The overall objective of the proposed research is to gain an understanding of the regulation of bile acid synthesis, the predominant pathway through which cholesterol and its major metabolite (i.e. bile acids) are eliminated from the body. Moreover, in keeping with the focus of our research efforts initiated during the first funding period we will pay particular attention to defining the structural determinants responsible in hepatic uptake of lipoproteins and subsequent catabolism of the cholesterol to bile acids. Based upon data obtained during the first funding period, we hypothesize that HDL is taken up by hepatocytes via a process that may involve hepatic recognition of the more basic isoforms of apo E (i.e. desialylated). We also propose that bile acid synthesis is not regulated directly by bile acids. But, rather, it is regulated by the activity of 7Alpha-hydroxylase which is, in turn, determined by the free cholesterol content of hepatic microsomal membranes. We propose that free cholesterol regulates the activity of 7Alpha-hydroxylase by both altering microsomal membrane lipid structure and by supplying substrate to the enzyme, which is not saturated normally. To achieve these goals we will use the hepatocyte culture model to examine the structural determinants on HDL that promote bile acid synthesis. These studies will yield hypothesis regarding these structure determinants that can be tested by constructing appropriate lipoprotein particles and examining if selected determinants affect bile acid synthesis in the predicted manner. To evaluate the hypothesis regarding regulation of 7Alpha-hydroxylase, hepatic microsomes will be reconstituted to have specific content of free cholesterol and phospholipids using recently developed methods. The absolute activity of 7Alpha-hydroxylase will be quantitated by GLC-mass spectroscopy and the lipid structure will be evaluated using electron spin resonance probes (both anisotropic and isotropic). The data will be evaluated in regard to how microsomal membrane lipid composition determines lipid structure and the activity of 7Alpha-hydroxylase. The combined data will be used to formulate strategies in regard to promoting bile acid synthesis and thus, helping to eliminate cholesterol from the body.
| Dueland, S; France, D; Wang, S L et al. (1997) Cholesterol 7alpha-hydroxylase influences the expression of hepatic apoA-I in two inbred mouse strains displaying different susceptibilities to atherosclerosis and in hepatoma cells. J Lipid Res 38:1445-53 |
| Moore, G L; Drevon, C A; Machleder, D et al. (1997) Expression of human cholesterol 7alpha-hydroxylase in atherosclerosis-susceptible mice via adenovirus infection. Biochem J 324 ( Pt 3):863-7 |
| Dueland, S; Drisko, J; Graf, L et al. (1993) Effect of dietary cholesterol and taurocholate on cholesterol 7 alpha-hydroxylase and hepatic LDL receptors in inbred mice. J Lipid Res 34:923-31 |
