A plasma membrane pool of cholesteryl esters that may mediate the selective uptake of cholesteryl esters from high-density lipoproteins

Selective uptake of high-density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles occurs by a nonendocytotic pathway that requires no specific apolipoprotein and results in the net delivery of cholesteryl esters to cells. Here we examine a reversibly cell-associated pool of cholesteryl ester tracer and its relationship to selective uptake. A fraction of cholesteryl ester tracer selectively taken up from HDL by rat primary or mouse Y1-BS1 adrenocortical cells was chased from the cells by subsequent incubation with unlabeled HDL. This pool of cholesteryl ester tracer was distinct from that irreversibly internalized, and in excess of that accounted for by dissociation of labeled HDL particles bound to the cell surface. In response to various metabolic effectors, cholesteryl ester tracer in this reversibly cell-associated pool of Y1-BS1 cells correlated linearly with irreversible selective uptake. Both reversibly and irreversibly cell-associated pools of cholesteryl ester tracer displayed similar saturation kinetics for uptake from HDL, and both pools correlated inversely with cell-free cholesterol levels. Cholesteryl ester tracer in the reversible pool was shown to serve as a precursor for irreversible selective uptake. A pool with properties similar to the reversibly cell-associated pool was identified in plasma membrane fractions; enough tracer was incorporated into this pool to account for the reversibly cell-associated pool of intact cells. The data suggest that a pool of cholesteryl esters in the plasma membrane is involved in selective uptake at a step prior to irreversible internalization.     

Uptake of lipoproteins by in situ perfused rat ovaries: identification of binding sites for high density lipoproteins

We have examined the uptake and distribution of 125I-labeled human high density lipoprotein, apolipoprotein E-free (hHDL3), 125I-rat high density lipoprotein (HDL), and human HDL (hHDL) reconstituted with [3H]cholesteryl linoleate after their in situ vascular perfusion to ovaries of gonadotropin-primed immature rats on days 6-9 post human chorionic gonadotropin (hCG)-injection. Some rats were treated with 4-aminopyrazolopyrimidine to reduce plasma lipoproteins and ovarian cholesteryl ester stores. Perfused ovaries were analyzed biochemically and autoradiographically, and progestin content of the ovarian effluent was quantified. Infusion of ovine luteinizing hormone and hHDL increased ovarian progestin secretion severalfold, indicating that the perfused ovary was functional. After perfusion with HDL reconstituted with [3H]cholesteryl linoleate, radioactive progestin appeared in the effluent; thus, sterol carried by exogenous HDL was converted to steroid. At 37 degrees C, uptake of 125I-hHDL3 was greatest after 15 min of perfusion with label. This was decreased by 80% when the perfusion was carried out at 4 degrees C and by 70-95% when excess unlabeled hHDL, but not human low density lipoprotein (hLDL), was included in the perfusate with 125I-hHDL. Aminopyrazolopyrimidine treatment enhanced 125I-hHDL uptake twofold. After perfusion for 15 min with 125I-hHDL3, radioactivity in the ovary was high for 3-30 min of HDL-free wash, then declined 75% by 30-60 min. With light and electron microscope autoradiography, 125I-hHDL3 was localized to corpora lutea, both along luteal cell surfaces and over their cytoplasm. The plasma membrane grains appeared to be associated with segments that lacked bristle coats. Perfusion with 125I-rat HDL produced a similar pattern of labeling. In ovaries perfused with 125I-BSA, silver grains were concentrated over macrophage-like cells but were sparse over luteal cells. We conclude that the in situ perfused rat ovary takes up 125I-hHDL3 by a temperature-dependent, lipoprotein-specific process, and that this lipoprotein is accumulated by luteal cells.     

Human plasma cholesteryl ester transfer protein enhances the transfer of cholesteryl ester from high density lipoproteins into cultured HepG2 cells

The role of human plasma cholesteryl ester transfer protein (CETP) in the cellular uptake of high density lipoprotein (HDL) cholesteryl ester (CE) was studied in a liver tumor cell line (HepG2). When HepG2 cells were incubated with [3H]cholesteryl ester-labeled HDL3 in the presence of increasing concentrations of CETP there was a progressive increase in cell-associated radioactivity to levels that were 2.8 times control. The CETP-dependent uptake of HDL-CE was found to be saturated by increasing concentrations of both CETP and HDL. The CETP-dependent uptake of CE radioactivity increased continuously during an 18-h incubation. In contrast to the effect on cholesteryl ester, CETP failed to enhance HDL protein cell association or degradation. Enhanced uptake of HDL cholesteryl ester was shown for the d greater than 1.21 g/ml fraction of human plasma, partially purified CETP, and CETP purified to homogeneity, but not for the d greater than 1.21 g/ml fraction of rat plasma which lacks cholesteryl ester transfer activity. HDL cholesteryl ester entering the cell under the influence of CETP was largely degraded to free cholesterol by a process inhibitable by chloroquine. CETP enhanced uptake of HDL [3H]CE in cultured smooth muscle cells and to a lesser extent in fibroblasts but did not significantly influence uptake in endothelial cells or J774 macrophages. These experiments show that, in addition to its known role in enhancing the exchange of CE between lipoproteins, plasma CETP can facilitate the in vitro selective transfer of CE from HDL into certain cells.     

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.     

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.     

Selective uptake of cholesteryl ester from high density lipoproteins by plasma membranes of adipose tissue

The interaction between high density lipoproteins (HDL) and adipose tissue is an important pathway for cholesterol and cholesteryl ester flux. In intact fat cells, a disproportionately greater net uptake of cholesteryl ester occurs subsequent to lipoprotein binding than would have been predicted from a consideration of holoparticle uptake alone. To characterize the early events in this process, cholesteryl hexadecyl ether, a nonmetabolizable, accumulative marker of cholesteryl ester, was incorporated into canine HDL2, and its uptake by omental adipocyte plasma membranes was measured in relation to the binding of HDL2, which in this animal species is enriched in apolipoprotein A-I and free of apolipoprotein E. The dose-response profile for HDL2 binding was consistent with a single lipoprotein binding site at all concentrations of HDL2, whereas uptake of cholesteryl ester from HDL2 was biphasic, suggesting a high affinity site at low HDL2 concentrations and a low affinity site at high lipoprotein concentrations. Pronase treatment stimulated binding twofold and this was accompanied by a parallel twofold stimulation of cholesteryl ester uptake. EDTA, on the other hand, reduced binding and uptake of cholesteryl ester by 20%, indicating partial dependence upon divalent cations. The proportion of HDL2 cholesteryl ester accumulated by plasma membranes relative to HDL2 protein bound was not altered by either pronase or EDTA, despite the fact that these agents had opposite effects upon binding. In dissociation studies, a portion of membrane-associated HDL2 did not equilibrate with exogenous HDL2 and a greater proportion of the cholesteryl ester failed to dissociate. A stepwise mechanism for cholesteryl ester uptake, involving (i) saturable, high affinity HDL2 binding to cell surface sites, (ii) vectoral, HDL2 concentration-dependent delivery of cholesteryl ester to the membrane, and (iii) cholesteryl ester sequestration into a nonexchangeable membrane compartment, appears to be independent of metabolic energy or cell processing.     

Evaluation of cholesteryl ester transfer in the seminiferous tubule cells of immature rats in vivo and in vitro

Sertoli cells and germ cells are separated from the interstitial blood capillaries by an extracellular matrix and the peritubular cells, which constitute a barrier to the movement of plasma lipoproteins. The present study was undertaken to evaluate in vivo and in vitro the high density lipoprotein (HDL) cholesteryl ester transfer from plasma to seminiferous tubule cells in the testis of 30-day-old rats. Firstly, the transfer of HDL cholesteryl oleate from plasma to testicular compartments was evaluated and, secondly, the role of apolipoproteins A-I and E in the uptake of cholesteryl ester by Sertoli cells was investigated. At 2 h after the administration of HDL reconstituted with [3H]cholesteryl ester, dimyristoyl phosphatidylcholine and apolipoproteins, the tissue space in the interstitial cells (740 +/- 60 microliters g-1 cell protein) was fourfold higher than that in the seminiferous tubule cells (170 +/- 10 microliters g-1). Sertoli cells were isolated and incubated with [3H]cholesteryl ester HDL reconstituted with apolipoprotein A-I or E to evaluate the mechanisms of cholesteryl ester influx. At the same apolipoprotein concentration (50 micrograms apolipoprotein ml-1 medium), the uptake of [3H]cholesteryl oleate from phospholipid-apolipoprotein E vesicles was twofold higher than that with phospholipid-apolipoprotein A-I vesicles. The presence of heparin reduced the uptake of cholesteryl ester from apolipoprotein E vesicles but not with apolipoprotein A-I vesicles, indicating that uptake of apolipoprotein A-I vesicles via a secretion of apolipoprotein E by the cells themselves was not involved. These results demonstrate that plasma lipoprotein cholesterol is able to cross the testis lamina propria and that Sertoli cells take up cholesteryl ester for seminiferous tubule cell metabolism mainly via an apolipoprotein E pathway.     

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.     

Catabolism of HDL1 cholesteryl ester in the rat. Effect of ethinyl estradiol treatment.

The present study was performed in control and ethinyl estradiol-treated rats in order to determine the mechanisms involved in the catabolism of HDL1 cholesteryl ester. Ligand blottings on liver membranes showed that purified HDL1, containing about 70% apolipoprotein E and 10% apolipoprotein AI, bind to the LDL receptor (130 kDa) and not to HB2 (100 kDa) or SR-BI (82 kDa), candidate HDL receptors. Immunoblots showed that the treatment increased the hepatic level of the LDL receptor five- to ten-fold, strongly decreased that of SRBI and did not change that of HB2. An in vivo kinetic study showed that the turnover of HDL1 cholesteryl ester is more rapid in treated than control rats. The liver participation (60%) in this clearance was not modified by the treatment. Therefore, it can be concluded that the catabolism of HDL1 cholesteryl ester, in control as in treated rats, is essentially ensured by the uptake of entire particles in the hepatocytes via LDL receptors.     

A low density lipoprotein-sized particle isolated from human atherosclerotic lesions is internalized by macrophages via a non-scavenger-receptor mechanism

A lipoprotein particle designated A-LDL, which contains apolipoprotein B (apoB) and which is the size of plasma low density lipoproteins (LDL), was isolated from homogenates of human aortic athersclerotic plaques by a combination of affinity chromatography and gel-filtration. Compared to plasma LDL, A-LDL was more electronegative, its hydrated density was lower and more heterogeneous, and its protein-to-lipid ratio was lower. In addition, apoB in A-LDL was highly degraded, and A-LDL was recognized by mouse peritoneal macrophages (MPM) as indicated by its ability to stimulate cholesterol esterification. Cholesterol esterification was saturable with an apparent Km of 100 micrograms of A-LDL cholesterol/ml. Stimulation of cholesterol esterification was linear with time, leading to extensive accumulation of cholesteryl ester in MPM over a 48-hr time interval. The uptake or degradation of acetyl-LDL (radiolabeled either in the protein with 125I or hydrophobic core with [3H]cholesteryl ether) was markedly decreased by excess unlabeled acetyl-LDL but not by A-LDL, and excess acetyl-LDL did not inhibit the uptake or degradation of labeled A-LDL. However, a 10-fold excess of A-LDL also failed to inhibit the uptake of labeled A-LDL. This finding was consistent with the observation that, unlike the saturable stimulation of cholesterol esterification in MPM induced by A-LDL, the uptake of cholesteryl ether-labeled A-LDL was almost linear over a 0-400 micrograms cholesterol/ml range. This discrepancy between dose response curves for A-LDL, which did not occur for acetyl-LDL, could be eliminated by a 24-hr postincubation period in the absence of lipoprotein, suggesting that A-LDL is catabolized less efficiently than acetyl-LDL following internalization. In summary, we conclude that A-LDL uptake by MPM occurs via a low affinity-high capacity process. Although the uptake of A-LDL is not readily saturated, it is of sufficient affinity to lead to lipid loading of macrophages even when A-LDL is present at relatively low concentrations. If these mechanisms are operative in vivo, they could explain how foam cells in human fatty streak lesions develop.     

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.     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.     

A hepatocyte receptor for high-density lipoproteins specific for apolipoprotein A-I

Apolipoprotein (apo) E-deficient rat high-density lipoproteins (HDL) bind to isolated rat hepatocytes at 4 degrees C by a process shown to be saturable and competed for by an excess of unlabeled HDL. Uptake (binding and internalization) at 37 degrees C was also saturable and competed for by an excess of unlabeled HDL. At 37 degrees C the HDL apoprotein was degraded as evidenced by the appearance of trichloroacetic acid-soluble radioactivity in the incubation media. The binding of a constant amount of 125I-apo-E-deficient HDL was measured in the presence of increasing concentrations of various lipoproteins. HDL and dimyristoyl phosphatidylcholine (DMPC) X apo-A-I complexes decreased binding by 80 and 65%, respectively. Human low-density lipoproteins, DMPC X apo-E complexes, and DMPC vesicles alone did not compete for apo-E-deficient HDL binding. However, DMPC X apo-E complexes did compete for the binding of the total HDL fraction that contained apo-E but to a lesser extent than did DMPC X apo-A-I. DMPC X 125I-apo-A-I complexes also bound to hepatocytes, and this binding was competed for by excess HDL (70%) and DMPC X apo-A-I complexes (65%), but there was no competition for binding by DMPC vesicles or DMPC X apo-E complexes. It thus appears that hepatocytes have a specific receptor for HDL and that apo-A-I is the ligand for this receptor.     

The kinetic parameters of the uptake of very-low-density lipoprotein remnant cholesteryl esters by perfused rat livers

The aim of this study was to determine the kinetic parameters of the hepatic uptake of VLDL remnant cholesteryl esters. Rat livers were perfused in situ with a broad range of remnant [3H]cholesteryl ester concentrations of known specific radioactivity. Following exactly 3 min of perfusion, hepatic lipids were extracted and labelled cholesteryl esters were separated by thin-layer chromatography and counted. The rate of cholesteryl ester uptake was a saturable process and the apparent kinetic parameters were determined from the Lineweaver-Burk plot of the data. Km and Vmax were calculated to be 72 microM and 35 nmol cholesteryl ester/min per g liver, respectively. For the purpose of comparison, we have expressed our kinetic parameters in terms of number of particles (Vmax = 0.022 nmol particles/min per g liver and Km = 45 nM) and compared our values with those obtained with chylomicron remnants by another group of investigators (Sherrill, B.C., Innerarity, T.L. and Mahley, R.W. (1980) J. Biol. Chem. 255, 1804-1807). We found that the maximal capacity for the removal of VLDL particles was similar to what was observed with rat chylomicron remnants. In contrast, the Km for the uptake process of VLDL remnant particles was approximately four times higher than that of rat chylomicron remnant particles. Our results are consistent with the hypothesis that hepatic removal of both chylomicron and VLDL remnants is mediated by the same receptor, but suggest that the affinity of VLDL remnants for the hepatic removal process is substantially lower, possibly due to structural differences between the two remnant particles.     

Metabolism of high density lipoproteins by the perfused rabbit liver

The role of the liver in the catabolism of high density lipoproteins (HDL) was examined in isolated perfused rabbit livers. Using 125I-labeled rabbit HDL the disappearance of labeled apolipoproteins from the perfusate was biphasic with 7% of the label removed after 20 min and a further 6% between 20 and 90 min. In contrast, with HDL labeled with [3H]cholesteryl esters 35% of label had been removed after 90 min. The effect of liver perfusion on HDL size and composition was further studied by recirculating rabbit HDL for 120 min. In control experiments HDL was incubated at 37 degrees C for 120 min with nonperfused media and with media that had been liver perfused. The added HDL was predominantly particles of 4.8-4.9-mm radius, and incubation with nonperfused and preperfused media produced no significant change in size. However, liver perfusion resulted in particles predominantly 4.2-4.3-mm radius. Hepatic perfusion also significantly reduced HDL cholesteryl ester composition as a percentage of lipoproteins mass from 13.3 +/- 2.2% in control incubations to 10.7 +/- 3.1% (p less than 0.001), and cholesteryl ester:protein mass ratio was reduced from 0.31 +/- 0.06 in control to 0.24 +/- 0.10 (p less than 0.001) after 120 min of liver perfusion. Thus interaction of rabbit HDL with rabbit liver results in smaller HDL particles significantly depleted of core cholesteryl esters.     

The preferential uptake of very-low-density lipoprotein cholesteryl ester by rat liver in vivo.

The removal from the blood and the uptake by the liver of injected very-low-density lipoprotein (VLDL) preparations that had been radiolabelled in their apoprotein and cholesteryl ester moieties was studied in lactating rats. Radiolabelled cholesteryl ester was removed from the blood and taken up by the liver more rapidly than sucrose-radiolabelled apoprotein. Near-maximum cholesteryl ester uptake by the liver occurred within 5 min of the injection of the VLDL. At this time, apoprotein B uptake by the liver was only about 25% of the maximum. Maximum uptake of the injected VLDL apoprotein B label was not achieved until at least 15 min after injection, by which time the total uptakes of cholesteryl ester and apoprotein B label were very similar. The results suggest that preferential uptake of the lipoprotein cholesteryl ester by the liver occurred before endocytosis of the entire lipoprotein complex. The fate of the injected VLDL cholesteryl ester after its uptake by the liver was also monitored. Radiolabel associated with the hepatic cholesteryl ester fraction fell steadily from its early maximum level, the rate of fall being faster and more extensive when the fatty acid, rather than the cholesterol, moiety of the ester was labelled. By 30 min after the injection of VLDL containing [3H]cholesteryl ester, over one-third of the injected label was already present as [3H]cholesterol in the liver. When VLDL containing cholesteryl [14C]oleate was injected, a substantial proportion (about 25%) of the injected radiolabelled fatty acid appeared in the hepatic triacylglycerol fraction within 60 min: very little was present in the plasma triacylglycerol fraction at this time.     

Role of cholesteryl ester transfer protein in selective uptake of high density lipoprotein cholesteryl esters by adipocytes

Previous reports attributed cholesteryl ester transfer protein (CETP)-mediated HDL cholesteryl ester (CE) selective uptake to the CETP-mediated transfer of CE from HDL to newly secreted apolipoprotein B-containing lipoproteins, which are then internalized by the LDL receptor (LDL-R). CETP has also been implicated in the remodeling of HDL, which renders it a better substrate for selective uptake by scavenger receptor class B type I (SR-BI). However, CETP-mediated selective uptake of HDL3-derived CE was not diminished in LDL-R null adipocytes, SR-BI null adipocytes, or in the presence of the receptor-associated protein. We found that monensin treatment or energy depletion of the SW872 liposarcoma cells with 2-deoxyglucose and NaN3 had no effect on CETP-mediated selective uptake, demonstrating that endocytosis is not required. This is supported by data indicating that CETP transfers CE into a compartment from which it can be extracted by unlabeled HDL. CETP could also mediate the selective uptake of HDL3-derived triacylglycerol (TG) and phospholipid (PL). The CETP-specific kinetics for TG and CE uptake were similar, and both reached saturation at approximately 5 microg/ml HDL. In contrast, CETP-specific PL uptake did not attain saturation at 5 microg/ml HDL and was approximately 6-fold greater than the uptake of CE. We propose two possible mechanisms to account for the role of CETP in selective uptake.     

Uptake and degradation of 125I-labelled high density lipoproteins in rat liver cells in vivo and in vitro.

1. The uptake of 125I-labelled high density lipoproteins (HDL) in various organs of the rat was determined after an intravenous injection. The uptake of 125I-labelled polyvinylpyrrolidone in the same organs was determined in order to assess uptake by fluid endocytosis. The uptake/organ was highest for the liver. The adrenals showed the highest uptake/unit weight of the organs studied. The liver, the kidneys and the spleen showed comparable values for uptake/g of tissue. The uptake of 125I-labelled HDL exceeded by far that of 125I-labelled polyvinylpyrrolidone in the liver, the kidneys, the spleen and the adrenals, indicating that the uptake of 125I-labelled HDL was mediated by adsorptive endocytosis. 2. The in vivo uptake of 125I-labelled HDL was determined in purified hepatocytes and non-parenchymal cells prepared by collagenase perfusion of livers from animals after intravenous injections of 125I-labelled HDL. When expressed per cell, the hepatocytes and the non-parenchymal liver cells took up about the same amount of 125I-labelled HDL. 3. The in vitro uptake and degradation of 125I-labelled HDL in isolated rat hepatocytes was studied. The uptake at increasing concentrations of 125I-labelled HDL was saturable indicating uptake mediated through binding sites. 125I-labelled HDL were easily degraded by contaminating proteases from the perfusate. 4. Subcellular fractionation by isopycnic centrifugation indicated that the accumulation of 125I-labelled HDL did not take place in the lysosomes, but rather on the plasma membrane and possibly in the endosomes (phagosomes). 5. 125I-labelled HDL were internalized into the cells and degraded in the lysosomes. Leupetin and chloroquine, inhibitors of the lysosomal function effectively inhibited the formation of 125I-labelled acid-soluble radioactivity by the cells. Chloroquine, but not the protease inhibitor leupeptin, reduced the hydrolysis of the cholesteryl ester moiety of HDL.     

Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I.

The transport of HDL cholesteryl esters (CE) from plasma to the liver involves a direct uptake pathway, mediated by hepatic scavenger receptor B-I (SR-BI), and an indirect pathway, involving the exchange of HDL CE for triglycerides (TG) of TG-rich lipoproteins by cholesteryl ester transfer protein (CETP). We carried out HDL CE turnover studies in mice expressing human CETP and/or human lecithin:cholesterol acyltransferase (LCAT) transgenes on a background of human apoA-I expression. The fractional clearance of HDL CE by the liver was delayed by LCAT transgene, while the CETP transgene increased it. However, there was no incremental transfer of HDL CE radioactivity to the TG-rich lipoprotein fraction in mice expressing CETP, suggesting increased direct removal of HDL CE in the liver. To evaluate the possibility that this might be mediated by SR-BI, HDL isolated from plasma of the different groups of transgenic mice was incubated with SR-BI transfected or control CHO cells. HDL isolated from mice expressing CETP showed a 2- to 4-fold increase in SR-BI-mediated HDL CE uptake, compared to HDL from mice lacking CETP. The addition of pure CETP to HDL in cell culture did not lead to increased selective uptake of HDL CE by cells. However, when human HDL was enriched with TG by incubation with TG-rich lipoproteins in the presence of CETP, then treated with hepatic lipase, there was a significant enhancement of HDL CE uptake. Thus, the remodeling of human HDL by CETP, involving CE;-TG interchange, followed by the action of hepatic lipase (HL), leads to the enhanced uptake of HDL CE by cellular SR-BI.These observations suggest that in animals such as humans in which both the selective uptake and CETP pathways are active, the two pathways could operate in a synergistic fashion to enhance reverse cholesterol transport.     

Regulation of the selective uptake of high density lipoprotein-associated cholesteryl esters

We have previously shown in rats that the cholesteryl ester component of high density lipoproteins (HDL) is taken up at a greater fractional rate than is the apolipoprotein A-I component (selective uptake) by liver and steroidogenic tissues. Selective uptake was also exhibited by cultured cells from these organs as well as by a wider range of cells in vitro (e.g., rat and human fibroblasts). We report here regulation of this pathway according to the cholesterol status of cells. Uptake of HDL cholesteryl esters by rat fibroblasts was decreased by prior loading of the cells with cholesterol, even while uptake of HDL-associated apoA-I actually increased. At high levels of cholesterol, the two were taken up about in parallel, i.e., selective uptake was suppressed. A similar regulation of selective uptake in primary rat hepatocytes in culture was not observed. To examine regulation of selective uptake in vivo, hypocholesterolemia was induced in rats using either 4-aminopyrazolo[3,4-d]pyrimidine or 17 alpha-ethinyl estradiol. Rat HDL, doubly labeled in both the apoprotein A-I and cholesteryl ester moieties with intracellularly trapped tracers, were injected into untreated and treated rats. The plasma decay kinetics and the tissue sites of uptake were then determined. Hypocholesterolemia increased the plasma fractional catabolic rates of both tracers. Selective uptake was observed in tissues of treated rats that did not exhibit selective uptake in untreated rats (muscle, adipose tissue, and skin). Similarly, hypocholesterolemia increased the contribution of selective uptake to total HDL cholesteryl ester uptake by adrenal and ovary. In contrast, regulation of selective uptake by liver could not be demonstrated under these conditions. Thus, selective uptake of HDL cholesteryl esters can be regulated in extrahepatic tissues of rats in vivo and in vitro, suggesting a role for selective uptake in the maintenance of cholesterol homeostasis in these tissues.