Figure 3

PKM2 activity decreased as a result of subunit dissociation upon insulin treatment, in a PI3K/mTOR independent manner. (A) 100 nM insulin treatment of 15 minutes reduced PKM2 activity in HepG2 cells by ~30%. Pretreatment with PI3K/mTOR inhibitors (50 μM LY294002 or 20 nM rapamycin for 30 minutes) did not significantly alter PKM2 activity. For activity measurement: NADH/LDH coupled assay was used to monitor decrease in OD due to oxidation of NADH at 340 nm. (B) Glycerol density gradient centrifugation was used to separate oligomeric forms of PKM2. Activity was assessed in different fractions to detect any shift in peaks i.e. subunit dissociation. Rise in peak I with concomitant fall in peak II is suggestive of disruption of highly oligomerised PKM2 (tetramer) in less oligomerised forms. Activity in a fraction corresponds to the respective amount of particular oligomeric form. (C) Relative peak areas show significant shift in peaks I and II. As tetrameric PKM2 has optimal activity [15], it can be concluded that PKM2 activity decreased as a result of dissociation of active tetramers. (D) Dose-dependent decrease in PKM2 activity upon insulin treatment. Data is expressed as mean ± SE. *P ≤ 0.05.