FIGURE 2.

Oleic acid increases intracellular levels of cAMP and PKA phosphorylation activity. A, oleic acid increases cAMP levels. Forskolin (FSK; 10 μm) and BSA-conjugated various fatty acids (0.5 mm) were treated for 30 min either in C₂C₁₂ myotubes or myoblasts. Values represent mean ± S.D. of three independent experiments in duplicate. Statistical analysis was performed using Student's t test. *, p < 0.05 and **, p < 0.01. B, oleic acid induces PKA activity. The PKA biosensors, cytosolic AKAR3 (n = 16, upper panel), and nuclear AKAR3 (n = 17, middle panel), were expressed in C₂C₁₂ cells. Cells were treated with 0.6 mm oleic acid or 10 μm foskolin as a positive control (n = 12, lower panel) at the indicated time points. Changes in PKA FRET ratio (an indication of PKA activity) were measured. C, oleic acid but not other long chain fatty acids induces phosphorylation of PKA substrates. Indicated fatty acids were used at 0.5 mm to treat C₂C₁₂ myotubes. Phosphorylation of PKA substrates was detected using p-PKA substrate and p-CREB antibodies. D, oleic acid induces phosphorylation of PKA substrates at early time points. Experiments were performed as indicated described in C. E, concentration-dependent effects of oleic acid and stearic on p-CREB.