Influence of apolipoproteins AI, AII, and Cs on the metabolism of membrane and lysosomal cholesterol in macrophages.

We have demonstrated previously that HDL-mediated efflux of plasma membrane cholesterol is independent of specific binding of apolipoproteins to the high density lipoprotein (HDL) receptor in either control or cholesterol-enriched cells (Karlin, J. B., Johnson, W. J., Benedict, C. R., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1987) J. Biol. Chem. 262, 12557-12564 and Johnson, W. J., Mahlberg, F. H., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1988) J. Biol. Chem. 263, 14099-14106). The present studies were conducted to determine if the process for removal of intracellular (lysosomal) cholesterol is similar to that of membrane cholesterol or if, in contrast, it is selectively regulated by specific apolipoproteins of HDL. For these reasons, we examined the influence of each of the major apolipoproteins of human HDL, apoAI, apoAII, and apoCs on the metabolism of membrane and lysosomal cholesterol in a macrophage foam cell model. We developed an experimental system which allows, for the first time, the simultaneous determination of lysosomal hydrolysis of cholesteryl ester and efflux and esterification of both lysosomal and membrane cholesterol. J774 and elicited mouse peritoneal macrophages were loaded with cholesteryl ester within lysosomes through phagocytosis of sonicated lipid droplets. Membrane and lysosomal pools of cholesterol were differentially radiolabeled. Discoidal complexes of egg phosphatidylcholine and purified apolipoproteins having a similar size and composition were used as cholesterol acceptors. Our results demonstrate that lysosomal hydrolysis of cholesteryl ester is independent of the presence of extracellular acceptors. Lysosomal production of cholesterol stimulates the esterification by acyl-CoA:cholesterol acyltransferase of membrane and lysosomal cholesterol. All the particles tested induce the efflux of both pools of cholesterol at a similar ratio. As efflux is stimulated, esterification by acyl-CoA:cholesterol acyltransferase is reduced. We conclude that none of these apolipoproteins selectively influences the efflux or the esterification of membrane of lysosomal cholesterol. In addition, we observe that particles containing apoAI are the most efficient acceptors, but this effect is not linked to specific binding to the HDL receptor.     

Expression of scavenger receptor BI facilitates sterol movement between the plasma membrane and the endoplasmic reticulum in macrophages

Scavenger receptor BI influences multiple aspects of cellular sterol metabolism. In this series of studies, we evaluated the effect of scavenger receptor BI expression on the distribution and movement of sterol between the plasma membrane and the endoplasmic reticulum in macrophages, by comparing control J774 cells to J774 cells in which SR-BI expression was constitutively increased 3-fold. J774 cells with increased expression of SR-BI (J774-SRBI cells) esterified plasma membrane cholesterol more rapidly as compared to control cells. The esterification of endogenously synthesized cholesterol was also more rapid in cells with increased SR-BI expression; this could be partially suppressed by removing cholesterol from the plasma membrane. The increased plasma membrane sterol esterification in J774-SRBI cells was not due to increased acyl-coA:cholesterol acyltransferase activity and was observed even though J774-SRBI cells manifested a smaller free cholesterol pool in the endoplasmic reticulum. Cholesterol ester hydrolysis was also more rapid in J774-SRBI cells. Increased expression of SR-BI also facilitated the clearance of cellular cholesterol ester to HDL(3). This latter observation, combined with the measurement of the smaller ER free cholesterol pool in J774-SRBI cells, suggests that the free cholesterol derived from the hydrolysis of cholesterol ester was rapidly transported back to the plasma membrane. It is concluded that expression of SR-BI in macrophages increases the rate of free cholesterol transport, and modulates free cholesterol distribution between the plasma membrane and the internal membrane compartments in macrophages.     

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.     

Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes

The relationship of cholesteryl ester hydrolysis to the physical state of the cholesteryl ester in J774 murine macrophages was explored in cells induced to store cholesteryl esters either in anisotropic (ordered) inclusions or isotropic (liquid) inclusions. In contrast to other cell systems, the rate of cholesteryl ester hydrolysis was faster in cells containing anisotropic inclusions than in cells containing isotropic inclusions. Two contributing factors were identified. Kinetic analyses of the rates of hydrolysis are consistent with a substrate competition by co-deposited triglyceride in cells with isotropic inclusions. In addition, hydrolysis of cholesteryl esters in cells with anisotropic droplets is mediated by both cytoplasmic and lysosomal lipolytic enzymes, as shown by using the lysosomotropic agent, chloroquine, and an inhibitor of neutral cholesteryl ester hydrolase, umbelliferyl diethylphosphate. In cells containing anisotropic inclusions, hydrolysis was partially inhibited by incubation in media containing either chloroquine or umbelliferyl diethylphosphate. Together, chloroquine and umbelliferyl diethylphosphate completely inhibited hydrolysis. However, when cells containing isotropic inclusions were incubated with umbelliferyl diethylphosphate, cholesteryl ester hydrolysis was completely inhibited, but chloroquine had no effect. Transmission electron microscopy demonstrated a primarily lysosomal location for lipid droplets in cells with anisotropic droplets and both non-lysosomal and lysosomal populations of lipid droplets in cells with isotropic droplets.These results support the conclusion that there is a lysosomal component to the hydrolysis of stored cholesteryl esters in foam cells.     

Induction of cellular cholesterol efflux to lipid-free apolipoprotein A-I by cAMP

In the present study apolipoprotein-mediated free cholesterol (FC) efflux was studied in J774 macrophages having normal cholesterol levels using an experimental design in which efflux occurs in the absence of contributions from cholesteryl ester hydrolysis. The results show that cAMP induces both saturable apolipoprotein (apo) A-I-mediated FC efflux and saturable apo A-I cell-surface binding, suggesting a link between these processes. However, the EC50 for efflux was 5-7-fold lower than the Kd for binding in both control and cAMP-stimulated cells. This dissociation between apo A-I binding and FC efflux was also seen in cells treated for 1 h with probucol which completely blocked FC efflux without affecting apo A-I specific binding. Thus, cAMP-stimulated FC efflux involves probucol-sensitive processes distinct from apo A-I binding to its putative cell surface receptor. FC efflux was also dramatically stimulated in elicited mouse peritoneal macrophages, suggesting that cAMP-regulated apolipoprotein-mediated FC efflux may be important in cholesterol homeostasis in normal macrophages. The presence of a cAMP-inducible cell protein that interacts with lipid-free apo A-I was investigated by chemical cross-linking of 125I-apo A-I with J774 cell surface proteins which revealed a Mr 200 kDa component when the cells were treated with cAMP.     

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.     

Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL

Helical apolipoproteins of high density lipoprotein (HDL) remove phospholipid and cholesterol from cells and generate HDL particles being mediated by ATP binding cassette transporter A1 (ABCA1). In murine macrophage cell line RAW264 cells, cAMP induced expression of ABCA1, release of cellular phospholipid and cholesterol by apolipoprotein A-I (apoA-I), and reversible binding of apoA-I to the cell. The apoA-I-dependent lipid release was directly proportional to the cAMP-induced binding of apoA-I, and was inhibited 70% by a monoclonal antibody selective to lipid-free apoA-I, 725-1E2. In contrast, apparent cellular cholesterol release to HDL was substantial even without ABCA1 induction, and it was increased only by 27% after the cAMP treatment. The antibody inhibited this increment by 70%. Lipid-free apoA-II liberated apoA-I from HDL by displacement and thereby markedly expanded the cAMP-induced part of the cholesterol release to the HDL-containing medium, and the antibody inhibited this part also by 70%. Binding experiments of the double-labeled reconstituted HDL showed that cAMP induced reversible binding of apoA-I but not the association of cholesteryl ester with the cells. The effect of the antibody on the cellular cholesterol release to the reconstituted HDL was similar to that of the HDL-mediated release. The data implicated that the ABCA1-dependent cholesterol release to HDL is mediated by apoA-I dissociated from HDL.     

Co-expression of scavenger receptor-BI and caveolin-1 is associated with enhanced selective cholesteryl ester uptake in THP-1 macrophages.

Scavenger receptor (SR)-BI mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. In Chinese hamster ovary (CHO) cells, SR-BI is predominantly associated with caveolae which we have recently demonstrated are the initial loci for membrane transfer of HDL cholesteryl esters. Because cholesterol accumulation in macrophages is a critical event in atherogenesis, we investigated the expression of SR-BI and caveolin-1 in several macrophage cell lines. Human THP-1 monocytes were examined before and after differentiation to macrophages by treatment with 200 nm phorbol ester for 72 h. Undifferentiated THP-1 cells expressed caveolin-1 weakly whereas differentiation up-regulated caveolin-1 expression greater than 50-fold. In contrast, both undifferentiated and differentiated THP-1 cells expressed similar levels of SR-BI. Differentiation of THP-1 cells increased the percent of membrane cholesterol associated with caveolae from 12% +/- 1.9% to 38% +/- 3.1%. The increase in caveolin-1 expression was associated with a 2- to 3-fold increase in selective cholesterol ether uptake from HDL. Two mouse macrophage cell lines, J774 and RAW, expressed levels of SR-BI similar to differentiated THP-1 cells but did not express detectable levels of caveolin-1. In comparison to differentiated THP-1 cells, RAW and J774 cells internalized 9- to 10-fold less cholesteryl ester. We conclude that differentiated THP-1 cells express both caveolin-1 and SR-BI and that their co-expression is associated with enhanced selective cholesteryl ester uptake.     

Cytoskeleton disruption in J774 macrophages: Consequences for lipid droplet formation and cholesterol flux

Macrophages store excess unesterified cholesterol (free, FC) in the form of cholesteryl ester (CE) in cytoplasmic lipid droplets. The hydrolysis of droplet-CE in peripheral foam cells is critical to HDL-promoted reverse cholesterol transport because it represents the first step in cellular cholesterol clearance, as only FC is effluxed from cells to HDL. Cytoplasmic lipid droplets move within the cell utilizing the cytoskeletal network, but, little is known about the influence of the cytoskeleton on lipid droplet formation. To understand this role we employed cytochalasin D (cyt.D) to promote actin depolymerization in J774 macrophages. Incubating J774 with acetylated LDL creates foam cells having a 4-fold increase in cellular cholesterol content (30-40% cholesterol present as cholesteryl ester (CE)) in cytoplasmic droplets. Lipid droplets formed in the presence of cyt.D are smaller in diameter. CE-deposition and -hydrolysis are decreased when cells are cholesterol-enriched in the presence of cyt.D or latrunculin A, another cytoskeleton disrupting agent. However, when lipid droplets formed in the presence of cyt.D are isolated and incubated with an exogenous CE hydrolase, the CE is more rapidly metabolized compared to droplets from control cells. This is apparently due to the smaller size and altered lipid composition of the droplets formed in the presence of cyt.D. Cytoskeletal proteins found on CE droplets influence droplet lipid composition and maturation in model foam cells. In J774 macrophages, cytoskeletal proteins are apparently involved in facilitating the interaction of lipid droplets and a cytosolic neutral CE hydrolase and may play a role in foam cell formation. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Upregulation of selective cholesteryl ester uptake pathway in mice with deletion of low-density lipoprotein receptor function

This study examines the effect of mutation of the low-density lipoprotein receptor (LDLR) on cholesterol metabolism, and especially lipoprotein-derived cholesteryl ester uptake, in murine ovarian granulosa cells. Although the tests were conducted on cells prepared by two different procedures, the results are similar. Deletion of LDLR function did not noticeably affect key enzymes of the steroidogenic pathway or affect progestin production and secretion in granulosa cells. No change was found in expression of LDL-related protein (LRP). These data suggested that cholesterol turnover in cells from the knockout animals is within normal limits and that the cells are not stressed to acquire more cholesterol. Both biochemical and morphological data indicate that unstimulated granulosa cells from LDLR^−/− mice are nonetheless programmed to take in double the amount of lipoprotein-derived cholesteryl ester (via the selective cholesteryl ester uptake pathway) and to process (hydrolyze, re-esterify, or utilize) more than twofold the cholesteryl ester processed by cells from wildtype (LDLR^+/+) animals. Bt₂cAMP stimulation of the murine granulosa cells increases the mass of cholesteryl ester taken up by the selective pathway by an additional 38%. To determine to what extent this increase is related to high-density lipoprotein (HDL) scavenger receptor protein (SR-BI) or caveolin function, Western blots and immunohistochemical studies were performed under a variety of conditions. SR-BI levels are found to be low in unstimulated cells of both LDLR^+/+ and LDLR^−/− animals, but highly expressed (∼20-fold increase over basal levels) in stimulated (Bt₂cAMP) cells of both animal models. Thus, the functional relationship between selective cholesteryl ester uptake and SR-BI receptor protein is not as tight as in previously reported studies, suggesting a requirement for other tissue factors. Caveolin expression did not change under any of the conditions tested and appears not to be functionally involved in this process. J. Cell. Physiol. 180:190–202, 1999. Published 1999 Wiley-Liss, Inc.     

Evidence for extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (Fu5AH): a model for delivery of high-density lipoprotein cholesterol

Rat hepatoma cells (Fu5AH) were studied as a model for the net delivery of apoE-free high-density lipoprotein (HDL) cholesterol to a cell. Incubating cells with HDL results in 1) a decrease in both media-free cholesterol and cholesteryl ester concentration; 2) decreased cell sterol synthesis; and 3) increased cell cholesteryl ester synthesis. HDL cholesteryl ester uptake is increased when cells are incubated for 18 hr in cholesterol poor media. Coincubation of 3H-cholesteryl ester-labeled low-density lipoprotein (LDL) with 50 microM chloroquine or 25 microM monensin results in a decrease in the cellular free cholesterol/cholesteryl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free cholesterol. In contrast, chloroquine and monensin do not alter the cellular FC/CE isotope ratio for 3H-CE HDL. This evidence indicates that acidic lysosomal cholesteryl ester hydrolase does not account for the hydrolysis of HDL-CE. Free cholesterol generated from 3H-cholesteryl ester of both LDL and HDL is reesterified intracellularly. At higher HDL concentrations (above 50 micrograms/ml) HDL cholesteryl ester hydrolysis is sensitive to chloroquine. We propose that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and that at higher HDL concentrations a lysosomal pathway may be functioning in addition to an extralysosomal pathway.     

Inhibition of acyl coenzyme A:cholesterol acyl transferase in J774 macrophages enhances down-regulation of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and prevents low density lipoprotein-induced cholesterol accumulation

Cholesteryl ester accumulation in arterial wall macrophages (foam cells) is a prominent feature of atherosclerotic lesions. We have previously shown that J774 macrophages accumulate large amounts of cholesteryl ester when incubated with unmodified low density lipoprotein (LDL) and that this is related to sluggish down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. To further explore intracellular cholesterol metabolism and regulatory events in J774 macrophages, we studied the effect of inhibitors of acyl-CoA:cholesterol acyl transferase (ACAT) on the cells' ability to accumulate cholesterol and to down-regulate receptor and reductase. Treatment of J774 cells with LDL in the presence of ACAT inhibitor 58-035 (Sandoz) prevented both cholesteryl ester and total cholesterol accumulation. Furthermore, 58-035 markedly enhanced down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-CoA reductase in the presence of LDL. In dose-response studies, down-regulation of the receptor by 58-035 paralleled its inhibition of ACAT activity. Compound 58-035 also increased the down-regulation of the J774 LDL receptor in the presence of 25-hydroxycholesterol and acetyl-LDL but not in the presence of cholesteryl hemisuccinate, which is not an ACAT substrate. The ability of 58-035 to enhance LDL receptor down-regulation was negated when cells were simultaneously incubated with recombinant high density lipoprotein3 discs, which promote cellular cholesterol efflux. In contrast to the findings with J774 macrophages, down-regulation of the human fibroblast LDL receptor was not enhanced by 58-035. These data suggest that in J774 macrophages, but not in fibroblasts, ACAT competes for a regulatory pool of intracellular cholesterol, contributing to diminished receptor and reductase down-regulation, LDL-cholesterol accumulation, and foam cell formation.     

Lipoprotein lipase enhances the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins

These studies were undertaken to examine the effects of lipoprotein lipase (LPL) and cholesteryl ester transfer protein (CETP) on the transfer of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins (VLDL). Human or rat VLDL was incubated with human HDL in the presence of either partially purified CETP, bovine milk LPL or CETP plus LPL. CETP stimulated both isotopic and mass transfer of cholesteryl esters from HDL into VLDL. LPL caused only slight stimulation of cholesteryl ester transfer. However, when CETP and LPL were both present, the transfer of cholesteryl esters from HDL into VLDL remnants was enhanced 2- to 8-fold, compared to the effects of CETP alone. The synergistic effects of CETP and LPL on cholesteryl ester transfer were more pronounced at higher VLDL/HDL ratios and increased with increasing amounts of CETP. In time course studies the stimulation of cholesteryl ester transfer activity occurred during active triglyceride hydrolysis. When lipolysis was inhibited by incubating LPL with either 1 M NaCl or 2 mM diethylparanitrophenyl phosphate, the synergism of CETP and LPL was reduced or abolished, and LPL alone did not stimulate cholesteryl ester transfer. These experiments show that LPL enhances the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. This property of LPL is related to lipolysis.     

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.     

Metabolism of cholesteryl ester lipid droplets in a J774 macrophage foam cell model

J774 macrophages rapidly incorporated [3H]cholesteryl oleate droplets by a non-saturable phagocytic process. In less than 2 h, foam cell morphology was acquired. The extent of loading obtained after 2 h was a linear function of the mass of cholesteryl oleate provided to the cells. The cholesteryl oleate incorporated was hydrolyzed in the cells at a linear rate over 24 h and the fractional hydrolysis was constant over a wide range of cellular esterified cholesterol contents. The rate of hydrolysis was influenced by the physical state of the cholesteryl ester; cholesteryl oleate in isotropic droplets was hydrolyzed 2-3-fold more rapidly than cholesteryl oleate in anisotropic droplets. The hydrolysis of both types of droplets was inhibited by lysosomotropic agents, indicating that hydrolysis occurred in the lysosomes. Only a small fraction (less than 10% after 24 h) of the free [3H]cholesterol generated in the lysosomes was esterified by ACAT resulting in a doubling of the cell free cholesterol content. Electron microscopy of cells treated with digitonin revealed the accumulation of free cholesterol in lipid-laden lysosomes. ACAT was active as endogenous free [14C]cholesterol was esterified in a linear manner over 24 h and was responsive to the presence of lysosomally-derived cholesterol, as the extent of esterification of the endogenous pool was directly proportional to the mass of [3H]cholesterol generated in the lysosomes.     

HDL modification by secretory phospholipase A(2) promotes scavenger receptor class B type I interaction and accelerates HDL catabolism

During inflammatory states plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) are reduced. Secretory group IIa phospholipase A(2) (sPLA(2)) is a cytokine-induced acute-phase enzyme associated with HDL. Transgenic mice overexpressing sPLA(2) have reduced HDL levels. Studies were performed to define the mechanism for the HDL reduction in these mice. HDL isolated from sPLA(2) transgenic mice have a significantly lower phospholipid content and greater triglyceride content. In autologous clearance studies, (125)I-labeled HDL from sPLA(2) transgenic mice was catabolized significantly faster than HDL from control mice (4.24 +/- 1.16 vs. 2.84 +/- 0.1 pools per day, P < 0.008). In both sPLA(2) transgenic and control mice, the cholesteryl ester component of HDL was more rapidly catabolized than the protein component, indicating a selective uptake mechanism. In vitro studies using CHO cells transfected with scavenger receptor class B type I (SR-BI) showed that sPLA(2)-modified HDL was nearly twice as efficient as a substrate for cholesteryl ester transfer. These data were confirmed in in vivo selective uptake experiments using adenoviral vector overexpression of SR-BI. In these studies, increased hepatic selective uptake was associated with increased (125)I-labeled apolipoprotein uptake in the kidney.We conclude that during inflammation sPLA(2) hydrolysis of HDL phospholipids alters the lipid composition of the particle, allowing for more efficient SR-BI-mediated selective cholesteryl ester uptake. This enhanced SR-BI activity generates HDL remnants that are preferentially catabolized in the kidney.     

Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester

Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied. Addition of [3H]CE-HDL to cells pretreated with adrenocorticotrophin in lipoprotein poor media resulted in a time- and concentration-dependent accumulation of [3H]cholesteryl ester and production of [3H]cholesterol and [3H]corticosterone. HDL-CE metabolism could be described as the sum of a high affinity ([ HDL-cholesterol]1/2 max = 16 micrograms/ml) and low affinity ([ HDL-cholesterol]1/2 max greater than 70 micrograms/ml) process. [3H]Cholesterol was found both intracellularly and in the media. Accumulation of [3H]cholesteryl ester could not be attributed to uptake and re-esterification of unesterified cholesterol since addition of Sandoz 58-035, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, did not prevent ester accumulation. Moreover, addition of chloroquine did not inhibit cholesteryl ester hydrolysis indicating that hydrolysis was not lysosomally mediated. Aminoglutethimide prevented conversion of [3H]CE-HDL to steroid hormones but did not inhibit [3H]cholesteryl ester uptake. Cellular accumulation of [3H] cholesteryl ester exceeded accumulation of 125I-apoproteins 5-fold at 1 h and 35-fold at 24 h indicating selective uptake of cholesteryl ester moiety. We conclude that rat adrenal cells possess a mechanism for selective uptake of HDL cholesteryl esters which provides substrate for steroidogenesis. These results constitute the first direct demonstration that cholesteryl esters in HDL can be used as steroidogenic substrate by the rat adrenal cortex.     

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.     

The effects of apolipoprotein F deficiency on high density lipoprotein cholesterol metabolism in mice

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20-25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/-0.9 mg/dl vs. WT: 1.2+/-0.3 mg/dl, p<0.05). No differences were observed in ABCG1-mediated cholesterol efflux capacity in either sex. Interestingly, ApoB-depleted serum from male KO mice was less effective at promoting ABCA1-mediated cholesterol efflux from J774 macrophages relative to WT controls.     

Regulation of the uptake of high-density lipoprotein-originated cholesteryl ester by HepG2 cells: role of low-density lipoprotein and plasma lipid transfer protein.

Cholesteryl ester uptake by the human hepatoma cell line HepG2 was studied in vitro by using radiolabeled cholesteryl ester as a tracer. After the cells were incubated in a lipoprotein deficient condition, the rate of radio labeled cholesteryl ester uptake from low-density lipoprotein (LDL) was estimated to be some 25-times higher than that from high-density lipoprotein (HDL). LDL-cholesteryl ester uptake was suppressed by preincubation of the cells with LDL, but pretreatment of the cells with HDL did not show significant effect. HDL-cholesteryl ester uptake was only slightly suppressed by pretreatment of the cells with LDL, and there was no effect with HDL pretreatment. HDL-cholesteryl ester uptake was not affected either by the presence of LDL or human plasma lipid transfer protein alone in the medium under our experimental conditions. Lipid transfer protein enhanced the uptake of radiolabeled cholesteryl ester originating from HDL by the cells only in the presence of LDL. Thus, lipid transfer protein catalyzes a bypass to LDL for the uptake by HepG2 cells of cholesteryl ester molecules which originate in HDL, and this pathway is much more efficient than direct uptake of cholesteryl ester originating in HDL by these cells.