Leucine and insulin independently regulate mTOR signalling. Primary human myotubes were untreated or incubated in the presence of leucine (Leu; 5 mM), insulin (Ins; 100 nM) or co-treated with leucine and insulin (Leu + Ins) for 30 min (A), 3 h (B) or 24 h (C). Phosphorylation levels of mTOR at Ser2448 were determined by Western blot analysis relative to total levels of mTOR. Data are presented as mean ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated.