Lipoprotein lipase mediates an increase in selective uptake of HDL-associated cholesteryl esters by cells in culture independent of scavenger receptor BI.

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl esters (CEs) by the liver. LPL promotes this selective lipid uptake independent of lipolysis. In this study, the role of SR-BI in the mechanism of this LPL-mediated increase in selective CE uptake was explored. Baby hamster kidney (BHK) cells were transfected with the SR-BI cDNA, and significant SR-BI expression could be detected in immunoblots, whereas no SR-BI was visualized in control cells. Y1-BS1 murine adrenocortical cells were cultured without or with adrenocorticotropic hormone, and cells with no detectable or with SR-BI were obtained. These cells incubated without or with LPL in medium containing 125I/[3H]cholesteryl oleyl ether- labeled HDL3; tetrahydrolipstatin inhibited the catalytic activity of LPL. In BHK and in Y1-BS1 cells without or with SR-BI expression, apparent HDL3 selective CE uptake ([3H]CEt - 125I) was detectable. Cellular SR-BI expression promoted HDL3 selective CE uptake by approximately 250-1,900%. In BHK or Y1-BS1 cells, LPL mediated an increase in apparent selective CE uptake. Quantitatively, this stimulating LPL effect was very similar in control cells and in cells with SR-BI expression. The uptake of radiolabeled HDL3 was also investigated in human embryonal kidney 293 (HEK 293) cells that are an established SR-BI-deficient cell model. LPL stimulated [3H]cholesteryl oleyl ether uptake from labeled HDL3 by HEK 293 cells substantially, showing that LPL can induce selective CE uptake from HDL3 independent of SR-BI. To explore the role of cell surface proteoglycans on lipoprotein uptake, we induced proteoglycan deficiency by heparinase treatment. Proteoglycan deficiency decreased the LPL-mediated promotion of HDL3 selective CE uptake. In summary, evidence is presented that the stimulating effect of LPL on HDL3 selective CE uptake is independent of SR-BI and lipolysis. However, cell surface proteoglycans are required for the LPL action on selective CE uptake. It is suggested that pathways distinct from SR-BI mediate selective CE uptake from HDL.

Selective uptake of low-density lipoprotein-associated cholesteryl esters by human fibroblasts, human HepG2 hepatoma cells and J774 macrophages in culture.

High-density lipoprotein-(HDL) associated cholesteryl esters (CE) are taken up by hepatic and extrahepatic cells at a higher rate than HDL apolipoproteins. This selective uptake of HDL CE is independent from HDL particle uptake. For low-density lipoprotein (LDL), receptor-mediated endocytosis by cells is well established. In this study, the question was addressed if LDL-associated CE are also taken up by cells independently from LDL particles, i.e., selectively. Human LDL (d = 1.02-1.05 g/ml) was doubly radiolabeled with intracellularly trapped tracers: [125I]Tyramine-Cellobiose ([125I]TC) traced apolipoprotein B, [3H]cholesteryl oleyl ether ([3H]CEt) traced CE. The uptake of doubly radiolabeled LDL by normal and LDL receptor-negative human skin fibroblasts, human HepG2 hepatoma cells and murine J774 macrophages was investigated. Each cell type took up LDL particles as indicated by [125I]TC. However, in fibroblasts, HepG2 cells and J774 macrophages the rate of uptake for LDL-associated [3H]CEt was greater than that according to [125I]TC. These results indicate that extrahepatic and hepatic cells selectively take up LDL CE and this uptake is independent from LDL receptor-mediated endocytosis. Loading cells with cholesterol down-regulated selective uptake of LDL CE. In summary, human skin fibroblasts, human HepG2 cells and murine J774 macrophages selectively take up LDL CE, i.e., CE are taken up independently from LDL particles.

Selective uptake of high-density lipoprotein-associated cholesteryl esters by human hepatocytes in primary culture.

High-density lipoprotein cholesteryl esters are taken up by many cells without simultaneous uptake of high-density lipoprotein apolipoproteins. This selective uptake was investigated in human hepatocytes in primary culture. Human high-density lipoprotein-3 (density, 1.125 to 1.21 gm/ml) was radiolabeled in both its apolipoprotein and in its cholesteryl ester moiety; uptake of these high-density lipoprotein3 tracers by hepatocytes was investigated. Apparent high-density lipoprotein3 particle uptake as measured with the cholesteryl ester tracer was in excess of that from the apolipoprotein tracer, indicating selective uptake of high-density lipoprotein3 cholesteryl esters by hepatocytes. This selective uptake is a regulated pathway in hepatocytes, as demonstrated by an inverse relationship between cell cholesterol and the rate of selective uptake. Studies on the mechanism of selective uptake have used inhibitors such as monensin, chloroquine, heparin, and a monoclonal antibody directed against low-density lipoprotein receptors. These experiments provide no evidence for a role of cell-secreted apolipoprotein E, endocytosis or retroendocytosis in selective uptake. The intracellular fate of high-density lipoprotein3-associated cholesteryl esters was investigated with [3H]cholesteryl oleate-labeled high-density lipoprotein3. Hepatocytes hydrolyzed [3H]cholesteryl oleate internalized from labeled high-density lipoprotein3; this catabolism was not inhibited by the presence of chloroquine. In parallel hepatocytes were incubated with [3H]cholesteryl oleate-labeled low-density lipoprotein. Cells hydrolyzed [3H]cholesteryl oleate taken up with low-density lipoprotein; however, this hydrolysis was inhibited by chloroquine, indicating lysosomal low-density lipoprotein cholesteryl ester catabolism. These experiments show that high-density lipoprotein3 cholesteryl esters selectively taken up by hepatocytes are hydrolyzed independently from the classical lysosomal catabolic pathway. The question was addressed if selective uptake mediates a net mass uptake of cholesterol rather than an isotope exchange phenomenon. Incubation of hepatocytes with high-density lipoprotein-3 suppressed endogenous sterol synthesis from sodium [14C]acetate. Hepatocytes were incubated in the presence of high-density lipoprotein3; medium cholesteryl esters decreased as a result of incubation with hepatocytes. These results show a net mass delivery of high-density lipoprotein cholesteryl esters to hepatocytes. In conclusion, the pathway for selective uptake of high-density lipoprotein cholesteryl esters could be demonstrated in human hepatocytes in primary culture. A role for selective uptake in high-density lipoprotein-mediated cholesterol delivery to the liver in human beings in vivo is proposed.