2C12 myotube hypertrophy increases shunting of carbon from glucose into protein. Second, reduction of glycolysis through 2-deoxy-D-glucose (2DG) lowers C2C12 and primary myotube size 16-40%. Third, reducing the cancer metabolism-associated enzyme PHGDH decreases C2C12 and primary myotube size 25-52%, whereas PHGDH overexpression increases C2C12 myotube size ≈20%. Fourth, the muscle hypertrophy-promoting kinase AKT regulates PHGDH expression. Together these results suggest that glycolysis is important for hypertrophying C2C12 myotubes by reprograming their metabolism similar to cancer cells.