Summary Hexokinase-II (HK-II) catalyzes the first step of glycolysis and also functions as a protective molecule, however, its role in protective autophagy has not been determined. Results showed that inhibition of HK-II diminished, while overexpression of HK-II potentiated, autophagy induced by glucose deprivation in cardiomyocyte and non-cardiomyocyte cells. Immunoprecipitation studies revealed that HK-II binds to and inhibits the autophagy suppressor, mTOR complex 1 (TORC1), and this binding was increased by glucose deprivation. The TOS motif, a scaffold sequence responsible for binding TORC1 substrates, is present in HK-II and mutating it blocked its ability to bind to TORC1 and regulate protective autophagy. The transition from glycolysis to autophagy appears to be regulated by a decrease in glucose-6 phosphate. We suggest that HK-II binds TORC1 as a decoy substrate and provides a previously unrecognized mechanism for switching cells from a metabolic economy based on plentiful energy, to one of conservation, under starvation.

Backgound: Hexokinase II binds to mitochondria and promotes cell survival.Results: Akt phosphorylates HK-II but not the threonine 473 mutant. The phosphomimetic T473D mutant decreases its dissociation from mitochondria induced by G-6P and increases cell viability against stress.Conclusion: Akt phosphorylates HK-II at Thr-473, resulting in increased mitochondrial HK-II and cell protection.Significance: The Akt-HK-II signaling nexus is important in cell survival.

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Activation of RhoA, a low molecular-weight G-protein, plays an important role in protecting the heart against ischemic stress. Studies using non-cardiac cells demonstrate that the expression and subsequent secretion of the matricellular protein CCN1 is induced by GPCR agonists that activate RhoA. In this study we determined whether and how CCN1 is induced by GPCR agonists in cardiomyocytes and examined the role of CCN1 in ischemic cardioprotection in cardiomyocytes and the isolated perfused heart. Methods and results In neonatal rat ventricular myocytes (NRVMs), S1P, lysophosphatidic acid (LPA) and endothelin-1 induced robust increases in CCN1 expression while phenylephrine, isoproterenol and carbachol had little or no effect. The ability of agonists to activate the small G-protein RhoA correlated with their ability to induce CCN1. CCN1 induction by S1P was blocked when RhoA function was inhibited with C3 exoenzyme or a pharmacological RhoA inhibitor. Conversely overexpression of RhoA was sufficient to induce CCN1 expression. To delineate the signals downstream of RhoA we tested the role of MRTF-A (MKL1), a co-activator of SRF, in S1P-mediated CCN1 expression. S1P increased the nuclear accumulation of MRTF-A and this was inhibited by functional inactivation of RhoA. In addition, pharmacological inhibitors of MRTF-A or knockdown of MRTF-A significantly diminished S1P-mediated CCN1 expression, indicating a requirement for RhoA/MRTF-A signaling. We also present data indicating that CCN1 is secreted following agonist treatment and RhoA activation, and binds to cells where it can serve an autocrine function To determine the functional significance of CCN1 expression and signaling, simulated ischemia/reperfusion(sI/R)-induced apoptosis was assessed in NRVMs. The ability of S1P to protect against sI/R was significantly reduced by inhibition of RhoA, ROCK or MRTF-A or by CCN1 knockdown. We also demonstrate that ischemia/reperfusion induces CCN1 expression in the isolated perfused heart and that this functions as a cardioprotective mechanism, evidenced by the significant increase in infarct development in response to I/R in the cardiac specific CCN1 KO relative to control mice. Conclusion Our findings implicate CCN1 as a mediator of cardioprotection induced by GPCR agonists that activate RhoA/MRTF-A signaling.

Introduction: There is emerging evidence that the metabolic pathway interplays with the survival pathway to preserve cellular homeostasis. Hexokinases (HKs) catalyze the first step of glucose metabolism and hexokinase-II (HK-II) is the predominant isoform in the heart. Our recent study revealed that HK-II positively regulates general autophagy in the absence of glucose. Mitochondrial HK-II (mitoHK-II) is regulated by Akt and provides cardioprotection while it is decreased in the ischemic heart. Hypothesis: We tested the hypothesis that mitoHK-II dissociation triggers mitochondria specific autophagy (mitophagy). Results: As previously reported, mitoHK-II levels were decreased by ~40% in the perfused mouse heart subjected to global ischemia and in neonatal rat ventricular myocytes (NRVMs) subjected to simulated ischemia. To assess the role of mitoHK-II dissociation, mitoHK-II dissociating peptide (15NG) was expressed in NRVMs. MitoHK-II was decreased by 40% in NRVMs expressing 15NG which was accompanied with Parkin translocation to mitochondria and ubiquitination of mitochondrial proteins. This response was attenuated by Parkin knockdown and reversed by the recovery of mitoHK-II by co-expression of HK-II but not by that of mitochondria binding deficient mutant. 15NG expression did not induce mitochondrial membrane depolarization nor PINK1 stabilization at mitochondria, suggesting that the effects of mitoHK-II dissociation is not dependent on the previously established mitochondria depolarization/PINK1 pathway. This was confirmed by the experiments using PINK1 siRNA. Modest dissociation of mitoHK-II (by 20%) did not induce mitophagic responses but remarkably enhanced FCCP induced mitophagy, indicating that these two pathways are synergetic. We will be analyzing 15NG transgenic mice generated in our lab to determine the mitophagic role of mitoHK-II dissociation in vivo. Conclusions: These results suggest that mitoHK-II dissociation can regulate Parkin dependent mitophagy, in conjunction with depolarization dependent mechanisms and that HK-II could confer cardioprotection by switching the cell from an energy production to an energy conservation mode under ischemia.