Abstract 634: Glucose Phosphorylation And Mitochondrial Binding Are Required For The Protective Effects Of Hexokinase I And II
Alterations in glucose metabolism have been demonstrated in heart failure and cardiac hypertrophy. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. Overexpression of HKI and HKII in tissue culture protects against oxidant-induced cell death. The protective effects of these enzymes are thought to be due to either an increase in glucose phosphorylation or closure of the mitochondrial permeability transition pore (mPTP) as a result of HK binding to the voltage dependent anion channel (VDAC) on the mitochondria. In order to determine the relative contribution of mitochondrial binding and glucose phosphorylating activities of HKs to their overall protective effects, we expressed full length (FL) HKI and HKII, their truncated (Tr) proteins lacking the mitochondrial binding domains, and catalytically inactive mutant (Mu) proteins in tissue culture. Overexpression of FL HKI and HKII resulted in complete protection against cell death (96±9% and 95±5% survival for FL-HKI and FL-HKII, respectively), decreased levels of reactive oxygen species, and inhibited mitochondrial permeability transition in response to H2O2. However, the truncated and mutant proteins only exerted partial effects (83±1% and 85±1% survival for Tr-HKI and Tr-HKII, and 83±1% and 77±2% for M-HKI and M-HKII, respectively). Similar results were obtained in primary neonatal rat cardiomyocytes. To explore the role of VDAC in the protective effects of HKs, we then measured the phosphorylation level of this protein in cells overexpressing HK. Overexpression of FL-HKI and FL-HKII resulted in a 5–10 fold increase in VDAC phosphorylation, while the mutant and truncated proteins resulted in smaller increases. The mechanism for VDAC phosphorylation appears to be through PKCϵ, as inhibitors of this enzyme led to a reversal of this process. These results suggest that both glucose phosphorylation and mitochondrial binding contribute to the protective effects of HKI and HKII, possibly through VDAC phosphorylation by PKCϵ. These findings imply that HK overexpression and binding to the mitochondria may be potential therapeutic targets in various forms of heart disease.