Abstract P060: VDAC2 and 3, but Not VDAC1, Are Required for HK2-Mediated Protection Against Cell Death

Abstract

The glycolytic enzyme hexokinase-2 (HK2) has been shown to protect several cell types including cardiomyocytes against death. However, the mechanisms of this HK2-mediated protection have not been elucidated. HK2 is normally associated with the mitochondrial voltage-dependent anion channels (VDACs) on the outer mitochondrial membrane, but it is unknown if association with VDACs is required for HK2’s protective functions. In addition, Akt has been shown to phosphorylate HK2, resulting in its translocation to the outer mitochondrial membrane. The purpose of this study was to investigate the role of the three VDAC isoforms, as well as Akt signaling, on HK2-mediated protection. Studies were conducted using cultured murine embryonic fibroblasts (MEFs) from wildtype (WT), VDAC1-/-, and VDAC3-/- mice. WT MEFs were also transfected with a VDAC2 siRNA to reduce VDAC2 protein expression. Murine HK2 was overexpressed by infection with adenovirus. Akt signaling was inhibited by 2 μM Akt Inhibitor VIII. Cell death was induced by 500 μM H2O2 for 4 hours, and cells were stained by Sytox green to identify dead cells and Bis-Benzimide to label all cell nuclei. Protein expression as well as subcellular localization was determined by Western Blot. The HK2 adenovirus resulted in 50-60% increased HK2 expression. In WT MEFs, HK2 overexpression resulted in roughly 50% less death compared to control transfected cells. Surprisingly, although VDAC1 appears to bind the greatest percentage of HK2, cells lacking VDAC1 were still protected by HK2 to a similar degree as WT cells. However, cells with reduced levels of VDAC2, or cells lacking VDAC3 were no longer protected from cell death by HK2. Additionally, HK2 overexpression remained protective in cells treated with the Akt inhibitor. In conclusion, association with the less abundant VDAC2 and VDAC3 isoforms, not the more abundant VDAC1 isoform, appears to be central to HK2-mediated cytoprotection. It also appears that active Akt is not essential to HK2-mediated protection against cell death.