Cholesterol and bile acids have been implicated in playing important roles in several major diseases of """"""""Western Society"""""""" including: arteriosclerosis, cholesterol gallstone formation, cholestatic liver disease and colon carcinogenesis. The overall goal of this renewal application is aimed at a more detailed understanding of the role bile acids play in the regulation of cholesterol and bile acid homeostasis, liver/intestinal physiology, and cholesterol gallstone disease. The overall goal will be accomplished through the following specific aims: [1] a) Determine which isoform(s) of protein kinase C is involved in the regulation of cholesterol 7 alpha-hydroxylase; b) Determine the mechanism of activation of protein kinase C isoforms by bile acids; c) Define protein kinase C and the bile acid responsive element of the cholesterol 7 alpha-hydroxylase promoter; d) Determine if bile acids activate protein kinase C isoforms in vivo in liver and ileum (Vlahcevic, Stravitz, Heuman, Hylemon); [2] a) Quantify adsorption of bile salts to model membranes; b) Develop and validate a general quantitative model of bile salt-membrane adsorption; c) Determine the effects of lecithin and cholesterol on toxicity of bile salts toward membranes; d) Determine the effect of biliary lipid composition on biliary bile salt toxicity in animal models of bile salt induced liver injury; e) Determine if protein kinase C activation by bile salts are consequences of the accumulation of bile salts on the membrane surface that can be predicted by quantitative modeling of bile salt-membrane adsorption (Heuman, Stravitz, Valhcevic ; [3] a) Selective overexpression of cholesterol 7 alpha- hydroxylase, sterol 27-hydroxylase, cholesterol ester hydrolase and acyl CoA:cholesterol acyltransferase in vitro (Hep G2 and Chinese hamster ovary cells); b) Assess the role each enzyme plays in maintaining cellular and whole body cholesterol homeostasis using recombinant adenovirus vectors in vivo (hamsters); c) Investigate the regulation of cholesterol ester hydrolase and acyl CoA:cholesterol acyltransferase in primary rat hepatocyte cultures and in vivo by bile acids, cholesterol and hormones (Pandak, Vlahcevic); [4] a) Complete the cloning, sequencing and analysis of a large bile acid inducible operon (bai) from the intestinal Eubacterium sp. VPI 12708; b) Determine the function that each gene product encoded by this operon plays inbile acid 7 alpha/beta-dehydroxylation; c) Isolate, characterize and identify bile acid 7 alpha-dehydroxylating bacteria from cholesterol gallstone patients having high (>30%) levels of deoxycholic acid and determine if these patients are colonized by unique 7 alpha-dehydroxylating bacterial species (Hylemon, Berr).
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