Insulin increased PKM 2 expression via HIF1α up-regulation, in a PI3K/mTOR dependent manner. (A) Serum starved HepG2 cells were treated with 100 nM insulin for 0–8 hours, maximum increase in PKM2 protein observed after 8 hours of 100 nM treatment. (B) Dose dependent increase in PKM2 expression. (C) Total RNA and protein was extracted and subjected to real time PCR and immunoblotting respectively, PKM2 mRNA increased by ~2 fold in a PI3K/mTOR dependent manner (as LY294002 and rapamycin decreased PKM2 mRNA). For PI3K/mTOR inhibition, cells were pretreated with 50 μM LY294002 or 20 nM rapamycin for 30 minutes before stimulating cells with insulin. (D) Representative immunoblot showing insulin-induced up-regulation of PKM2 and its transcriptional regulator HIF1α in a PI3K/mTOR sensitive manner. However, PKL expression remained unchanged on insulin treatment. For PI3K/mTOR inhibition, cells were pretreated with 50 μM LY294002 or 20 nM rapamycin for 30 minutes. (E) Densitometric analysis was done using Alpha Imager EP densitometer (Alpha Innotech Corp., USA). (F) CoCl2 treatment (100 μM for 8 hours) resulted in accumulation of HIF1α with concomitant increase in PKM2 expression, siRNA mediated HIF1α silencing resulted in decreased PKM2 expression. These results suggest that insulin promoted PKM2 expression through HIF1α induction, in a PI3K/mTOR dependent manner. β actin was used as endogenous control in real time experiments and loading control in immunoblotting. mRNA data is expressed as mean ± SE. *P ≤ 0.05.