The long range objective of the proposed research is to gain a complete understanding of the role of oxygenated sterols (oxysterols) in the complex mechanisms by which cholesterol metabolism is regulated. Two key unresolved aspects of the putative participation of oxysterols in this important area of biomedical research will be investigated. First, the role of oxysterols will be more precisely defined by correlating both their positive and negative effects in different cellular compartments with changes in activities of key enzymes of cholesterol metabolism as a function of time. This will be accomplished by studies of the effects of a cholesterol-enriched diet, and cessation thereof, on the concentrations of oxysterols in rat liver subcellular fractions in relation to the corollary effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate limiting enzyme of cholesterol synthesis, as an index for negative response, and on cholesterol 7 alpha-hydroxylase, the rate limiting enzyme of bile acid synthesis, as an index for positive response. In addition, the effects of mevalonic acid, as a precursor of sterols and oxysterols in vivo, on these same factors, will be investigated. Second, the hypothesis will be explored that oxysterols, specifically those identified as potential signal molecules in rat liver, are associated with specific proteins in 1) the cytosol, and 2) the nucleus, by identifying endogenously bound oxysterols in protein fractions separated by isoelectric focusing. Any putative receptor proteins thus identified will be isolated, purified, and characterized. Initial exploration of the possible significance of the fact that a major portion of the intracellular oxysterols exist in esterified form also will be conducted. All of these planned experiments have as their fundamental goal a better understanding of the centrally important issue of how cholesterol metabolism is regulated. Such understanding will contribute to achieving the ability either to prevent or reverse atherosclerosis. In addition, detailed knowledge of how cholesterol metabolism is regulated will be important in efforts to prevent or control other pathological conditions, such as cholesterol gallstone disease or malignancies.
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