The major objective of this proposal is to examine the extent to which individual bile acids and selected bile acid analogues effect synthesis of bile acids and cholesterol. Several in vitro and in vivo models will be used (cultured rat and human hepatocytes, freshly suspended rat hepatocytes and chronic bile fistula rats) to study the effects of individual bile acids on bile acid and cholesterol synthesis under controlled experimental conditions and without interference from other bile acids. Experiments described in the first part of the proposal were designed to 1) measure relative potency of free and conjugatedd bile acids and bile acid analogues on bile acid synthesis; 2) to examine the relationship between bile acid structure and their inhibitory potency; 3) to determine quantitatively the contribution of cholesterol substrate from newly synthesized cholesterol and from the plasma lipoprotein cholesterol and 4) to establish whether bile acids affect their own synthesis directly, i.e. by acting on the cholesterol 7 alpha-hydroxylase enzyme or, indirectly, i.e., by regulating the availability of free cholesterol substrate in the liver. The regulation of the latter could occur at the intracellular level due to effects of bile acid on cholesterol synthesis, esterification or hydrolysis and/or by bile acids modulating (stimulating/inhibiting) cholesterol uptake from the different lipoprotein fractions. In the later phase of these studies, we plan to determine in cultured rat and human hepatocytes to what extent different lipoprotein fractions contribute free or esterified cholesterol substrate to bile acid synthesis. These experiments will be conducted in the LDL receptor stimulated and non-stimulated cultured rat hepatocytes. We plan to study the contribution of cholesterol to bile acid synthesis from all lipoprotein fractions (inclusive of chylomicron and chylomicron remnants). The conversion of labeled preformed free and esterified cholesterol and labeled mevalonate (newly synthesized cholesterol) to bile acids using two different labels (3H and 14C) will be used as an index of bile acid synthesis rates. These isotope measurements will be coupled with determinations of bile acid mass in the culture media. In addition, we plan to determine whether individual bile acids differ in their effect on the hepatic uptake of cholesterol (free and esterified) from different plasma lipoprotein fractions.
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