Mechanism of the hepatic lipase-induced accumulation of high-density lipoprotein cholesterol by cells in culture

Publisher Website
Google Scholar
Abstract
Hepatic lipase can enhance the delivery of high-density lipoprotein (HDL) cholesterol to cells by a process which does not involve apoprotein catabolism. The incorporation of HDL-free (unesterified) cholesterol, phospholipid and cholesteryl ester by cells was compared to establish the mechanism of this delivery process. Human HDL was reconstituted with 3H-free cholesterol and [14C]sphingomyelin, treated with hepatic lipase in the presence of albumin to remove the products of lipolysis, reisolated and then incubated with cultured rat hepatoma cells. Relative to control HDL, modification of HDL with hepatic lipase stimulated both the amount of HDL-free cholesterol taken up by the cell and the esterification of HDL-free cholesterol but did not affect the delivery of sphingomyelin. Experiments utilizing HDL reconstituted with 14C-free cholesterol and [3H]cholesteryl oleoyl ether suggest that hepatic lipase enhances in incorporation of HDL-esterified cholesterol. The amount of free cholesterol delivered as a result of treatment with hepatic lipase was 4-fold that of esterified cholesterol. On the basis of HDL composition, the cellular incorporation of free cholesterol was about 10 times that which would occur by the uptake and degradation of intact particles. The preferential incorporation of HDL-free cholesterol did not require the presence of lysophosphatidylcholine. To correlate the events observed at the cellular level with alterations in lipoprotein structure, high-resolution, proton-decoupled 13C NMR spectroscopy (0.55 MHz) was performed on HDL3 in which the cholesterol molecules were replaced with [4-13C]cholesterol by particle reconstitution. The loss of HDL phospholipid following incubation with hepatic lipase or phospholipase A2 in the presence of albumin caused cholesterol molecules at the surface of the HDL particle to exhibit greater segmental motion as indicated by a decrease in line width of the 4-13C resonance at a chemical shift of 41.70 ppm. Removal of phospholipid molecules was also associated with some redistribution of cholesterol molecules from the core to the surface of the HDL particle and an increased polar group segmental motion of the phospholipid molecules remaining in the surface. Phospholipid and cholesterol molecules are apparently more widely spaced in the surface of modified HDL. The hypothesis that hepatic lipase, via its phospholipase activity, shifts the equilibrium of free cholesterol between HDL and the plasma membrane, resulting in a net delivery of free cholesterol to the cell by a surface transfer process is supported.