Creatine enhances differentiation of myogenic C2C12 cells by activating both p38 and Akt/PKB pathways

In myogenic C2C12 cells, 5 mM creatine increased the incorporation of labeled [S-35] methionine into sarcoplasmic (+ 20%, P < 0.05) and myofibrillar proteins (+ 50%, P < 0.01). Creatine also promoted the fusion of myoblasts assessed by an increased number of nuclei incorporated within myotubes (+ 40%, P < 0.001). Expression of myosin heavy chain type II (+ 1,300%, P < 0.001), troponin T (+65%, P < 0.01), and titin (+40%, P < 0.05) was enhanced by creatine. Mannitol, taurine, and beta-alanine did not mimic the effect of creatine, ruling out an osmolaritydependent mechanism. The addition of rapamycin, the inhibitor of mammalian target of rapamycin/70-kDa ribosomal S6 protein kinase (mTOR/p70s6k) pathway, and SB 202190, the inhibitor of p38, completely blocked differentiation in control cells, and creatine did not reverse this inhibition, suggesting that the mTOR/p70s6k and p38 pathways could be potentially involved in the effect induced by creatine on differentiation. Creatine upregulated phosphorylation of protein kinase B (Akt/PKB; +60%, P < 0.001), glycogen synthase kinase-3 (+70%, P < 0.001), and p70s6k (+50%, P < 0.001). Creatine also affected the phosphorylation state of p38 (+50% at 24 h and +70% at 96 h, P < 0.05) as well as the nuclear content of its downstream targets myocyte enhancer factor-2 (+55% at 48 h and +170% at 96 h, P < 0.05) and MyoD (+60%, P < 0.01). In conclusion, this study points out the involvement of the p38 and the Akt/PKB-p70s6k pathways in the enhanced differentiation induced by creatine in C2C12 cells.