The objective of this work is to understand the role of malic enzyme in the development of ethanol-induced fatty liver and heptotoxicity. Malic enzyme provides NADPH for fatty acid synthesis, microsomal drug hydroxylation, microsomal ethanol oxidation, and reduction of oxidized glutathione, each of which has been implicated in the production or prevention of fatty liver in animals fed highlevels of ethanol. The functional role of malic enzyme in these pathways will be assessed by comparing these pathways in hepatocytes from normal and mutant mice fed a liquid diet with or without ethanol. We have isolated and are characterizing genomic DNAs for mouse malic enzyme. We will use homologous recombination to creat a line of pluripotent stem cells that harbors a null mutattion in the gene for malic enzyme. ells containing the target mutation will be selected by the positive-negative selection procedure or by a PCR/sib-selection method. Recombinant stem cells will be introduced into the blastocoel cavity of 3.5-day mouse embryos. The blastocysts will be surgically transferred into the uterus of a pseudopregnant mouse. Chimeric off-spring will be detected by coat- color and crossbred to obtain homozygous mutant mice. We will measure hepatic total lipids and triglycerides, and plasma ethanol and triglycerides. In hepatocytes, we will measure flux of carbon through the fatty acid synthesis pathway, activity of the dehydrogenases of the phosphogluconate oxidative pathway and flux through the phosphogluconate oxidative pathway to assess compensatory mechanisms for NADPH production; ethanol oxidation and microsomal drug hydroxylation; and concentrations of total and reduced gluthathione to determine if malic enzyme furnishes NADPH for the reduction of glutathione. These experiments will test the hypothesis that NADPH production by malic enzyme plays a role in the ethanol-induced hepatotoxicity; either a contributor role, furnishing the NADPH necessary for the accumulation of heaptic fat via de novo synthesis, or an inhibitory role, providing the NADPH necessary for eliminating toxic metabolites. Id=f these pilot studies establish the feasibility of this approach, future studies will analyze metabolic function in lines of mice expressing different levels of malic enzyme and in mice made null for other enzymatic functions.
