ATP Regulation of FGF21 in Muscle

To determine the effect of ATP (Adenosine 5′-triphosphate, Sigma-Aldrich Corp, St. Louis, MO, USA) on either FGF21 protein or mRNA levels, isolated fiber cultures or FDB muscles were previously serum-starved for 2 h (DMEM culture medium without horse serum). Subsequently, muscles were stimulated with exogenous ATP at selected concentrations (0.1-100 µM), for different times (30-360 min). When blockers or inhibitors were used, they were incubated 30 min before and during the stimulation with ATP. Evaluation of changes in mRNA and protein levels in response to ATP was performed in the presence of 100 μM Suramin (Sigma-Aldrich Corp, St. Louis, MO, USA), 25 μM Nifedipine (Sigma-Aldrich Corp, St. Louis, MO, USA), 100 nM Rapamycin (Sigma-Aldrich Corp, St. Louis, MO, USA), 50 μM LY294002 (Cell Signaling Technology, Danvers, MA, EEUU), 10 μM Akt VIII (Sigma-Aldrich Corp, St. Louis, MO, USA) 30 μM Cycloheximide (Sigma-Aldrich Corp, St. Louis, MO, USA) or 0.5 μM actinomycin-D (Sigma-Aldrich Corp, St. Louis, MO, USA).