FIGURE 5.

Effects of PI3K and AKT inhibition on leptin signaling in HSCs. Primary rat HSCs were cultured in serum-containing medium as described in Fig. 2. HSCs were pretreated with LY294003 (25 μm) or vehicle (dimethyl sulfoxide) for 30 min (to inhibit PI3K), Ad5dnAkt (multiplicity of infection 100) or Ad5GFP (multiplicity of infection 100) for 24 h (to inhibit Akt), or cyclopamine (5 μm) or tomatidine (5 μm) for 24 h (to inhibit Hh pathway signaling) prior to the addition of leptin (100 ng/ml) or vehicle. To inhibit AKT, HSCs were treated with Ad5dnAkt prior to treatment with leptin(100 ng/ml) or vehicle. A, protein was harvested, and changes in gene expression were confirmed by Western blot analysis. Results are representative of triplicate experiments. B, densitometry was performed on all treatment groups. Leptin-treated groups are demonstrated as mean ± S.D. (error bars) of triplicate experiments. *, p < 0.05; **, p < 0.01; †, p < 0.005.