FIGURE 1.

Effects of palmitate and oleate on insulin-stimulated tyrosine phosphorylation of IRS-2 and serine phosphorylation of Akt and GSK-3 in H4IIEC3 hepatocytes. A, H4IIEC3 cells were incubated in the presence or absence of palmitate (Pal) or oleate (Ole) for 16 h prior to stimulation with insulin (1 ng/ml, 15 min). Total cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted (IB) with the indicated antibodies. Total cell lysates were subjected to immunoprecipitation (IP) with phosphotyrosine antibody prior to SDS-PAGE to examine tyrosine phosphorylation of IRS-2. Detection was by enhanced chemiluminescence. Representative blots are shown. B, the values from densitometry of three (p-IRS-2), eight (p-Akt), or five (p-GSK-3α) independent experiments were normalized to the level of total IRS-2, Akt, or GSK-3α protein, respectively, and expressed as the mean -fold increase over control ± S.E. *, p < 0.05 versus insulin treatment alone. **, p < 0.01 versus insulin treatment alone.