The role of apolipoproteins of HDL in the selective uptake of cholesteryl linoleyl ether by cultured rat and bovine adrenal cells

Rat adrenal cells in culture were used to study the uptake of cholesteryl linoleyl ether [( 3H]cholesteryl linoleyl ether), a nonhydrolyzable analog of cholesteryl ester. When [3H]cholesteryl linoleyl ether was added in the form of liposomes, its uptake was enhanced by adrenocorticotropin (ACTH) and by addition of milk lipoprotein lipase and interfered by heparin. When the adrenal cells were incubated with homologous [3H]cholesteryl linoleyl ether-HDL, ACTH treatment also resulted in an increase in [3H]cholesteryl linoleyl ether uptake. The uptake of [3H]cholesteryl linoleyl ether was in excess of the uptake and metabolism of 125I-labeled HDL protein and was not sensitive to heparin. Unlabeled HDL or delipidated HDL reduced very markedly the uptake of [3H]cholesteryl linoleyl ether, while addition of phosphatidylcholine liposomes had little effect. Attempts were made to deplete and enrich the adrenal cells in cholesterol and, while depletion resulted in a decrease in [3H]cholesteryl linoleyl ether-HDL uptake, enrichment of cells with cholesterol had no effect. Among the individual apolipoproteins tested, apolipoprotein A-I and the C apolipoproteins reduced [3H]cholesteryl linoleyl ether uptake, while apolipoprotein E was not effective. Since the labeled ligand studied was a lipid, these effects could not be due to an exchange of apolipoproteins, but indicated competition for binding sites. Preferential uptake of human [3H]cholesteryl linoleyl ether-HDL3 by bovine adrenal cells was found when compared to the uptake and metabolism of 125I-labeled HDL. The present results suggest that the preferential uptake of HDL cholesteryl ester (as studied with [3H]cholesteryl linoleyl ether) requires an interaction between the apolipoproteins of HDL and cell surface components.     

Uptake of rat plasma HDL subfractions labeled with [3H]cholesteryl linoleyl ether or with 125I by cultured rat hepatocytes and adrenal cells

Rat plasma low- and high-density lipoproteins were labeled with [3H]cholesteryl linoleyl ether and isolated by rate-zonal ultracentrifugation into apolipoprotein B-containing LDL, apolipoprotein E-containing HDL1 and apolipoprotein E-poor HDL2. These fractions were incubated with cultured rat hepatocytes and comparable amounts of all lipoproteins were taken up by the cells. Rat HDL was isolated at d 1.085-1.21 g/ml and apolipoprotein E-free HDL was prepared by heparin Sepharose chromatography. The original HDL and the apolipoprotein E-free HDL were labeled with 125I or with [3H]cholesteryl linoleyl ether and incubated with rat hepatocytes or adrenal cells in culture. The uptake of apolipoprotein E-free [3H]cholesterol linoleyl ether HDL by the cultured hepatocytes was 20-40% more than that of the original HDL. Comparison of uptake of cholesteryl ester moiety (represented by uptake of [3H]cholesteryl linoleyl ether) and of protein moiety (represented by metabolism of 125I-labeled protein) was carried out using both original and apolipoprotein E-free HDL. In experiments in which low concentrations of HDL were used, the ratio of 3H/125I exceeded 1.0. In cultured adrenal cells, the uptake of [3H]cholesteryl linoleyl ether-labeled HDL was stimulated 3-6-fold by 1 X 10(-7) M ACTH, while the uptake of 125I-labeled HDL increased about 2-fold. The ratio of 3H/125I representing cellular uptake was 2-3 and increased to 5 in ACTH-treated cells. The present results indicate that in cultured rat hepatocytes the uptake of homologous HDL does not depend on the presence of apolipoprotein E. Evidence was also presented for an uptake of cholesteryl ester independent of protein uptake in cultured rat adrenal cells and to a lesser extent in rat hepatocytes.     

Behaviour of phospholipase-modified HDL towards cultured hepatocytes. I. Enhanced transfers of HDL sterols and apoproteins

Human HDL subfractions (HDL2, HDL3, or HDL separated by heparin affinity chromatography) were labelled either on their apolipoprotein moiety with 125I or on their sterols: unesterified [14C]cholesterol and [3H]cholesteryl linoleyl ether, a non-hydrolysable analog of esterified cholesterol. HDL subfractions were then treated with or without phospholipase A2 from Crotalus adamanteus in presence of albumin leading to a 72-82% phosphatidylcholine degradation. Control and treated HDL were reisolated and then addressed to cultured rat hepatocytes. (A) During incubations, unesterified [14C]cholesterol from HDL3 readily appeared in hepatocytes. The specific uptake of HDL esterified cholesterol calculated from [3H]cholesteryl ether was 2-4-times less important. Uptake of HDL cholesterol tended to saturate at 150-200 micrograms/ml HDL protein. A prior phospholipase treatment of HDL3 stimulated by 2-5-fold the uptake of [3H]cholesteryl ether, whereas the transfer of free [14C]cholesterol was minimally increased. The uptake of 3H/14C-labelled sterols from HDL2 was 2-3-times higher than from HDL3. (B) Parallel experiments were conducted with 125I-labelled HDL subfractions. At 37 degrees C, the specific uptake and degradation of HDL3 125I-apolipoprotein were about 2-fold enhanced following treatment of HDL3 with phospholipase A2. Uptakes of apolipoprotein and of esterified cholesterol were compared, indicating a preferential delivery of the sterol over apoprotein (X5). The dissociation was still more pronounced with phospholipase-treated HDL3. Competition experiments showed that 12-times more unlabelled HDL3 were required to half reduce the uptake of HDL3 [3H]cholesteryl ether than to impede similarly the HDL 125I-apolipoprotein recovered in cells. Uptake of 125I-labelled apolipoprotein from HDL2 was quantitatively comparable to that from HDL3. (C) Binding of 125I-HDL subfractions was followed at 4 degrees C. A specific binding was observed for HDL2 and HDL3, although kinetic parameters were quite different (KD of 9 and 25 micrograms/ml, respectively). Following phospholipolysis, both the specific and non-specific contributions to total binding were increased. Hence, hepatocytes take up more 125I-labelled apolipoprotein and 3H/14C-labelled sterols from lipolysed HDL than from unmodified particles. This is associated to changes in the binding characteristics.     

Metabolism of HDL-cholesteryl ester in the rat, studied with a nonhydrolyzable analog, cholesteryl linoleyl ether

Intralipid was sonicated with [3H]cholesteryl linoleyl ether (a nonhydrolyzable analog of cholesteryl linoleate) and incubated with rat HDL and d greater than 1.21 fraction of rabbit serum at a ratio of 0.012 mg triacylglycerol to 1 mg HDL protein. 25% of [3H]cholesteryl linoleyl ether was transferred to HDL. The labeled HDL was injected into donor rats and was screened for 4 h. [125I]HDL was subjected to the same protocol as the 3H-labeled HDL, including screening. The screened, labeled sera were injected into acceptor rats and the disappearance of radioactivity from the circulation was compared. The t1/2 in the circulation of [125I]HDL was about 10.5 h, while that of [3H]cholesteryl linoleyl ether-HDL was about 8 h. The liver and carcass were the major sites of uptake of [3H]cholesteryl linoleyl ether-HDL and accounted for 29-41% (liver) and 30% (carcass) of the injected label. Maximal recovery of [3H]cholesteryl linoleyl ether in the liver was seen 48 h after injection, and thereafter there was a progressive decline of radioactivity, which reached 7.8% after 28 days. The maximal recovery of [125I]HDL in the liver was about 9%. Pretreatment of the acceptor rats with estradiol for 5 days resulted in a 20% increase in the hepatic uptake of [3H]cholesteryl linoleyl ether-HDL and a 5-fold increase in adrenal uptake. The present findings indicate that in the rat the liver is the major site of uptake of HDL cholesteryl ester and that part of the HDL cholesteryl ester may be cleared from the circulation separately from the protein moiety. On the basis of our previous findings (Stein, Y., Kleinman Y, Halperin, G., and Stein, O. (1983) Biochim. Biophys. Acta 750, 300-305) the loss of the [3H]cholesteryl linoleyl ether from the liver after 14-28 days was interpreted to indicate that the labeled [3H]cholesteryl linoleyl ether had been taken up by hepatocytes.     

Effect of lipid transfer protein on plasma lipids, apolipoproteins and metabolism of high-density lipoprotein cholesteryl ester in the rat

The role of human plasma lipid transfer protein (LTP) in lipoprotein metabolism was studied in the rat, a species without endogenous cholesteryl ester and triacylglycerol transfer activity. Partially purified human LTP was injected intravenously into rats. The plasma activity was between 1.5- and 4-fold that of human plasma during the experiments. 6 h after the injection of LTP, a significant increase in serum apoB, and no significant changes in serum total cholesterol, free cholesterol, triacylglycerols, apoA-I, apoE, or apoA-IV were noted. Cholesterol was increased in very-low density and low-density lipoproteins (VLDL and LDL) and decreased in large-sized apoE-rich HDL. ApoA-I-containing particles with a size smaller than in normal rats were present in serum of LTP-treated rats. The mean diameter of HDL particles decreased and apoE, normally present on large-sized HDL, was present on smaller sized particles. The metabolic fate of cholesteryl ester, originally associated with HDL, was studied by injection of [3H]cholesteryl linoleyl ether-labelled apoA-I-rich HDL in the absence and in the presence of LTP. The disappearance of [3H]cholesteryl linoleyl ether, injected as part of apoA-I-rich HDL, from serum was increased in the LTP-treated rats; the t1/2 changed from 3.9 to 2.2 h, resulting in an increased accumulation of [3H]cholesteryl linoleyl ether in the liver. This can be explained by the redistribution of HDL [3H]cholesteryl linoleyl ether to VLDL and LDL in the presence of LTP, leading to the combined contribution of VLDL, LDL and HDL to the hepatic uptake. The present findings show profound effects of LTP on the chemical composition of HDL subspecies, the size of HDL and on the plasma turnover and hepatic uptake of cholesteryl esters originally present in apo A-I-rich HDL.     

Putative role of cholesteryl ester transfer protein in removal of cholesteryl ester from vascular interstitium, studied in a model system in cell culture

A model system to study the putative role of cholesteryl ester transfer protein in the egress of interstitial cholesteryl ester is described. Confluent cultures of bovine aortic smooth muscle cells were labeled for 24 h with [3H]cholesteryl linoleyl ether and [14C]cholesteryl linoleate by incubation with bovine milk lipoprotein lipase. This method of labeling results in the transfer of cholesteryl linoleyl ether and cholesteryl ester to three compartments: a trypsin-releasable, trypsin-resistant and catabolic compartment (Stein, O., Halperin, G., Leitersdorf, E., Olivecrona, T. and Stein, Y. (1984) Biochim. Biophys. Acta 795, 47-59). The efflux of labeled cholesteryl linoleyl ether and cholesteryl ester from the extracellular and cell-surface related compartments into a serum-free culture medium containing 1% bovine serum albumin was studied during 24 h of postincubation. The efflux was expressed as a percentage of pulse value, i.e., radioactivity retained by the cell culture at the end of the labeling period. The efflux of [3H]cholesteryl linoleyl ether, [14C]cholesteryl ester and 14C-labeled free cholesterol (formed by cellular hydrolysis of cholesterol ester) into the culture medium with 1% bovine serum albumin was about 5% of the pulse value. Addition of human lipoprotein-deficient serum resulted in a 3-10-fold increase in the efflux of [3H]cholesteryl linoleyl ether and [14C]cholesteryl ester, but did not change markedly the efflux of 14C-labeled free cholesterol. Rat lipoprotein-deficient serum which does not contain cholesteryl ester transfer protein did not increase the efflux of [3H]cholesteryl linoleyl ether or [14C]cholesteryl ester. The rate of cholesteryl ester efflux in the presence of human lipoprotein-deficient serum was linear for about 6 h and increased further up to 24 h. Addition of Intralipid to medium containing human lipoprotein-deficient serum further enhanced the efflux of [3H]cholesteryl linoleyl ether and, to a lesser extent, that of cholesteryl ester. A similar effect was observed also by addition of rat VLDL to medium containing human lipoprotein-deficient serum. Inhibition of cholesteryl linoleyl ether and cholesteryl ester efflux and marked enhancement of free cholesterol efflux occurred when rat HDL was added to medium containing human lipoprotein-deficient serum, while human HDL was only slightly inhibitory. The results obtained with human lipoprotein-deficient serum were reproduced with partially purified cholesteryl ester transfer protein. Using the partially purified cholesteryl ester transfer protein, the efflux of cholesteryl linoleate was compared to that of cholesteryl oleate and was found to be the same.     

Uptake of high-density lipoprotein-associated apoprotein A-I and cholesterol esters by 16 tissues of the rat in vivo and by adrenal cells and hepatocytes in vitro

The uptake of high-density lipoprotein (HDL)-associated apolipoprotein A-I and cholesterol esters was estimated in 16 tissues of the rat using rat HDL doubly labeled with nondegradable tracers; covalently attached 125I-tyramine-cellobiose traced apo-A-I, and [3H]cholesteryl linoleyl ether traced cholesterol esters. Both labels remained associated with the HDL fraction in the plasma, adequately traced their unlabeled counterparts, and were well trapped at their sites of uptake. Cholesteryl ether was taken up at a greater fractional rate than apo-A-I by adrenal, ovary, and liver: 7-fold, 4-fold, and 2-fold greater, respectively. The rates of uptake of cholesteryl ether and apo-A-I were about equal in the other tissues (except kidney). The disproportionate uptake of HDL cholesteryl ether relative to HDL apo-A-I was also observed in primary cultures of rat adrenal cells and hepatocytes. Uptake of both moieties in both cell types showed saturability. Both the absolute rate of uptake of [3H]cholesteryl ether and the ratio of ether uptake to apo-A-I uptake were greater in adrenal cells than in hepatocytes, consonant with the in vivo observations. Very similar results were obtained using HDL biologically labeled with [3H]cholesterol esters. The disproportionate uptake of [3H]cholesteryl ether was not significantly decreased by depletion of apo-E from the HDL nor by reductive methylation of the apo-E to block its recognition by receptors. However, apo-A-I uptake was decreased, suggesting that apo-E mediates the uptake of particles containing apo-A-I but does not contribute to the disproportionate uptake of [3H]cholesteryl ether.     

Cholesterol removal by peritoneal lavage with phospholipid-HDL apoprotein mixtures in hypercholesterolemic hamsters

Syrian hamsters were rendered hypercholesterolemic by supplementation of their diet with 1% cholesterol and 15% butter. The hamsters were injected intraperitoneally (i.p.) with about 20 mg of phospholipid liposomes containing trace amounts of [3H]cholesteryl linoleyl ether ([ 3H]CLE) alone or combined with 10 mg delipidated high-density lipoprotein (apoHDL). After 2 h the peritoneal cavity was washed repeatedly with up to 15 ml phosphate-buffered saline. 60%-70% of [3H]CLE were retained after i.p. injection without apoHDL, 30-50% in the presence of apoHDL. The amount of free cholesterol recovered in the peritoneal lavage was significantly higher when apoHDL was combined with 18:2 sphingomyelin or dilinoleyl phosphatidylcholine liposomes, when compared to either liposomes or apoHDL alone. It is suggested that supplementation of dialysate with HDL apolipoproteins and phospholipids in patients undergoing continuous peritoneal dialysis could be of use in a cholesterol depletion regimen.     

Removal of cholesteryl ester from hepatic reticuloendothelial cells in vivo is not enhanced by plasma cholesteryl ester transfer protein

The putative role of cholesteryl ester transfer protein (CETP) in the removal of cholesteryl ester from hepatic reticuloendothelial cells in vivo was studied in hamsters. The parameter tested was retention of [3H]cholesteryl linoleyl ether ([3H]CLE), a nonhydrolysable analog of cholesteryl ester, in the liver after injection of [3H]CLE labeled acetylated LDL, which is targetted to nonparenchymatous littoral cells. In hamsters fed laboratory chow, plasma cholesteryl ester transfer activity (CETA) was 10.6 +/- 0.9 units and the retention of [3H]CLE in the liver 28 days after injection was 86% of the 4 h value. It was about 55% in rats fed the same diet, in which CETA was not detectable. When the diet was supplemented with 2% cholesterol and 15% margarine, CETA activity in hamsters increased 2-fold, yet no change in retention of [3H]CLE in liver was seen after 28 days. In rats, the retention of [3H]CLE in the liver was also not changed by the dietary fat supplementation. These results do not support the role of CETP in vivo in removal of cholesteryl ester from intact reticuloendothelial cells.     

Angiotensin II stimulates receptor-mediated uptake of LDL by bovine adrenal cortical cells in primary culture

Bovine adrenal cells were isolated from the subcapsular region of the gland to obtain cultures enriched in cells of the zona glomerulosa. The cells kept in primary cultures were shown to respond to angiotensin II and adrenocorticorticotropin (ACTH) by a significant increase in aldosterone production. These primary adrenal cultures were used to study the effect of angiotensin II on LDL metabolism. Addition of angiotensin II for 48 h to the culture medium resulted in a 200-300% increase in LDL metabolism, and the lowest effective concentration was 10(-8) -10(-9) M. The angiotensin II effect became evident after 12-16 h of incubation. To compare the metabolism of the 125I-labeled protein moiety to that of cholesteryl ester of LDL, the lipoprotein was labeled also with cholesteryl linoleyl ether, a nonhydrolyzable analog of cholesteryl ester. Under basal conditions and in the presence of angiotensin II or ACTH the ratio of [3H]cholesteryl linoleyl ether to 125I indicate some preferential uptake of the cholesteryl ester moiety. Stimulation of specific LDL binding at 4 degrees C and LDL metabolism at 37 degrees C by 10(-7) M angiotensin II occurred at all concentrations of LDL studied. Linearization of the kinetic data showed that angiotensin II increased the LDL receptor number significantly but not the affinity of the LDL receptor for its ligand. The present findings indicate that in analogy to ACTH, angiotensin II can influence receptor-mediated uptake of LDL by adrenal cortical cells. It remains to be shown whether the angiotensin II effect on LDL metabolism is limited to adrenal cells or will affect other cells which express the angiotensin II receptor.     

Caveolin-1 negatively regulates SR-BI mediated selective uptake of high-density lipoprotein-derived cholesteryl ester.

The class B, type I scavenger receptor (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. SR-BI is predominantly associated with caveolae in Chinese hamster ovary cells. The caveola protein, caveolin-1, binds to cholesterol and is involved in intracellular cholesterol trafficking. We previously demonstrated a correlative increase in caveolin-1 expression and the selective uptake of HDL cholesteryl esters in phorbol ester-induced differentiated THP-1 cells. The goal of the present study was to determine if the expression of caveolin-1 is the causative factor in increasing selective cholesteryl ester uptake in macrophages. To test this, we established RAW and J-774 cell lines that stably expressed caveolin-1. Transfection with caveolin-1 cDNA did not alter the amount of 125I-labeled HDL that associated with the cells, although selective uptake of HDL [3H]cholesteryl ether was decreased by approximately 50%. The amount of [3H]cholesterol effluxed to HDL was not affected by caveolin-1. To directly address whether caveolin-1 inhibits SR-BI-dependent selective cholesteryl ester uptake, we overexpressed caveolin-1 by adenoviral vector gene transfer in Chinese hamster ovary cells stably transfected with SR-BI. Caveolin-1 inhibited the selective uptake of HDL [3H]cholesteryl ether by 50-60% of control values without altering the extent of cell associated HDL. We next used blocking antibodies to CD36 and SR-BI to demonstrate that the increase in selective [3H]cholesteryl ether uptake previously seen in differentiated THP-1 cells was independent of SR-BI. Finally, we used beta-cyclodextrin and caveolin overexpression to demonstrate that caveolae depleted of cholesterol facilitate SR-BI-dependent selective cholesteryl ester uptake and caveolae containing excess cholesterol inhibit uptake. We conclude that caveolin-1 is a novel negative regulator of SR-BI-dependent selective cholesteryl ester uptake.     

High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages

Binding of high density lipoprotein (HDL) to its receptor on cultured fibroblasts and aortic endothelial cells was previously shown to facilitate sterol efflux by initiation of translocation of intracellular sterol to the plasma membrane. After cholesterol-loaded human monocyte-derived macrophages were incubated with either [3H]mevalonolactone or lipoprotein-associated [3H]cholesteryl ester to radiolabel intracellular pools of sterol, incubation with HDL3 led to stimulation of 3H-labeled sterol translocation from intracellular sites to the cell surface which preceeded maximum 3H-labeled sterol efflux. A similar pattern was demonstrated for macrophages that were preloaded with cholesterol derived from either low density lipoprotein (LDL), acetyl-LDL, or phospholipase C-modified LDL. However, in macrophages that were not loaded with cholesterol, HDL3 stimulated net movement of 3H-labeled sterol from the plasma membrane into intracellular compartments, the opposite direction from that seen for cholesterol-loaded cells. A similar influx pattern was found in nonloaded macrophages and fibroblasts that were labeled with trace amounts of exogenous [3H]cholesterol. Cholesterol translocation from intracellular pools to the cell surface of cholesterol-loaded macrophages appeared to be stimulated by receptor binding of HDL, since chemical modification of HDL with tetranitromethane (TNM), which abolishes its receptor binding, reduced its ability to stimulate 3H-labeled sterol translocation and efflux. In nonloaded cells, however, the ability of HDL3 to stimulate sterol efflux and movement of sterol from the plasma membrane into intracellular pools was unaffected by TNM modification. Thus, binding of HDL to its receptor on cholesterol-loaded macrophages appears to promote translocation of intracellular cholesterol to the plasma membrane followed by cholesterol efflux into the medium. However, in nonloaded macrophages, HDL stimulates sterol movement from the plasma membrane into intracellular pools by a receptor-independent process.     

Lipoprotein lipase mediated uptake of non-degradable ether analogues of phosphatidylcholine and cholesteryl ester by cultured cells

Lipoprotein lipase mediated transfer of cholesteryl ester and its ether analog, cholesteryl linoleyl ether, from unilamellar liposomes, prepared from a nonhydrolyzable ether analog of 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleyl ether-sn-glycero-3-phosphocholine (DOEPC), was studied in various cells in culture. It was found that lipoprotein lipase enhanced the uptake of cholesteryl linoleyl ether and of DOEPC. These findings provided a definitive proof that hydrolysis of liposomal PC is not needed for the lipoprotein lipase catalyzed transfer of cholesteryl linoleyl ether and cholesteryl ester to cells. The lipids transferred by lipoprotein lipase to cells were localized in three compartments, trypsin-releasable, resistant and metabolic; the latter was a chloroquine-sensitive pool as evidenced by inhibition of cholesteryl ester hydrolysis. Labeled PC and, to a lesser extent DOEPC, in the trypsin-releasable pool was able to return to the medium, while cholesteryl linoleyl ether and cholesteryl ester required cholesteryl ester transfer protein for release. The transfer of cholesteryl linoleyl ether and cholesteryl ester into a trypsin-resistant compartment did not require metabolic energy and occurred also in formaldehyde-fixed cells. Metabolic energy was needed for the translocation of cholesteryl linoleyl ether and cholesteryl ester into the lysosomal compartment, presumably by a process of endocytosis. The physiological relevance of the present findings is that as intravascular hydrolysis of triacylglycerol-rich lipoproteins is mediated by lipoprotein lipase attached to endothelial cells, the latter can provide a very extensive surface for removal and metabolism of phospholipids and cholesteryl ester by a mechanism mediated by lipoprotein lipase.     

Tissue distribution of [3H]cholesteryl linoleyl ether-labeled human Lp(a) in different rat organs

The sites of tissue uptake of human lipoprotein(a) (Lp(a] were studied in rats using [3H]cholesteryl linoleyl ether [( 3H]CLE) as a marker. Since rat plasma has no cholesteryl ester transfer activity, the amount of label in various tissues should reflect the quantitative uptake of Lp(a). Isolated Lp(a) was labeled with [3H]CLE by incubation overnight of Lp(a), a source of cholesteryl ester transfer activity (1.23 g/ml infranate of human plasma), and [3H]CLE-labeled Intralipid. Following labeling, the homogeneity and integrity of Lp(a) was shown by agarose electrophoresis and immunoblotting. Intact Lp(a) was injected via the tail vein of rats (120-170 g, n = 4 at each time point), and tissues were collected at various times thereafter (4-48 h). The disappearance curve of [3H]CLE-labeled Lp(a) from rat plasma was bimodal and had an initial rapid t1/2 of 1.8 h followed by a slower component, t1/2 = 13.3 h. Tissue uptake at all sampling times was greatest in liver (28.5% at 48 h of total dpm injected), followed by the intestine (9-12%), with less than 3% uptake by spleen. The small intestine was divided into four segments, and while the 3H radioactivity was similar in the proximal segments, a time-related increase in [3H]CLE was seen in its most distal portion. These studies indicate that the tissue sites of degradation in the rat of human Lp(a) are similar to human low-density lipoproteins (LDL); the increase in label in the distal portion of the small intestine with time may represent [3H]CLE excreted through the bile and absorbed by the mucosal cells.     

Hepatic uptake and metabolism of phosphatidylcholine associated with high density lipoproteins

Background

Phosphatidylcholine (PC) is the predominant phospholipid associated with high density lipoproteins (HDL). Although the hepatic uptake of cholesteryl esters from HDL is well characterized, much less is known about the fate of PC associated with HDL. Thus, we investigated the uptake and subsequent metabolism of HDL-PC in primary mouse hepatocytes.

Methods and results

The absence of scavenger receptor-BI resulted in a 30% decrease in cellular incorporation of [³H]PC whereas [³H]cholesteryl ether uptake was almost completely abolished. Although endocytosis is not involved in the uptake of cholesteryl esters from HDL, we demonstrate that HDL internalization accounts for 40% of HDL-PC uptake. Extracellular remodeling of HDL by secretory phospholipase A₂ significantly enhances HDL lipid uptake. HDL-PC taken up by hepatocytes is partially converted to triacylglycerols via PC-phospholipase C-mediated hydrolysis of PC and incorporation of diacylglycerol into triacylglcyerol. The formation of triacylglcerol is independent of scavenger receptor-BI and occurs in extralysosomal compartments.

Conclusions and general significance

These findings indicate that HDL-associated PC is incorporated into primary hepatocytes via a pathway that differs significantly from that of HDL-cholesteryl ester, and shows that HDL-PC is more than a framework molecule, as evidenced by its partial conversion to hepatic triacylglycerol.     

Regulation of the selective uptake of high density lipoprotein-associated cholesteryl esters by human fibroblasts and Hep G2 hepatoma cells

We have previously shown that the liver and steroidogenic tissues of rats in vivo and a wider range of cells in vitro, including human cells, selectively take up high density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles. This process is regulated in tissues of rats and in cultured rat cells according to their cholesterol status. In the present study, we examined regulation of HDL selective uptake in cultured human fibroblasts and Hep G2 hepatoma cells. The cholesterol content of these cells was modified by a 20-hr incubation with either low density lipoprotein (LDL) or free cholesterol. Uptake of HDL components was examined in a subsequent 4-6-hr assay using intracellularly trapped tracers: 125I-labeled N-methyl-tyramine-cellobiose-apoA-I (125I-NMTC-apoA-I) to trace apoA-I, and [3H]cholesteryl oleyl ether to trace cholesteryl esters. In the case of fibroblasts, pretreatment with either LDL or free cholesterol resulted in decreased selective uptake (total [3H]cholesteryl ether uptake minus that due to particle uptake as measured by 125I-NMTC-apoA-I). In contrast, HDL particle uptake increased with either form of cholesterol loading. The amount of HDL that was reversibly cell-associated (bound) was increased by prior exposure to free cholesterol, but was decreased by prior exposure to LDL. In the case of Hep G2 cells, exposure to free cholesterol only slightly increased HDL particle uptake; selective uptake decreased after both forms of cholesterol loading, and reversibly bound HDL increased after exposure to free cholesterol, but either did not change or decreased after exposure to LDL. It was excluded that either LDL carried over into the HDL uptake assay or that products secreted by the cultured cells influenced these results. Thus, selective uptake by cells of both hepatic and extrahepatic origin was down-regulated by cholesterol loading, under which conditions HDL particle uptake increased. Total HDL binding was not directly correlated with either the rate of selective uptake or the rate of HDL particle uptake or the cholesterol status of the cells, suggesting more than one type of HDL binding site.     

Utilization of cholesterol-rich lipoproteins by perfused rat adrenals

This study describes high density lipoprotein (HDL) uptake in the rat adrenal using a newly developed nonrecycling perfusion technique to control both the quality and quantity of the supplied lipoprotein. The aim of the study was to quantify a nonendocytic (alternative) pathway in the delivery of HDL-cholesterol. All experiments were conducted using an acute lipoprotein-deficient rat model (24 h 4-aminopyrazolo-[3, 4-d]-pyrimidine, 4-APP) in which circulating levels of cholesterol were reduced by one half, but various adrenal gland measurements of cholesterol metabolism were unchanged. Both rat HDL (rHDL) and affinity-purified human HDL3 (hHDL3) were used throughout the study. Microscopic autoradiographs (ARGs) indicate that both ligands bind avidly and exclusively to cells of the adrenal fasciculata and reticularis zones. Despite differences in binding affinity, both ligands deliver approximately the same total cholesterol to the cell interior as estimated by double-labeled residualizing tags on HDL (i.e., 125I-labeled dilactitol tyramine-[3H]cholesteryl linoleyl ether (DTT-CLE) HDL). The internalized cholesterol can account for much of the corticosterone produced during the 90-min time frame; however, only a small fraction of this cholesterol could have been provided via the endocytic pathway. Data obtained with the use of 125I-labeled DTT-[3H]CLE-HDL show that only 8.0% (or 0.7%) of corticosterone produced with rHDL (or hHDL3) could have come from cholesterol internalized as a component of intact HDL (i.e., via the endocytic pathway). These calculations strengthen the electron microscopy autoradiographic data that show that few exposed silver grains (representing the localization of the 125I-isotope) are found within the cell cytoplasm. Thus, despite differences in the uptake characteristics of the two ligands, most of the HDL-cholesterol internalized and used for corticosterone production during adrenal perfusion apparently comes from a pathway in which intact HDL are not internalized.     

The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes

Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL3). This binding was independent of apolipoprotein E as HDL3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL3. To examine the importance of apolipoprotein A-I, the major HDL3 apolipoprotein, in the specific binding of HDL3 to human adipocytes, HDL3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL3, a 92 +/- 3.1% reduction (mean +/- S.E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [3H]cholesteryl ether labeled HDL3, was also significantly reduced (about 35% reduction, P less than 0.005) by substituting apolipoprotein A-II for A-I in HDL3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I.     

A nonendocytotic mechanism for the selective uptake of high density lipoprotein-associated cholesterol esters

We have previously described in rats the selective uptake of HDL-associated cholesterol esters (traced by [3H]cholesteryl oleyl ether) in excess of the uptake of HDL-associated apoA-I. In the present studies we show that the mechanism also exists in cultured cells of human and mouse origin as well. This selective uptake represents a net uptake of cholesterol esters and not an isotope exchange, as shown by mass flux studies in adrenal cells. Inhibitors of receptor recycling, chloroquine, monensin, and colchicine, inhibited uptake of apoA-I from HDL by Hep G-2 human hepatoma cells to about the same extent as a reference protein, asialofetuin, but inhibited uptake of the cholesteryl ether tracer much less. Levels of NaN3 which effectively inhibited sucrose pinocytosis inhibited uptake of apoA-I to about the same extent but did not inhibit uptake of the cholesteryl ether at all. Thus, not only receptor recycling, but endocytosis as well, appears not to be involved in selective uptake. This conclusion was supported by studies in which synthetic HDL particles were made to contain two neutral lipid core tracers; one of them, the [3H]cholesteryl ether previously used, was selectively taken up, whereas the other, [14C]sucrose octaoleate, was excluded from selective uptake. Thus, selective uptake cannot involve endocytosis of the entire lipid core, but may involve other specific transfer mechanisms.     

Intravascular metabolism of the cholesteryl ester moiety of rat plasma lipoproteins

The intravascular metabolism of the cholesteryl ester moiety of rat plasma LDL, HDL1, and HDL2 was determined in intact male rats. Biosynthetically labeled lipoproteins were prepared by zonal ultracentrifugation from the plasma of rats injected with [3H]cholesterol. The lipoproteins were concentrated by vacuum ultrafiltration as other procedures were found to alter the biological properties of the lipoproteins. After injection of labeled LDL, [3H]cholesteryl esters remained with the injected lipoprotein and decayed from plasma with a t1/2 of 7-8 hours. [3H]Cholesteryl esters in HDL1 behaved similarly and decayed with a t1/2 of 10.5 hours. With HDL2, however, a different metabolic pattern was observed with intraplasma conversion of some [3H]cholesteryl ester HDL2 particles to HDL1. Since such conversion of HDL2 to HDL1 was not observed after in vitro incubations of rat plasma, this process seems to depend on metabolic events that occur in vivo. [3H]Cholesteryl esters disappeared from HDL2 with a t1/2 of 6-7 hours, while the esters that were transferred to HDL1 decayed with a t1/2 of 10-11 hours, similar to labeled cholesteryl esters injected with HDL1. The study demonstrated that the high apoE content of rat plasma HDL1 is not associated with rapid catabolism of the lipoprotein and that a major source of HDL1 in the rat is the intraplasma conversion of HDL2 particles to HDL1.