NUTRITIONAL REGULATION OF APOPROTEIN B GENE EXPRESSION

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Research is proposed to study the nutritional regulation of apolipoprotein B (ApoB) gene expression. The overall goal of this proposal is to define the molecular mechanism(s) involved in the regulation of ApoB gene expression by cholesterol and fatty acids in HepG2 and Caco-2 cells representing human liver and intestine, respectively. The major emphasis of this proposal is to identify the factors which regulate the synthesis and secretion of ApoB at the posttranslational level. The specific aims of this proposal are: 1) To investigate the effect of oleate and cholesterol on the intracellular degradation of ApoB. 2) To establish the existence of metabolically distinct intracellular pools of ApoB and their rate of turnover as influenced by fatty acids and cholesterol. 3) To determine the role of cholesteryl ester synthesis through the activation of acyl- CoA:cholesterol acyltransferase (ACAT) in regulation of ApoB synthesis and secretion. 4) To examine the possible existence of factors which might regulate ApoB gene expression at transcriptional and, in particular, posttranscriptional mRNA stability levels. To achieve this goal, the concentration of ApoB in the conditioned media of HepG2 and Caco-2 cells incubated with cholesterol and oleate, respectively, will be determined by electroimmunoassay, microELISA and immunoblotting using monospecific polyclonal or monoclonal antibodies to ApoB. The potential effects of cholesterol and oleate on editing of ApoB mRNA in Caco-2 cells will be determined by electrophoresis of conditioned medium on gradient SDS polyacrylamide gels and immuno-blotting with monoclonal antibodies specific for N- and C-terminal domains of ApoB. The rate of intracellular degradation of ApoB, the existence and the turnover rates of distinct intracellular pools of ApoB and the regulatory role of ACAT in these processes will be determined by pulse-chase studies and subcellular fractionation followed by immunoprecipitation, electrophoresis, immunoblotting and/or autoradiography in conjunction with the use of specific proteases, inhibitors of ACAT and high resolution electron microscopic immunocytochemistry. To elucidate the molecular mechanism(s) involved in the regulation of ApoB gene expression by fatty acids and cholesterol, cellular mRNA measurement, mRNA stability determination, gel migration retardation and/or nuclear run-on assay, and transient expression of hybrid ApoB/CAT construct in HepG2 and Caco-2 cells will be employed. Information obtained from these studies may be used for a new approach to treatment and control of hyperlipoproteinemias and atherosclerosis.