Increased glycolytic pooling and NADPH accumulation due to insulin-induced PKM2 activity inhibition. Serum starved HepG2 cells were treated with or without 100 nM insulin for 15 minutes or pretreated with 50 μM LY294002 or 20 nM rapamycin or 5 mM NAC or 200 μM for 30 minutes followed by 100 nM insulin treatment. Intracellular PEP, FBP and NADPH were extracted and measured as described in materials and methods section. (A) Increased accumulation of PEP (~1.7 fold) and FBP (~1.6 fold) was observed in insulin stimulated cells. LY294002 and rapamycin decreased glycolytic pooling modestly, however ROS scavengers NAC and ascorbate substantially decreased glycolytic pooling of FBP and PEP. (B) NADPH accumulated in insulin treated cells, suggesting enhanced anabolic synthesis via PPP [8, 9]. Pretreatment with PI3K/mTOR inhibitors decreased NADPH accumulation; however, pretreatment with ROS scavengers reduced NADPH accumulation to greater extent, bringing NADPH levels similar to that of control. These results suggest that insulin promoted accumulation of glycolytic intermediates mainly by decreasing PKM2 activity, and also indicates the diversion of glucose flux towards PPP [8, 9]. Actual change in intracellular concentrations is expressed as nmol per million cells. Data is expressed as mean ± SE. *P ≤ 0.05.