Abstract 16738: Hk-II Regulates Autophagy Under Glucose Deprivation Through TORC1 Inhibition in Cardiomyocytes
Rationale Hexokinase-II (HK-II) catalyzes phosphorylation of glucose, the first step of glycolysis and has been demonstrated to confer cardioprotection. Objective To test the hypothesis that HK-II plays a regulatory role in starvation induced autophagy thereby contributing to cardioprotection. Results Glucose deprivation (GD) increased LC3-II formation, a hallmark of autophagy, in neonatal rat ventricular myocytes (NRVMs). This response was reversed by addition of 2-deoxy-glucose (2-DG), implying a role of HK-II in GD induced autophagy. This was confirmed by the finding that autophagy induced by GD was blocked by HK-II knockdown by siRNA and conversely potentiated by overexpression of HK-II. Knockdown or overexpression of HK-I alone failed to induce autophagy suggesting a specific role of HK-II in autophagy elicited by removal of its substrate. The extent of apoptosis induced by GD was augmented when autophagy was inhibited, confirming a protective role of autophagy against GD. Addition of 2-DG or HK-II siRNA increased, while overexpression of HK-II diminished, apoptosis induced by GD. To elucidate signaling mechanisms, we examined regulation of TORC1, a key regulator of autophagy. The activities of AMPK and Akt, two TORC1 upstream kinases, were not altered by manipulation of HK-II expression under GD. In contrast phosphorylation of p70S6K and 4E-BP1 were markedly reduced suggesting that GD decreased activity of TORC1. The decreased phosphorylation of TORC1 substrates by GD was further enhanced by overexpression of HK-II and inhibited by HK-II knockdown. These data suggest that HK-II negatively regulates TORC1 during GD. Immunoprecipitation study revealed that HK-II binds to mTOR through raptor and this association is increased by GD. HK-II, but not HK-I, has a TOS motif, a scaffold sequence responsible for binding TORC1 substrates to raptor and subsequent phosphorylation by mTOR. Mutating the TOS motif in HK-II blocked its binding to mTOR, its ability to regulate TORC1 activity and its effect on autophagy induction by GD. Conclusions These results suggest that HK-II inhibits TORC1 function by its binding as a decoy substrate and reveal a previously unrecognized role of HK-II in autophagy through inhibition of TORC1 activity and cell protection.
- © 2012 by American Heart Association, Inc.