The synthesis and incorporation of apo B into chylomicrons and VLDL within the intestinal cell is required for the secretion of these lipoproteins. Fatty acids, by stimulating cellular triglyceride synthesis, drive intestinal lipoprotein production and secretion. Cholesterol is also a necessary component of the triglyceride-rich lipoprotein particle. It is likely that in association with an influx of fatty acids, the availability of cholesterol plays a role in the regulation of apo B metabolism and triglyceride transport by the gut. This proposal will investigate the regulation of apo B metabolism by luminal, intracellular, and serosal factors which alter cholesterol flux and/or cholesterol metabolism in the human intestinal cell line CaCo-2. Cholesterol flux will be altered by incubating cells with: bile salt micelles with or without cholesterol and/or fatty acids, micelles containing 25-hydroxycholesterol, or phospholipid vesicles (luminal factors); lovastatin, mevalonate, mevalonate plus an inhibitor of squalene cyclase, or a combination of these agents (cellular factors); or, delipidate fetal calf serum with or without individual lipoproteins added back (serosal factors). Documentation of changes in cholesterol metabolism will be characterized by HMG-CoA reductase mass and activity, as well as at the level of gene expression of the LDL receptor and reductase. Mechanisms for the regulation of apo B metabolism by cholesterol and fatty acid flux will be addressed by analyzing: apo B mass within cells and that secreted into the basolateral medium (ELISA); apo B synthesis and degradation rates (immunoprecipitation following pulse-chase experiments with [35S]-methionine); apo B mRNA steady-state abundance of apo B (Northern and dot blot hybridizations). Antisense oligonucleotide-targeting of apo B mRNA will also be investigated. CaCo-2 cells will be incubated with one or four phosphoramidate-modified oligonucleotides which complement different sites of apo B mRNA. After full characterization of the effect of the oligonucleotide on apo B100 and B48 synthesis, degradation, and secretion, the regulation of triglyceride-rich lipoprotein secretion, cholesterol absorption, and cholesterol metabolism will be studied. The results of these studies will provide new and important information on the regulation of intestinal apo B metabolism by fatty acid-driven lipoprotein production and changes in cholesterol flux.
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