Apolipoprotein E (apo E) is a major plasma apolipoprotein that is associated with very low density lipoprotein (VLDL) and with subfractions of high density lipoprotein (HDL). Apo E plays an important role in cholesterol metabolism by virtue of its interaction with cell surface receptors. This activity appears to be crucial for the rapid removal by the liver of apo E-containing remnant particles derived from lipoprotein metabolism in peripheral tissues. This process of reverse cholesterol transport which moves cholesterol from peripheral tissues to the liver is important in preventing the accumulation of cholesterol and cholesterol esters in the arterial wall as occurs in atherosclerosis. In contrast to most apolipoproteins which are synthesized primarily at the major sites of lipoprotein production in the liver and small intestine, apo E is synthesized at high rates in a variety of peripheral tissues including adrenal gland, kidney, skin, and brain. Apo E synthesis shows complex patterns of tissue-specific regulation which are influenced by cellular cholesterol metabolism, an array of regulatory molecules, and the developmental stage of the cell. Two of the projects in this proposal are focused on the regulation of apo E expression. In the first, experiments are proposed to determine how apo E synthesis is altered in response to changes in cholesterol metabolism in rat adrenal gland. These experiments include measurements of apo E synthesis, apo E mRNA, and apo E gene transcription to establish the level of regulation. Adrenal cells responding to altered cholesterol metabolism will be identified by immunocytochemical and in situ hybridization procedures. Primary cultures of adrenal cells will be used to explore the mechanisms through which apo E expression is altered. A second project will determine the DNA elements in the apo E. gene that are required for metabolic and developmental regulation. These studies will use transient transfection assays and stable transformation protocols to introduce test genes carrying apo E regulatory sequences into cultured cells. Subsequent alterations of cellular cholesterol metabolism or the developmental stage of the cell will test which apo E DNA sequences are required to respond to these stimuli. These experiments will also specifically test the role of the 5' non-coding region of apo E mRNA in the regulation of apo E synthesis. A third project will examine the influence of dietary fat and cholesterol on the expression of genes coding for the major plasma apolipoproteins (apo AI, apo AII, apo B100, apo B48, apo E) and the low density lipoprotein (LDL) receptor. These studies will involve the determination of steady-state mRNA concentrations for each of these proteins in the livers and small intestines of animals fed diets containing different amounts of cholesterol in combination with saturated fat or diets containing fats which differ in the degree of saturation. These studies ask about the mechanisms through which dietary fat and cholesterol alter the concentrations of plasma LDL and HDL. The plasma concentrations of these lipoproteins are important factors in the development of atherosclerotic heart disease.
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