Oxidized lipoproteins are believed to play a crucial role in the development of atherosclerosis. Dietary oxidized lipids likely contribute to the generation of oxidized lipoproteins. Indeed studies in humans and animals suggest that dietary oxidized lipids are taken up by the small intestine and secreted as lipoproteins. Whether oxidized lipids alter the composition of lipoproteins or interfere with the normal assembly and secretion of intestinal lipoproteins is unknown. Because the intestine contributes directly to the plasma pool of atherogenic lipoproteins, information regarding mechanisms by which oxidized lipids might regulate lipoprotein assembly and secretion would be important. MTP, apo b, and the influx of lipids are all recognized mediators of lipoprotein secretion by intestinal cells. Additionally, recent data have implicated p-glycoprotein as a facilitator of cholesterol trafficking and lipoprotein secretion in intestinal cells. This proposal will address, therefore, mechanisms by which oxidized lipids might alter lipoprotein assembly and secretion and secretion by regulating the availability of lipid flux through the intestine or by regulating the activities, mass or gene expression of MTP, apo B, or p-glycoprotein. Experiments will be performed in CaCo-2 cells grown on semi-permeable supports and results supported by experiments performed in everted gut sacs from rats. Cells will be incubated with the oxidized lipid of interest and the incorporation of this lipid into cellular lipids and its eventual secretion in a triacylglycerol-rich lipoprotein particle will be estimated. Moreover, the effect of the oxidized lipid on triacylglycerol-rich lipoprotein assembly and secretion will be determine by estimating the synthesis and secretion of apo B and lipids, translocation efficiency and degradation of apo B, and apo B mRNA. MTP and p-glycoprotein activities and mass will also be estimated in cells during influx of oxidized lipids. The effects of oxidized lipids on the atherogenicity of secreted lipoprotein particles will be addressed by either co-culturing CaCo-2 cells with macrophages or by incubating remnant particles obtained from CaCo-2 cells with mouse peritoneal macrophages and assessing the propensity for foam cell formation.
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