Ascorbate or NAC pretreatment decreases insulin induced ROS and increases PKM2 activity. (A) Serum starved HepG2 cells, pretreated with 200 μM ascorbate or 5 mM NAC for 1 hour , were incubated with DCFH-DA for 30 minutes at 37°C (in dark) followed by 100 nM insulin treatment for 15 minutes. DCFH-DA fluorescence was measured to assess production of ROS (see Materials and methods). Insulin increased ROS production by ~60% as compared to serum starved control. However, ascorbate or NAC pretreatment significantly decreased insulin induced ROS. (B) PKM2 activity increased in ascorbate or NAC pretreated cells compared to untreated control. (C) Dose dependent increase in ROS production by insulin. (D) PKM2 activity from insulin treated cells in absence and presence of 1mM DTT. Reversal of insulin-induced decrease in PKM2 activity by DTT suggests the possibility of oxidation of PKM2 by ROS. Data is expressed as mean ± SE. *P ≤ 0.05.