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.

Impaired lipid metabolism is a common risk factor underlying several metabolic diseases such as metabolic syndrome and type 2 diabetes. Branched-chain amino acids (BCAAs) that include valine, leucine and isoleucine have been proven to share a role in lipid metabolism and hence in maintaining metabolic health. We have previously introduced a hypothesis suggesting that BCAA degradation mechanistically connects to lipid oxidation and storage in skeletal muscle. To test our hypothesis, the present study examined the effects of BCAA deprivation and supplementation on lipid oxidation, lipogenesis and lipid droplet characteristics in murine C2C12 myotubes. In addition, the role of myotube contractions on cell metabolism was studied by utilizing in vitro skeletal-muscle-specific exercise-like electrical pulse stimulation (EPS). Our results showed that the deprivation of BCAAs decreased both lipid oxidation and lipogenesis in C2C12 myotubes. BCAA deprivation further diminished the number of lipid droplets in the EPS-treated myotubes. EPS decreased lipid oxidation especially when combined with high BCAA supplementation. Similar to BCAA deprivation, high BCAA supplementation also decreased lipid oxidation. The present results highlight the role of an adequate level of BCAAs in healthy lipid metabolism.

Objective: Proliferating cancer cells shift their metabolism to anaerobic glycolysis even in the presence of oxygen to generate glycolytic intermediates as substrates for anabolic reactions. We hypothesise that a similar metabolic remodelling also occurs during physiological skeletal muscle hypertrophy. Methods: We used mass spectrometry in hypertrophying muscles both in C2C12 muscle cells in vitro and plantaris mice muscle in vivo and assessed metabolomic changes and the incorporation of stable isotope [U-13C6] glucose tracer compared with non-treated controls. We also performed enzyme inhibition for further mechanistic analysis and a systematic review to align any changes in metabolomics during muscle growth with previous findings. Results: Both our metabolomics analysis and the systematic review reveal altered metabolite concentrations in anabolic pathways such as in the pentose phosphate and serine synthesis pathway to build up biomass, as well as in the hexosamine biosynthetic pathway that serves as a basis for the post-translational so-called O-linked glycosylation modification. We further demonstrated that labelled carbon from [U-13C6]glucose is increasingly incorporated into the non-essential amino acids in hypertrophying myotubes. The inhibition of the key enzyme phosphoglycerate dehydrogenase (Phgdh) supresses muscle protein synthesis by 75% highlighting the importance of the serine pathway for maintaining muscle size. Conclusion: Understanding the mechanisms that regulates skeletal muscle mass will help in developing effective treatments against muscle weakness. Our results provide evidence for metabolic rewiring of glycolytic intermediates into anabolic pathways during muscle growth, such as in the serine synthesis and hexosamine biosynthetic pathways.