Metformin regulates myoblast differentiation through an AMPK-dependent mechanism

This study aims to investigate how metformin (Met) affects muscle tissue by evaluating thedrug effects on proliferating, differentiating, and differentiated C2C12 cells. Moreover, wealso investigated the role of 5’-adenosine monophosphate-activated protein kinase (AMPK)in the mechanism of action of Met. C2C12 myoblasts were cultured in growth medium withor without Met (250μM, 1mM and 10mM) for different times. Cell proliferation was evaluatedby MTT assay, while cell toxicity was assessed by Trypan Blue exclusion test and LactateDehydrogenase release. Fluorescence Activated Cell Sorting analysis was performed tostudy cell cycle. Differentiating myoblasts were incubated in differentiation medium (DM)with or without 10mM Met. For experiments on myotubes, C2C12 were induced to differentiatein DM, and then treated with Met at scalar concentrations and for different times. Westernblotting was performed to evaluate the expression of proteins involved in myoblastdifferentiation, muscle function and metabolism. In differentiating C2C12, Met inhibited celldifferentiation, arrested cell cycle progression in G2/M phase and reduced the expression ofcyclin-dependent kinase inhibitor 1. These effects were accompanied by activation ofAMPK and modulation of the myogenic regulatory factors. Comparable results wereobtained in myotubes. The use of Compound C, a specific inhibitor of AMPK, counteractedthe above-mentioned Met effects. We reported that Met inhibits C2C12 differentiation probablyby blocking cell-cycle progression and preventing cells permanent exit from cell-cycle.Moreover, our study provides solid evidence that most of the effects of Met on myoblastsand myotubes are mediated by AMPK.