Alcoholic liver disease (ALD) is the leading cause of liver failure. A primary role for hepatic alcohol metabo- lism in ALD is generally accepted. Thus, a correct understanding of hepatic adaptations for ethanol metabolism is paramount to understanding ALD pathophysiology and for developing effective new therapeutic strategies. All charged, hydrophilic mitochondrial metabolites must cross mitochondrial outer membranes (MOM) through voltage dependent anion channels (VDAC). Our recent data support the hypothesis that hepatic ethanol me- tabolism to acetaldehyde (AcAld) in the cytosol leads to closure of VDAC, decreased permeability of MOM, and suppression of mitochondrial function. By inhibiting mitochondrial entry of competing respiratory sub- strates, VDAC closure promotes selective more rapid oxidation of reactive AcAld, which freely permeates mito- chondrial membranes independently of VDAC to be detoxified to acetate in the mitochondrial matrix. In the short term, VDAC closure is adaptive to promote more ethanol metabolism, but in the longer term VDAC clo- sure and consequent suppression of normal mitochondrial metabolism may become be maladaptive. VDAC closure after ethanol depends on AcAld formation, but the link from AcAld to VDAC closure remains unknown. Accordingly, using a high throughput assay of ethanol-induced VDAC closure based on ethanol-induced inhibi- tion of ureagenic respiration, our high risk, high reward project is to screen compound libraries for agents that block ethanol-induced VDAC closure. The pharmacological targets of these agents will identify candidate pathways mediating VDAC closure after ethanol exposure of hepatocytes. We have two specific aims. In Spe- cific Aim 1, we will screen panels of protein kinase and phosphatase inhibitors, acetylation/deacetylation inhibi- tors and the 50,000-compound ChemBridge Library for agents that prevent ethanol-dependent inhibition of ureagenic respiration using multi-well respirometry (Seahorse).
In Specific Aim 2, we will validate lead com- pounds (hits) from screening in several ways, including determining whether hits can both prevent and reverse ureagenic inhibition after directly added AcAld. For hit compounds with known pharmacological actions (e.g., inhibition of a specific kinase), we will examine chemically unrelated agents having the same pharmacological target for their ability to prevent and reverse ethanol-dependent inhibition of ureagenic respiration. Overall this high risk, high yield project should identify agents that prevet ethanol-dependent inhibition of ureagenic respi- ration, a measure of VDAC closure in intact hepatocytes, and implicate specific signaling pathways and other new targets in the metabolic adaptation of hepatocytes to ethanol metabolism. The reward of this high risk, high reward project will be to implicate specific signaling pathways leading to VDAC inhibition from ethanol for future rigorous mechanistic studies. Moreover, small molecules identified that modulate ethanol-dependent VDAC closure may have promise for therapeutic effect in ALD.
Alcoholic liver disease (ALD) is the leading cause of liver failure whose pathogenesis begins with liver me- tabolism of alcohol (ethanol) first to the toxic and reactive aldehyde, acetaldehyde (AcAld), and then to non- toxic acetate. This project examines the novel hypothesis that AcAld causes closure of voltage dependent ani- on channels (VDAC) in mitochondrial outer membranes and hence suppresses normal mitochondrial metabo- lism, events that on the one hand are adaptive in promoting selective detoxification of freely permeant AcAld but on the other hand are maladaptive in predisposing to ALD. By screening small molecule libraries for com- pounds that reverse ethanol- and AcAld-induced inhibition of urea-forming respiration (a measure of VDAC closure and mitochondrial dysfunction), we expect to identify new signaling pathways underlying the effects of AcAld on mitochondrial metabolism for rigorous mechanistic studies in the future.
|Maldonado, Eduardo N; Lemasters, John J (2014) ATP/ADP ratio, the missed connection between mitochondria and the Warburg effect. Mitochondrion 19 Pt A:78-84|