Fig. 3. HRE-Luc induction in S2 cells is mediated by the activation of the PI3K-AKT pathway. (A) Effect of a PI3K inhibitor on the insulin-dependent HRE response. To assess the effect of PI3K inhibition on the insulin-stimulated HRE response, cells were treated for 20 hours with insulin (10 µg/ml) in the presence of LY294002 () or the vehicle (DMSO) (). LY294002 was added 1 hour prior to insulin stimulation and luciferase activity was determined and normalized to protein concentration. LY294002 provoked a dose-dependent inhibition of the HRE response. (B) Effect of LY294002 on dLDH mRNA induction. To study the effect of PI3K inhibition on dLDH mRNA increase after insulin stimulation, S2 cells were treated for 16 hours with insulin (10 µg/ml) in the presence or not of 10 µM LY294002. Total RNA was prepared and analyzed by northern blot using a specific dLDH probe (upper panel) or 18S rRNA as a control (lower panel). Treatment with LY294002 provoked a clear reduction of dLDH mRNA levels. (C) Role of dAKT and dPDK1 in HRE-Luc induction by insulin. Cells were incubated in the presence or absence of dsRNA directed against GFP, dAKT or dPDK1. After 3 days, the cells were treated or not with insulin 10 µg/ml for 20 hours, lysed and luciferase activity was determined. dAKT and dPDK1 dsRNAs caused strong inhibition of HRE-Luc stimulation by insulin. (D) Effect of exogenous AKT on HRE-Luc expression. Cells were transiently transfected with a plasmid expressing AKT (pAC-AKT, 250 ng), together with 100 ng of a plasmid expressing Renilla luciferase (pRL). Twenty-four hours after transfection, cells were lysed and luciferase activity was determined. Results are shown as the ratio of firefly to Renilla luciferase activities; transfection of AKT led to strong induction of the HRE reporter. (E) Effect of dPTEN silencing. Cells were incubated in the presence or absence of dsRNA directed against GFP or dPTEN, lysed and luciferase activity was determined. dPTEN dsRNA treatment provoked strong induction of the HRE response.