Thermal stimulation promotes myogenic differentiation through enhanced glucose uptake and transient lipid droplet accumulation in C2C12 cells.

Mild thermal stimulation enhances skeletal muscle differentiation; however, its underlying metabolic basis remains unclear. Here, we demonstrate that thermal stimulation promotes myogenic differentiation through enhanced glucose uptake and transient lipid droplet (LD) accumulation in C2C12 myoblasts. Thermal stimulation at 39 °C induced a transient increase in LD formation during early differentiation. Early transient LD accumulation, particularly on days 1 and 2, was positively correlated with the day 5 fusion index, suggesting that early LD formation is associated with subsequent myogenic differentiation. Suppression of LD formation by Plin2 knockdown impaired myotube formation, indicating that LD formation is functionally required for myogenic differentiation. Thermal stimulation enhanced glucose uptake, accompanied by increased Slc2a4 and Ppargc1a expression and increased GLUT4 fluorescence intensity. Moreover, higher glucose availability further supported LD formation and myogenic differentiation even under serum-free conditions. Notably, mitochondrial mass, ATP content, and oxidative capacity remained largely unchanged, suggesting that enhanced glucose uptake is associated with LD accumulation without a corresponding increase in mitochondrial oxidative capacity. These findings identify transient LD formation as a key metabolic event driving myogenic differentiation under thermal stimulation.