Figure 6.

Insulin is sufficient to inactivate FOXO1. (A) C2C12 cells were incubated in serum-free complete medium (comp), in medium without glucose (-Glc), without amino acids (-aa), without vitamins (-Vit), or in medium without amino acids, glucose, and vitamins (salts) for 8 h. Insulin (200 mM) was added every 2 h (even lanes). (B) C2C12 cells were incubated for 8 h in HBSS or serum-free complete medium, and insulin was added every 2 h at 25 nM (lanes 1,6), 100 nM (lanes 2,7), 300 nM (lanes 3,8), or 1 μM (lanes 4,9). In lanes 5 and 10, no insulin was added. (Lanes 5,10) The appearance of a slower migrating species does not correlate with FOXO1 phosphorylation. (C) 293 cells were cotransfected with a luciferase reporter gene driven by the InR promoter, and plasmids expressing FOXO1 (all lanes), Akt (lanes 1-3), Akt KD (lanes 4-6), or pcDNA3.1 as control (lanes 7,8). In lanes 3 and 6, LY294002 at 20 μM was added 6 h before analyzing luciferase activity.