VDAC in Ethanol and Aldehyde-Induced Mitochondrial Dysfunction

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DESCRIPTION (provided by applicant): Liver pathology in alcoholic liver disease (ALD), non-alcoholic steatohepatitis (NASH) and forms of toxicant-associated steatohepatitis (TASH) is indistinguishable. ALD pathogenesis may be related to generation of toxic acetaldehyde (AcAld), whereas NASH and TASH are associated with generation of malondialdehyde (MDA), 4-hydroxynonenal (HNE), chloracetaldehyde (ClAcAld) and others by lipid peroxidation and/or toxicant metabolism. With the major exceptions of oxygen and short chain fatty acids, all mitochondrial metabolites cross mitochondrial outer membranes (MOM) via open voltage dependent anion channels (VDAC). The central hypothesis of this project is that ethanol and aldehydes close VDAC, decrease permeability of MOM and suppress normal mitochondrial function. Such VDAC closure allows the selective and more rapid mitochondrial oxidation of toxic aldehydes, which permeate mitochondria freely. Although VDAC closure is adaptive in promoting aldehyde detoxification, VDAC closure may be maladaptive in promoting steatosis and lipotoxicity. Accordingly, we propose to: 1) Characterize the effects of ethanol and aldehydes on ureagenesis in cultured hepatocytes, since ureagenesis is a major energy-consuming process that is dependent on exchange of metabolites across mitochondrial membranes. We will characterize the effects of ethanol, AcAld, MDA, HNE, ClAcAld and other aldehydes on ureagenic respiration and outer membrane permeability with the expectation that exogenous aldehydes and AcAld formed by ethanol metabolism will cause dose-dependent inhibition of ureagenesis and a decrease of MOM permeability to low molecular weight (≤ 3 kDa) solutes. Follow-on experiments identify kinase pathways mediating VDAC closure. 2) Determine the contributions of the three individual VDAC isoforms to suppression of ureagenesis in hepatocytes by ethanol and aldehydes using phosphorothioate siRNA single and double knockout/knockdowns of the VDAC isoforms. 3) Evaluate the role of aldehydes, kinases and VDAC in steatosis and lipotoxicity in vitro and in vivo. In preliminary experiments, aldehyde promoted steatosis dependent on c-Jun N-terminal kinase (JNK) after incubation of hepatocytes with Intralipid and sensitized to tumor necrosis factor-α (TNFα)-dependent apoptosis. We will evaluate mechanisms underlying this cell killing and the role of specific VDAC isoforms in promoting both steatosis and cell death. ALD and NASH are widely prevalent diseases in the U.S. for which therapy is largely ineffective. Lack of effective therapy reflects our ignorance of the underlying etiologies. In particular, the basis for the identical histopathology of ALD, NASH and TASH is unknown. Aldehyde-dependent VDAC closure provides a shared mechanism for the underlying pathophysiology of these diseases, which will likely lead to better strategies for treatment and prevention.