REGULATION OF HEPATIC LIPOPROTEIN SYNTHESIS

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Production of lipids and apoproteins by the liver is a key determinant of lipoprotein levels in the plasma. The overall goal of this proposal is to study the mechanisms of lipoprotein biosynthesis by the liver. The approach to achieve this goal will be divided in two objectives. Objective I is to study, in rat hepatocyte cultures, the regulation of apolipoprotein synthesis, cellular transport, and secretion. Regulatory mechanisms of apoprotein synthesis will be measured by 1) hybridization assays of cellular RNA with apoprotein cDNA probes, 2) immunoprecipitation of preapoproteins produced by translation of hepatocyte mRNA in cell free protein synthesis systems, and 3) immunoprecipitation of apoproteins synthesized by intact cells subsequent to pulse chase labeling of hepatocyte cultures. Cellular transport and secretion will be quantified by immunoprecipitation of apoproteins in cell homogenates, subcellular fractions, and culture media after biosynthetic labeling of cultures. Apoprotein contents of cells, subcellular fractions, and culture media will be measured by radioimmunoassays. Hepatocytes will be obtained from animals in which apoprotein production has been perturbed in vivo. Animals will be fed a carbohydrate-rich diet, a cholesterol-rich diet, and a diet containing cholesterol and propylthiouracil. These diets have well established consequences on plasma lipoprotein levels. They will be used to quantitate, by using the above techniques, pretranslational, translational, and post-translational events in the regulation of hepatic lipoprotein biosynthesis. Objective II is to study effects of long term perturbations in vitro on lipoprotein biosynthesis. For this purpose, rat hepatocytes have been immortalized by transfer of the early genes of SV40. Clonal lines already available will be screened for cellular and media contents of lipid and apoproteins at defined culture conditions to determine whether phenotypic expression of lipoprotein production differs among clones. Selected clones will be tested for genetic and phenotypic stability and studied in detail for apoprotein synthesis, intracellular processing, and secretion. These experiments should advance our knowledge on molecular events operating in lipoprotein biosynthesis.