Fig. 3. Gin A induces feedback activation of the PI3K pathway in 3T3-L1 adipocytes under insulin resistance conditions.

3T3-L1 adipocytes were starved in serum-free medium overnight and incubated in EBSS or EBSS containing 2X AA, then treated with or without Gin A (10 μM) (A) or with the indicated concentrations of Gin A or PF-4708671 (20 μM) (B) for 5 hr. The cells were left unstimulated or stimulated with insulin (100 nM) for 10 min. Cells were harvested and analyzed for the phosphorylation of S6K1^T389, AKT^S473, S6^S235/236, and IRS-1^S1101, followed by reprobing with their specific antibodies for total protein. β-actin was included as a loading control. Relative protein phosphorylation was determined by analyzing the density of the bands and presented as bar graphs. The results are the mean ± SD from three experiments. *p<0.05; **p<0.01; ^#p<0.05; ^##p<0.01, compared to insulin-stimulated control.