Dietary cholesterol suppresses receptor-dependent uptake of low density lipoprotein (LDL) by the liver and raises LDL cholesterol levels. However, the response to a given level of dietary cholesterol varies greatly among different species and among different individuals of the same species. The overall goal. of the proposed research is to understand the mechanistic basis for the marked variability in response to dietary cholesterol, using the rat and hamster as animal models that span the range of responses seen in humans. Potential mechanisms by which the liver may compensate for changes in net sterol input include induction of bile acid synthesis, suppression of de novo sterol synthesis and suppression of receptor- dependent LDL uptake. The first major group of studies will determine the mechanisms whereby dietary lipids regulate these three pathways in the hamster in vivo. Specifically, the transcriptional and/or post- transcriptional events involved in the regulation of these pathways by cholesterol, bile acids and fatty acids will be examined. In particular, mRNA and protein levels of 7alpha-hydroxylase, LDL receptor and 3-hydroxy- 3-methylglutaryl coenzyme A (HMG-CoA) reductase will be measured and correlated with assays of bile acid output, receptor-dependent LDL uptake, HMG-CoA reductase activity and absolute rates of sterol synthesis. Preliminary studies indicate that transcriptional regulation of 7alpha- hydroxylase by cholesterol differs in the rat and hamster. The possibility that other differences in the regulation of hepatic sterol metabolism also contribute to the overall response to dietary lipids will thus be addressed in the first group of studies. The second group of studies will be undertaken to determine the molecular mechanisms that control 7alpha- hydroxylase gene expression in the rat and hamster. The 5 '-flanking regions of the rat and hamster genes have been isolated and will be evaluated using a combination of transfection analysis, in vitro transcriptional analysis and transgenic approaches to identify the putative regulatory sequences that confer sensitivity to sterols and bile acids and the trans-acting factors that interact with these sequences. In particular, these studies will seek to identify the basis for differences in sensitivity to cholesterol-mediated induction of gene expression in the rat and hamster. Overall, these detailed experiments should provide information on the molecular mechanisms involved in the regulation of the major pathways that determine sterol balance across the liver and plasma LDL concentrations.
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