FIGURE 3.

Rapamycin suppresses oxygen consumption in myotubes. Oxygen consumption was measured using a Seahorse XF24 Flux Analyzer. C2C12 myoblasts were differentiated on Seahorse plates and pre-treated with rapamycin or vehicle (DMSO) for the indicated times. Glucose was maintained at 25 mM throughout the experiment. Acute addition of palmitate (0.5 mM final concentration, in complex with BSA) had only a minor effect on total oxygen consumption, which was reversed with etomoxir (50 μM), an inhibitor of carnitine palmitoyl transferase 1. Oligomycin was then added to inhibit ATP synthase and determine the rate of proton leak across the mitochondrial membrane. Finally, FCCP was added to uncouple mitochondria and measure maximal respiration rate. Decreased respiration in rapamycin-treated cells was verified in three independent experiments, consistent with previous reports (Schieke et al., 2006; Ramanathan and Schreiber, 2009). The length of rapamycin pre-treatment (1 h vs. 24 h) had a significant effect on respiration rate under basal, but not uncoupled conditions. *p < 0.05. Error bars show SEM.