The objective of this proposal is to elucidate the mechanisms whereby intravenous lipid emulsions, a commonly used energy substrate in patients requiring parenteral nutrition, deliver their triglyceride to cells, and once internalized, alter cell lipid synthesis, catabolism, and accumulation. A question to be addressed is how triglyceride delivery and cell metabolism are modified by the specific triglyceride species contained in the emulsions, i.e., medium chain, long chain, or omega-3 very long chain fish oil triglycerides. In vivo responses to intravenous infusion of different emulsions containing these three triglycerides in normal volunteers will be evaluated to determine the specific alterations in plasma lipids and lipoproteins on plasma cholesterol and triglyceride distribution. The effects of these alterations on uptake of emulsion particles and their remnants (as well as endogenous lipoproteins), and on cell triglyceride and cholesterol metabolism will be explored in cultured macrophages, fibroblasts, and hepatocyte-like HepG2 cells (Study A, Aim 1). To assess the role of apoprotein E in emulsion clearance and emulsion-cell interactions, effects of lipid infusion will be compared in subjects with varying apo E phenotypes, specifically: phenotypes that are associated with rapid post-prandial triglyceride-rich particle clearance (apo E3/3); and phenotypes associated with slower clearance (apo E3/2 or apo E2/2) (Study B.
Aim 1). The importance of particle size and lipid composition in modulating apoprotein E binding to emulsion particles, and in changing apoprotein E structural conformation (particularly in its receptor recognition domain) will be explored in vitro, using model emulsions and recombinant human apo E. Apo E interactions with different particles will be characterized by binding affinities, binding stoichiometries, and apo E structure (using circular dichroism and NMR) particularly in its receptor recognition domain; and then by distribution of apo E to different populations in particle mixtures (Aim 2). The effects of size, lipid composition and apoprotein E content on emulsion particle uptake and intracellular targeting (to different cell compartments) and on cell triglyceride and cholesterol metabolism will be delineated in studies proposed for Aim 3, using both biochemical approaches and fluorescence microscopy. The potential of triglyceride to modify intracellular cholesterol metabolism will also be explored.
Aim 4 will test the effects of free (non-esterified) medium, long, and omega-3 very long chain fatty acids on intracellular uptake, targeting and metabolism of lipoprotein and emulsion particles (+/- apoprotein E), and on lipid metabolism in cultured cells. These thematically linked studies are designed to clarify not only lipid emulsion metabolism, but also the impact of exogenous triglyceride-rich particles on endogenous lipoproteins, and on triglyceride and cholesterol metabolism in cell lines representative of the liver and macrophage systems. These studies will also provide well-characterized model systems for better understanding of human lipoprotein metabolism and atherogenesis.
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