The long term objective is to understand the molecular mechanisms involved in the biosynthesis of apoB48-containing lipoproteins. Our working model is that in post-prandial state apoB48-containing lipoproteins are synthesized first as HDL-like primordial lipoproteins and at a later stage the core of these particles is increased to accommodate large amounts of neutral lipids. We hypothesize that the coordinate induction of triglyceride and apoAIV synthesis results in the assembly and secretion of larger chylomicrons. To examine this hypothesis, we will: 1. Develop cell culture that can synthesize large quantities of apoB48- containing lipoproteins and study the effect of increased triglyceride synthesis on the assembly and secretion of these particles. 2. a) Analyze the role of apoAIV in the stabilization of the surface of intracellular nascent particles by expressing apoAIV in Caco-2 cells, cotransfected or not with apoB48, and studying the increase in size of the secreted lipoproteins and b) study the coordinate effects of increased neutral lipid and apoAIV synthesis on lipoprotein assembly and secretion. 3. Determine the rates of intracellular degradation and secretion of apoB48 by pulse-chase analysis of transfected cell lines, perform control experiments to eliminate the possibilities of extracellular degradation, uptake and sequestration of nascent particles and examine the effect of neutral lipid synthesis in conjunction with the overexpression of apoAIV on the rates of intracellular degradation and secretion of apoB48. These studies will provide information about the role of apoAIV in the stabilization of nascent particles and the effect of increased neutral lipid and apoAIV synthesis on intracellular degradation of apoB48. Such information will serve as the groundwork for in vivo studies to modulate lipoprotein secretion in the context of disorders such as hypertriglyceridemia, hypercholesterolemia, and atherosclerosis. The cell lines developed will be valuable in the study of intracellular mechanisms of lipoprotein assembly.
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