The combination of ursolic acid and leucine potentiates the differentiation of C2C12 murine myoblasts through the mTOR signaling pathway

Aging causes phenotypic changes in skeletal muscle progenitor cells that lead to the progressive loss of myogenic differentiation and thus a decrease in muscle mass. The naturally occurring triterpene, ursolic acid, has been reported to be an effective agent for the prevention of muscle loss by suppressing degenerative muscular dystrophy. Leucine, a branched-chain amino acid, and its metabolite, β-hydroxy-β-methylbutyric acid, have been reported to enhance protein synthesis in skeletal muscle. Therefore, the aim of the present study was to investigate whether the combination of ursolic acid and leucine promotes greater myogenic differentiation compared to either agent alone in C2C12 murine myoblasts. Morphological changes were observed and creatine kinase (CK) activity analysis was performed to determine the conditions through which the combination of ursolic acid and leucine would exert the most prominent effects on muscle cell differentiation. The effect of the combination of ursolic acid and leucine on the expression of myogenic differentiation marker genes was examined by RT-PCR and western blot analysis. The combination of ursolic acid (0.5 µM) and leucine (10 µM) proved to be the most effective in promoting myogenic differentiation. The combination of ursolic acid and leucine significantly increased CK activity than treatment with either agent alone. The level of Myosin heavy chain, a myogenic differentiation marker protein, was also enhanced by the combination of ursolic acid and leucine. The combination of ursolic acid and leucine significantly induced the expression of myogenic differentiation marker genes, such as myogenic differentiation 1 (MyoD) and myogenin, at both the mRNA and protein level. In addition, the number of myotubes and the fusion index were increased. These findings indicate that the combination of ursolic acid and leucine promotes muscle cell differentiation, thus suggesting that this combination of agents may prove to be beneficial in increasing muscle mass.