Although the liver is the major target organ for ethanol, only 10-15% who abuse alcohol develop cirrhosis. A number of factors in addition to alcohol have therefore been proposed to contribute to the risk. Based on our preliminary studies showing that alcohol prevents the adaptive regulatory response of System A transport to amino acid starvation, we propose that ethanol acts synergistically with malnutrition to alter intracellular metabolism and protein synthesis. Our working hypothesis states that ethanol and/or its metabolites decrease System A amino acid transport through alteration(s) of adaptive, hormonal and/or plasma membrane regulatory processes. We propose to examine 5 steps involved in regulation of A system amino acid transport including: (1) hormone receptors (glucagon and insulin), (2) intracellular signals (glucocorticoids and amino acid starvation), (3) synthesis, (4) processing and (5) plasma membrane lipid composition and fluidity. These studies will be performed in rats chronically fed liquid diets including ethanol for 1 to 4 weeks. Amino acid transport will be measured in vesicles from liver subfractions, and in isolated cultured hepatocytes, Glucagon and insulin binding will be determined, tyrosine kinase activity and production of cAMP by adenylate cyclase will be measured. The effect of ethanol on hormone stimulation of specific cytosolic enzymes will be used as an indirect measure of hepatocytes to synthesize proteins. Intracellular transit times and rates of glycosylation of secretory proteins and sinusoidal membrane proteins will be determined. These studies should provide important information about the specific steps altered in hepatocyte protein processing. The results will be compared to the defects produced by microtubule inhibitors (colchicine and nocodazole). Finally, the effect of lipid composition and membrane fluidity on amino acid transport will be analyzed using liposomes and fluidizing agents to perturb the lipid composition and bulk fluidity. Taken together these studies will dissect the possible steps involved in ethanol inhibition of the hepatic regulation of amino acid metabolism and transport. The results will provide a basis for the understanding of altered liver function with ethanol intake and potentially provide a rational basis for use (or non-use) of amino acid and hormonal therapy (steroids and insulin/glucagon) in alcoholic liver disease.
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