The series of experiments proposed in this research proposal will define for the first time the spectrum of nascent intracellular lipoproteins as isolated from within rat intestinal cells. Identification of these nascent forms will facilitate their subsequent isolation in the secretory output of the intestine - the lymph and portal vein. The intracellular particles will be isolated by rupturing epithelial cells and isolation of either a cytoplasmic or Golgi organelle fraction. Using methodology which was developed during preliminary studies to isolate intracellular HDL particles, all liberated lipoprotein classes will be isolated by ultracentrifugation and characterized by apoprotein radioimmunoassay, immunoelectrophoresis, electron microscopy and lipid thin layer chromotography. Particular attention will be paid to LDL and HDL fractions, as these classes of lipoprotein had not previously been found in the cell. The relative distribution of apoproteins and lipid in the cell among the lipoprotein classes, prior to any extracellular modification, will thus be characterized. The factors that influence the synthesis of the nascent lipoproteins will be studied by fat and cholesterol feeding, bile ligation, and induction of a hypocholesterolemic state. Both intracellular and secretory nascent particles will be isolated and characterized after each of these stimuli. The metabolism and catabolic fate of the lipoproteins synthesized by the intestine will then be examined by labeling the cholesterol ester core of the nascent HDL particle in a series of in vitro and in vivo experiments. Lipoproteins (particularly HDL) are implicated in the pathogenesis of atherosclerosis and heart disease. Attempts to modify circulating serum lipoprotein levels should be enhanced considerably by the characterization of the lipoproteins synthesized in the intestine and their metabolic fate.
