A novel amino acid mixture containing isoleucine, glycine, and cystine improves insulin sensitivity with restoring mitochondrial oxygen consumption in C2C12 myotubes

Background: Saturated fatty acids facilitate insulin insensitivity within peripheral tissues, which have been linked to the development of metabolic syndrome. Although some amino acids are involved in the regulation of lipid metabolism, their effect on insulin sensitivity remains unclear. This study investigated the effect of amino acids on the restoration of FFA-induced insulin insensitivity using C2C12 skeletal muscle cells. Methods: C2C12 myotubes were treated with 0.5 mM palmitate overnight. Then, 1 mM of each amino acid was added, and the cells were incubated for an additional 18 hours. Akt phosphorylation was assessed with a western blot after stimulation with 100 nM insulin. Under the same conditions, glucose uptake was measured using a glucose uptake assay kit. Mitochondrial function was measured using an extracellular flux analyzer. Results: Palmitate treatment reduced Akt phosphorylation to 40% of the control levels. However, among 17 amino acids evaluated, isoleucine (I), glycine (G), and cystine (C) restored Akt phosphorylation successfully. Under the same conditions, glucose uptake was reduced by palmitate. A mixture of I, G, and C (IGC) were found to restore glucose uptake. In addition, palmitate decreased oxygen consumption and ATP production in C2C12 mitochondria, as determined by the Seahorse system, which is an extracellular flux analyzer. IGC was also found to restore mitochondrial function. Conclusions: The data collected in this study suggest that an appropriate amino acid mixture consisting of I, G and C may restore insulin signaling and glucose utilization suppressed by FFA. This was also found to restore mitochondrial oxygen consumption. Novelty: This study uniquely demonstrates that a novel amino acid mixture—comprising isoleucine, glycine, and cystine (IGC)—effectively restores insulin sensitivity and mitochondrial oxygen consumption in palmitate-induced insulin-resistant C2C12 myotubes. This conclusion could be used to identify a potential therapeutic strategy for metabolic dysfunction. Keywords: Insulin sensitivity, Mitochondrial function, C2C12 myotubes, Amino acid mixtures, Isoleucine, Glycine, Cystine