Dietary lipid is a crucial nutrient for the neonatal mammal. However, there is a paucity of knowledge of the regulation of the mucosal phase of lipid absorption in the neonate. The PI has developed unique in vivo and in vitro model systems to study regulation of intestinal apolipoprotein expression and lipoprotein biogenesis in a species whose gastrointestinal development and lipoprotein metabolism are very similar to that of the human infant. Feeding studies in newborn swine have demonstrated regulation of apoB, AI, AIV and CIII expression by dietary and biliary lipid. Most noteworthy are recent results demonstrating coordinate pre-transitional induction of ApoAIV and III and expression by a variety of dietary fatty acids including medium chain fatty acids, which are neither re-esterified nor incorporated into chylomicrons. Results from nuclear runoff assays suggest that this regulation of apoAIV and CIII expression by lipid absorption occurs at the level of gene transcription. In contrast, studies in a novel newborn piglet intestinal cell line, IPEC-1, have demonstrated post-translational regulation of apoB and AI secretion by incubation with oleic acid, possibly involving mobilization of a pre-formed intracellular pool of apolipoprotein. Preliminary studies have also demonstrated regulation of apoAI, but not apoB, secretion by taurocholate and phosphatidylcholine, major components of bile. Further studies are proposed to 1) Define the cellular mechanisms of regulation of lipoprotein biogenesis and incorporation of apolipoproteins by fatty acids, as well as bile salt and phospholipid, in the IPEC-1 cell line. Lipid and apolipoprotein radiolabeling and cell fractionation techniques will be used to investigate regulation of lipoprotein assembly and secretion at the subcellular level; 2) Define the cellular mechanisms of the coordinate pre-translational regulation of intestinal expression of apoAIV and CIII genes, which are adjacent and share a common intergenic region, by fatty acids. The experimental approach will define the cellular mechanism for transcriptional activation of the apoAIV/CIII gene complex by fatty acids. Transfection with plasmids containing the entire apoAIV/CIII intergenic region, as well as deletion constructs, driving a CAT reporter gene, in IPEC-1 cells and in newborn swine small intestinal epithelium primary cultures, will define a lipid response element (LRE). Trans-acting factors interacting with the LRE will then be isolated and characterized. Results of these studies will ultimately guide the provision of dietary lipid to the neonate in a form which will optimize lipoprotein and apolipoprotein synthesis and secretion.
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