Porcine hepatocytes take up apoE-free high density lipoprotein (HDL) via a high affinity binding site on the cell surface. Part of the HDL is subsequently degraded in lysosomes and part of it is released in macromolecular and probably altered form. The major objectives of this proposal are to study the regulation and physiological consequences of high affinity uptake of apoE-free HDL and the relation between uptake, irreversible degradation and the release of macromolecular HDL. A homologous system of cultured porcine hepatocytes and porcine HDL will be used in the study.
The specific aims are to: a) determine whether high affinity HDL binding can influence cellular sterol synthesis, cholesterol esterification, and cholesterol level, b) whether HDL binding sites are regulated in cholesterol-loaded or -depleted cells; c) measure the comparative uptake, degradation and release in macromolecular form of HDL-apoprotein and -cholesteryl ester to determine whether one of these components is absorbed preferentially; d) to examine the effect of cytoskeletal and metabolic inhibitors on uptake and release; e) to distinguish HDL binding from internalization and estimate the fraction of internalized HDL that is degraded or released as a macromolecule; and f) examine released HDL immunochemically, electrophoretically, by gel filtration and density gradient ultracentrifugation to detect cell-mediated modifications in the size, density, or immunochemical reactivity of the lipoprotein. Cells will be loaded with cholesterol by incubation with cholesterol-lecithin dispersions and mevalonolactone, and will be depleted of sterol by incubation with lecithin and Compactin. Binding will be distinguished from internalization using pulse-chase experiments, trypsin treatment, metabolic inhibitors, and paraformaldehyde-treated cells. The uptake and release of HDL-apoAI and -core lipid will be assessed using double-labeled HDL (125I-apoAI, 3H-cholesteryl ether) and the proteolysis of apoAI will be examined using 125I-apoAI-labeled HDL. Both the irreversible catabolism and reversible high affinity association of HDL with pig liver cells may be relevant to the currently held hypothesis that HDL participates in the removal of cholesterol from peripheral cells and the return of the sterol to the liver by a """"""""shuttle"""""""" mechanism, and may have relevance to humans in view of the similarity of plasma lipoproteins and lipoprotein metabolism in both species.
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