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63 MITOCHONDRIAL BINDING AND GLUCOSE PHOSPHORYLATION ARE BOTH NEEDED FOR THE PROTECTIVE EFFECTS OF HEXOKINASE I AND II.
  1. L. Sun,
  2. S. Shukair,
  3. F. Moazed,
  4. T. Naik,
  5. H. Ardehali
  1. Northwestern University, Chicago, IL.

Abstract

Alterations in glucose metabolism have been demonstrated in diverse disorders, ranging from heart disease to cancer. 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. VDAC is believed to form part of mPTP, the opening of which causes cellular injury. The relative contribution of HK binding to the mitochondria and the increase in glucose phosphorylation to the overall protective effects of HKs are not clear. Furthermore, there is no clear evidence supporting the hypothesis that HK binding to mitochondria inhibits mPTP. To better understand the mechanism(s) behind the protective effects of HKs, we overexpressed full-length HKI and HKII (FL-HKI and FL-HKII, respectively), their truncated proteins lacking the N-terminal hydrophobic domains (Tr-HKI and Tr-HKII, respectively), and catalytically inactive proteins (Mut-HKI and mut-HKII, respectively) in human embryonic kidney (HEK293) cells. The truncated enzymes cannot bind to mitochondria but can phosphorylate glucose, whereas the catalytically inactive enzymes can bind to the mitochondria but do not phosphorylate glucose. The cells overexpressing these constructs were subjected to oxidant stress followed by measurement of mitochondrial membrane potential and cell death. Overexpression of FL-HKI and FL-HKII resulted in complete protection against oxidant-induced loss of mitochondrial membrane potential and cell death (survival percentage of 96 ± 9 and 95 ± 5 for FL-HKI and FL-HKII, respectively). Although overexpression of the truncated and mutant proteins reduced cell death, the degree of protection was about 40 to 50% less than that of the full-length proteins. Furthermore, FL-HKI and FL-HKII inhibited mitochondrial permeability transition (MPT) in the presence of H2O2, whereas the truncated and mutant forms only caused partial inhibition. Similar results were obtained when these proteins were expressed in primary neonatal rat cardiomyocytes using an adenoviral technique. To understand the mechanism for the protective effects of HKs, we measured VDAC phosphorylation in cells overexpressing these proteins. Overexpression of FL-HKI and FL-HKII resulted in a 5- to 10-fold increase in VDAC phosphorylation. The mechanism for VDAC phosphorylation appears to be through PKC-e as inhibitors of this enzyme led to a reversal of this process. These results suggest that both glucose phosphorylation and inhibition of mPTP contribute to the protective effects of HKI and HKII. Furthermore, overexpression of HKI and HKII leads to VDAC phosphorylation in a PKC-e-dependent pathway. These findings bear implications of HK overexpression and binding to the mitochondria as a potential clinical treatment strategy for various forms of human disease.

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