Cellular cholesterol flux studies: methodological considerations.

Reverse cholesterol transport (RCT) is the process in which peripheral cells release cholesterol to an extracellular acceptor such as high-density lipoprotein (HDL) which then mediates cholesterol delivery to the liver for excretion. RCT represents a physiological mechanism by which peripheral tissues are protected against excessive accumulation of cholesterol. The first step in RCT is the interaction of the cell with lipoprotein particles, a process that results in both the cellular uptake and release of cholesterol. The various components of this cholesterol flux can be viewed as efflux, influx and net flux. Experimental protocols for measuring each of these components of cholesterol flux are very different, and a number of considerations are required to design experimental approaches for the quantitation of flux parameters. Although many flux studies have been conducted in the past, the recent discoveries of the scavenger receptor B1 (SR-B1) and ATP binding cassette 1 (ABCA1), which mediate the movement of cholesterol between cells and extracellular acceptors, has led to increased interest in studies of cellular cholesterol flux. The aim of this review is to present a discussion of the methodological considerations that should be evaluated during the design and analysis of cellular cholesterol flux experiments.

Scavenger receptor class B type I affects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL.

Results from several laboratories clearly indicate that expression of scavenger receptor class B type I (SR-BI) enhances the bidirectional flux of cholesterol between cells and lipoproteins. Because the activity of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis, is regulated by cell cholesterol content, we designed experiments to investigate the effect of SR-BI expression on the activity of this enzyme and on net cellular cholesterol mass. In addition, we compared the function of SR-BI with its human homolog, CD36 and LIMPII analogous 1. Our experiments demonstrate that both receptors enhance the flux of unesterified or free cholesterol bidirectionally, down a concentration gradient. Receptor-mediated cholesterol flux can effectively modulate multiple aspects of cellular cholesterol metabolism, including the pool that regulates the activity of HMG-CoA reductase. We also found that constitutive expression of SR-BI alters the steady state level of cellular cholesterol and phospholipid when SR-BI-expressing cells are maintained in medium containing serum lipoproteins. All of these effects are proportional to the level of receptor on the cell surface. These data indicate that the level of SR-BI expression determines both the rate of free cholesterol flux and the steady state level of cellular cholesterol.

Analysis of chimeric receptors shows that multiple distinct functional activities of scavenger receptor, class B, type I (SR-BI), are localized to the extracellular receptor domain.

Scavenger receptor BI (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without degradation of the HDL particle. In addition, SR-BI stimulates the bi-directional flux of free cholesterol (FC) between cells and lipoproteins, an activity that may be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of FC from plasma HDL. SR-BI also increases cellular cholesterol mass and alters cholesterol distribution in plasma membrane domains as judged by the enhanced sensitivity of membrane cholesterol to extracellular cholesterol oxidase. In contrast, CD36, a closely related class B scavenger receptor, has none of these activities despite binding HDL with high affinity. In the present study, analyses of chimeric SR-BI/CD36 receptors and domain-deleted SR-BI have been used to test the various domains of SR-BI for functional activities related to HDL CE selective uptake, bi-directional FC flux, and the alteration of membrane cholesterol mass and distribution. The results show that each of these activities localizes to the extracellular domain of SR-BI. The N-terminal cytoplasmic tail and transmembrane domains appear to play no role in these activities other than targeting the receptor to the plasma membrane. The C-terminal tail of SR-BI is dispensable for activity as well for targeting to the plasma membrane. Thus, multiple distinct functional activities are localized to the SR-BI extracellular domain.

High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI.

The role of high density lipoprotein (HDL) phospholipid in scavenger receptor BI (SR-BI)-mediated free cholesterol flux was examined by manipulating HDL(3) phosphatidylcholine and sphingomyelin content. Both phosphatidylcholine and sphingomyelin enrichment of HDL enhanced the net efflux of cholesterol from SR-BI-expressing COS-7 cells but by two different mechanisms. Phosphatidylcholine enrichment of HDL increased efflux, whereas sphingomyelin enrichment decreased influx of HDL cholesterol. Although similar trends were observed in control (vector-transfected) COS-7 cells, SR-BI overexpression amplified the effects of phosphatidylcholine and sphingomyelin enrichment of HDL 25- and 2.8-fold, respectively. By using both phosphatidylcholine-enriched and phospholipase A(2)-treated HDL to obtain HDL with a graded phosphatidylcholine content, we showed that SR-BI-mediated cholesterol efflux was highly correlated (r(2) = 0.985) with HDL phosphatidylcholine content. The effects of varying HDL phospholipid composition on SR-BI-mediated free cholesterol flux were not correlated with changes in either the K(d) or B(max) values for high affinity binding to SR-BI. We conclude that SR-BI-mediated free cholesterol flux is highly sensitive to HDL phospholipid composition. Thus, factors that regulate cellular SR-BI expression and the local modification of HDL phospholipid composition will have a large impact on reverse cholesterol transport.

The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines.

Studies show that lipid-free apoA-I stimulates release of cholesterol and phospholipid from fibroblasts and macrophages. ATP-binding cassette 1 (ABC1) is implicated in this release and has been identified as the genetic defect in Tangier disease, evidence that ABC1 is critical to the biogenesis of high density lipoprotein. We quantified levels of ABC1 mRNA, protein, and cholesterol efflux from J774 mouse macrophages +/- exposure to a cAMP analog. Up-regulating ABC1 mRNA correlated to increased cholesterol efflux in a dose- and time-dependent manner. mRNA levels rose after 15 min of exposure while protein levels rose after 1 h, with increased efflux 2-4 h post-treatment. In contrast to cells from wild-type mice, peritoneal macrophages from the Abc1 -/- mouse showed a lower level of basal efflux and no increase with cAMP treatment. The stimulation of efflux exhibits specificity for apoA-I, high density lipoprotein, and other apolipoproteins as cholesterol acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin. We have studied a number of cell types and found that while other cell lines express ABC1 constitutively, only J774 and elicited mouse macrophages show a substantial increase of mRNA and efflux with cAMP treatment. ApoA-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment.

Human ApoA-IV overexpression in transgenic mice induces cAMP-stimulated cholesterol efflux from J774 macrophages to whole serum.

The role of apolipoprotein A-IV (apoA-IV) in lipoprotein metabolism has not been established. The aim of the present study was to investigate the role of apoA-IV in reverse cholesterol transport by comparing cellular cholesterol efflux to serum or serum fractions from control mice and from mice transgenic for human apoA-IV (HuA-IVTg mice). When Fu5AH hepatoma cells were used, the cholesterol efflux to serum from either control or transgenic mice was similar. When control J774 macrophage cells were used, a comparison of efflux to serum or lipoprotein-deficient serum (LPDS) failed to demonstrate any differences between control and transgenic mice. In contrast, when the J774 cells were pretreated with cAMP, there was a stimulation of efflux to whole serum or LPDS from HuA-IVTg mice. cAMP treatment had no effect on efflux to serum or LPDS from control mice. Pretreatment of the cells with cAMP did not enhance the efflux response to high density lipoprotein isolated from HuA-IVTg mouse serum. Our results suggest that apoA-IV, unassociated with high density lipoprotein particles, is responsible for enhanced cholesterol efflux. This study illustrates the role of lipid-free apolipoproteins in mediating cellular cholesterol efflux with use of a biological fluid and is potentially of physiological relevance, especially in apolipoprotein-rich extravascular fluids.

Efflux of cholesterol from different cellular pools.

Free cholesterol is very efficiently removed from cells by 2-hydroxypropyl-beta-cyclodextrins. The efflux of cholesterol occurs from two distinct kinetic pools: the half-times (t(1/2)) for the two pools in CHO-K1 cells are 15 +/- 5 s and 21 +/- 6 min and they represent 25% +/- 5% and 75% +/- 5% of the readily exchangeable cell cholesterol, respectively. In this study we have determined that the fast pool and the majority of the slow kinetic pool for cholesterol efflux are apparently present in the plasma membrane. Numerous agents that inhibit intracellular cholesterol trafficking are unable to affect either the size or the t(1/2) for efflux of either kinetic pool. In contrast, treatment of the cells with N-ethylmaleimide (NEM), exogenous lipases such as sphingomyelinase and phospholipase C, calcium ionophore A23187, or heat resulted in the dramatic increase in the size of the fast kinetic pool of cholesterol. These changes in the kinetics of cholesterol efflux are not specific to the nature of the extracellular acceptor indicating that they are a consequence of changes in the cell plasma membrane. The above treatments disrupt the normal organization of the lipids in the plasma membrane via either hydrolysis or randomization. The phosphatidylcholine and sphingomyelin present in the plasma membrane are critical for maintaining the two kinetic pools of cholesterol; any alteration in the amount or the location of these phospholipids results in an enhancement of efflux by redistributing cholesterol into the fast kinetic pool.

Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells.

Activation of volume-regulated anion current (VRAC) plays a key role in the maintenance of cellular volume homeostasis. The mechanisms, however, that regulate VRAC activity are not fully understood. We have examined whether VRAC activation is modulated by the cholesterol content of the membrane bilayer. The cholesterol content of bovine aortic endothelial cells was increased by two independent methods: (a) exposure to a methyl-beta-cyclodextrin saturated with cholesterol, or (b) exposure to cholesterol-enriched lipid dispersions. Enrichment of bovine aortic endothelial cells with cholesterol resulted in a suppression of VRAC activation in response to a mild osmotic gradient, but not to a strong osmotic gradient. Depletion of membrane cholesterol by exposing the cells to methyl-beta-cyclodextrin not complexed with cholesterol resulted in an enhancement of VRAC activation when the cells were challenged with a mild osmotic gradient. VRAC activity in cells challenged with a strong osmotic gradient were unaffected by depletion of membrane cholesterol. These observations show that changes in membrane cholesterol content shift VRAC sensitivity to osmotic gradients. Changes in VRAC activation were not accompanied by changes in anion permeability ratios, indicating that channel selectivity was not affected by the changes in membrane cholesterol. This suggests that membrane cholesterol content affects the equilibrium between the closed and open states of VRAC channel rather than the basic pore properties of the channel. We hypothesize that changes in membrane cholesterol modulate VRAC activity by affecting the membrane deformation energy associated with channel opening.

Expression of scavenger receptor BI in COS-7 cells alters cholesterol content and distribution.

Previous studies have shown that scavenger receptor BI (SR-BI) stimulates the bidirectional flux of free cholesterol (FC) between HDL and SR-BI-expressing cells. A major component of the enhanced FC flux appears to occur independently of HDL binding to SR-BI and may be due to changes in membrane lipid domains resulting from SR-BI expression (1). In the present study, the impact of SR-BI on cellular cholesterol metabolism was determined by examining SR-BI-mediated changes in cellular cholesterol mass, the esterification of HDL-derived FC, and changes in membrane lipid pools. Growth of SR-BI-expressing cells in medium containing HDL led to increased cellular cholesterol mass, most of which accumulated as ester. The esterification of HDL-derived FC was enhanced by SR-BI-expression to a far greater extent than the SR-BI mediated increase in FC uptake, suggesting an SR-BI-mediated effect on cholesterol utilization in the cell. This observation was tested by comparing FC esterification rates in SR-BI positive and negative cells when equivalent amounts of extracellular FC were taken up via cyclodextrins or apolipoprotein AI/phospholipid disks, neither of which contained cholesteryl ester. Under these conditions, SR-BI did not preferentially stimulate cholesterol esterification. These results indicate that the enhanced esterification of HDL-derived FC in SR-BI-expressing cells is due to the expanded pool of cellular FC and not to a specific effect of SR-BI on cholesterol utilization. Two approaches were used to test the effects of SR-BI expression on membrane lipid organization. In the first, the sensitivity of cellular FC to exogenous cholesterol oxidase was tested under conditions in which there is a preferential oxidation of caveolar cholesterol. SR-BI-expression was found to greatly increase the fraction of cellular cholesterol available to the oxidase as compared to either vector-transfected cells or cells expressing the related class B scavenger receptor CD36. These results suggest that SR-BI expression alters the distribution of membrane-free cholesterol to a caveolar fraction or alters the accessibility of this membrane fraction to exogenous cholesterol oxidase. In the second approach, the efflux of cellular FC to high concentrations of cyclodextrins was monitored under conditions where desorption of FC from the plasma membrane is rate limiting for efflux. SR-BI-expressing cells showed a shift in the distribution of FC between two kinetic pools with more FC in the fast pool and less in the slow pool. These data support a model in which SR-BI expression leads to a redistribution of cholesterol to membrane domains that serve to facilitate the flux of FC between cells and lipoproteins.

Cytotoxic cholesterol is generated by the hydrolysis of cytoplasmic cholesteryl ester and transported to the plasma membrane.

The present study examines the fate and effects of free cholesterol (FC) generated by the hydrolysis of cytoplasmic cholesteryl esters (CE) in model macrophage foam cells. J774 or elicited mouse peritoneal macrophages (MPM) were enriched with CE by incubating with acetylated low density lipoprotein (acLDL) and FC/phospholipid dispersions, thus creating model foam cells. Treatment of the foam cells with the acyl coenzyme-A:cholesterol acyltransferase (ACAT) inhibitor, CP-113,818, in the absence of any extracellular cholesterol acceptors, resulted in cellular toxicity. This was accompanied by an increase in the amount of FC available for oxidation by an exogenous cholesterol oxidase. Furthermore, cellular toxicity was proportional to the size of the oxidase susceptible pool of FC over time. Morphological analysis and in situ DNA fragmentation assay demonstrated the occurrence of apoptosis in the ACAT inhibited cells. Co-treatment with the hydrophobic amine U18666A, an intracellular cholesterol transport inhibitor, led to a dose dependent reduction in cytotoxicity and apoptosis, and blocked the movement of FC into the oxidase susceptible pool. In addition, treating model foam cells with CP-113,818 plus chloroquine, a compound that inhibits the function of acidic vesicles, also diminished cellular toxicity. Staining with the cholesterol binding dye filipin revealed that the macrophages treated with CP-113,818 contained a cholesterol oxidase accessible pool of FC in the plasma membrane. These results suggest that FC generated by the hydrolysis of cytoplasmic CE is transported through acidic vesicles to the plasma membrane, and accumulation of FC in this pool triggers cell death by necrosis and apoptosis.

Scavenger receptor BI and cholesterol trafficking.

Scavenger receptor BI (SR-BI) mediates the selective uptake of HDL cholesteryl ester into steroidogenic cells and the liver and is a major determinant of the plasma HDL concentration in the mouse. Recent studies indicate that SR-BI also alters the metabolism of apolipoprotein B-containing particles and influences the development of atherosclerosis in several animal models. These results and the similar pattern of SR-BI expression in humans emphasize that it is important to learn how this receptor influences lipoprotein metabolism and atherosclerosis in people.

Crystallization of free cholesterol in model macrophage foam cells.

-The present study examined free cholesterol (FC) crystallization in macrophage foam cells. Model foam cells (J774 or mouse peritoneal macrophages [MPMs]) were incubated with acetylated low density lipoprotein and FC/phospholipid dispersions for 48 hours, resulting in the deposition of large stores of cytoplasmic cholesteryl esters (CEs). The model foam cells were then incubated for up to 5 days with an acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor (CP-113,818) in the absence of an extracellular FC acceptor to allow intracellular accumulation of FC. FC crystals of various shapes and sizes formed in the MPMs but not in the J774 macrophages. Examination of the MPM monolayers by microscopy indicated that the crystals were externalized rapidly after formation and thereafter continued to increase in size. Incubating J774 macrophages with 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP) in addition to CP-113,818 caused FC crystal formation as a consequence of CPT-cAMP stimulation of CE hydrolysis and inhibition of cell growth. In addition, 2 separate cholesterol phases (liquid-crystalline and cholesterol monohydrate) in the plane of the membrane bilayer were detected after 31 hours of ACAT inhibition by the use of small-angle x-ray diffraction of J774 macrophage foam cells treated with CPT-cAMP. Other compounds reported to inhibit ACAT, namely progesterone (20 microgram/mL) and N-acetyl-D-sphingosine (c(2)-ceramide, 10 microgram/mL), induced cellular toxicity in J774 macrophage foam cells and FC crystallization when coincubated with CPT-cAMP. Addition of the extracellular FC acceptors apolipoproteins (apo) E and A-I (50 microgram/mL) reduced FC crystal formation. In MPMs, lower cell density and frequent changes of medium were conducive to crystal formation. This may be due to "dilution" of apoE secreted by the MPMs and is consistent with our observation that the addition of exogenous apoE or apoA-I inhibits FC crystal formation in J774 macrophage foam cells cotreated with CP-113,818 plus CPT-cAMP. These data demonstrate that FC crystals can form from the hydrolysis of cytoplasmic stores of CEs in model foam cells. FC crystal formation can be modulated by the addition of extracellular FC acceptors or by affecting the cellular rate of CE hydrolysis. This process may contribute to the formation of FC crystals in atherosclerotic plaques.

Comparison of the capacity of beta-cyclodextrin derivatives and cyclophanes to shuttle cholesterol between cells and serum lipoproteins.

Previous studies from this laboratory have demonstrated that low concentrations of cyclodextrins (<1.0 mm), when added to serum, act catalytically as cholesterol shuttles to accelerate the exchange of free cholesterol between cells and serum lipoproteins. As cholesterol shuttles, cyclodextrins have the potential to serve as pharmacological agents for modifying cholesterol metabolism. In the present study, we have quantitated the cholesterol-shuttling capacity of a series of newly synthesized beta-cyclodextrin derivatives (betaCDs), with varying structure, and two double-decker cyclophanes. The general protocol is as follows. [(3)H]cholesterol-labeled CHOK1 cells are incubated for 2 h with the test compounds alone or together with 5% human serum, and efflux of the cellular [(3)H]cholesterol is measured. As methyl beta-cyclodextrin (MbetaCD) served as the basis for comparison, initial experiments were conducted that demonstrated there was a dose-dependent stimulation of cell cholesterol efflux as the concentration of MbetaCD increased, with an EC(50) that was calculated to be 0.05 mm. To determine the cholesterol-shuttling capacity of the newly synthesized compounds, cell cholesterol efflux is measured when the compounds are present alone, at a concentration of 0.05 mm, or together with 5% human serum. Our results demonstrate that the double-decker cyclophanes are the most efficient cholesterol shuttles. Under our experimental conditions, methyl beta-cyclodextrin (MbetaCD) approximately doubles the efflux of cell cholesterol to serum, whereas one of the double-decker cyclophanes produces a 4-fold stimulation in efflux. Four of the beta-cyclodextrin derivatives (betaCDs) display shuttling ability similar to that of MbetaCD. Furthermore, there does not appear to be a structural pattern among the other betaCDs which could explain their shuttling capacity.

Cell cholesterol efflux: integration of old and new observations provides new insights.

Numerous studies using a variety of cell/acceptor combinations have demonstrated differences in cholesterol efflux among cells. These studies also show that different acceptors, ranging from simple molecules like cyclodextrins to serum, stimulate efflux through a variety of mechanisms. By combining early observations with data derived from recent studies, it is now possible to formulate a model for cell cholesterol efflux which proposes that an array of different mechanisms, including aqueous diffusion, lipid-free apolipoprotein membrane microsolubilization, and SR-BI-mediated cholesterol exchange contribute to cholesterol flux. In this model the relative importance of each mechanism would be determined both by the cell type and the nature of the extracellular cholesterol acceptor.

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.

Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.

In addition to its effect on high density lipoprotein (HDL) cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI) was recently reported to stimulate free cholesterol (FC) flux from Chinese hamster ovary (CHO) cells stably expressing mouse SR-BI, a novel function of SR-BI that may play a role in cholesterol removal from the vessel wall where the receptor can be found. It is possible that SR-BI stimulates flux simply by tethering acceptor HDL particles in close apposition to the cell surface thereby facilitating the movement of cholesterol between the plasma membrane and HDL. To test this, we used transiently transfected cells and compared the closely related class B scavenger receptors mouse SR-BI and rat CD36 for their ability to stimulate cholesterol efflux as both receptors bind HDL with high affinity. The results showed that, although acceptor binding to SR-BI may contribute to efflux to a modest extent, the major stimulation of FC efflux occurs independently of acceptor binding to cell surface receptors. Instead our data indicate that SR-BI mediates alterations to membrane FC domains which provoke enhanced bidirectional FC flux between cells and extracellular acceptors.

Apolipoprotein-mediated plasma membrane microsolubilization. Role of lipid affinity and membrane penetration in the efflux of cellular cholesterol and phospholipid.

Lipid-free apolipoprotein (apo) A-I contributes to the reverse transport of cholesterol from the periphery to the liver by solubilizing plasma membrane phospholipid and cholesterol. The features of the apolipoprotein required for this process are not understood and are addressed in the current study. Membrane microsolubilization of human fibroblasts is not specific for apo A-I; unlipidated apos A-II, C, and E incubated with the fibroblast monolayers at a saturating concentration of 50 micrograms/ml are all able to release cholesterol and phospholipid similarly. To determine the properties of the apolipoprotein that drive the process, apo A-I peptides spanning the entire sequence of the protein were utilized; the peptides correspond to the 11- and 22-residue amphipathic alpha-helical segments, as well as adjacent combinations of the helices. Of the 20 helical peptides examined, only peptides representing the N-and C-terminal portions of the protein had the ability to solubilize phospholipid and cholesterol. Cholesterol efflux to the most effective peptides, 44-65 and 209-241, was approximately 50 and 70%, respectively, of that to intact apo A-I. Deletion mutants of apo E and apo A-I were constructed that have reduced lipid binding affinities as compared with the intact molecule. The proteins, apo A-I (Delta222-243), apo A-I (Delta190-243), apo E3 (Delta192-299) and apo E4 (Delta192-299) all exhibited a decreased ability to remove cellular cholesterol and phospholipid. These decreases correlated with the reduced ability of these proteins to penetrate into a phospholipid monomolecular film. Overall, the results indicate that insertion of amphipathic alpha-helices between the plasma membrane phospholipid molecules is a required step in the mechanism of apolipoprotein-mediated cellular lipid efflux. Therefore the lipid binding ability of the apolipoprotein is critical for efficient membrane microsolubilization.

Removal of cellular cholesterol by pre-beta-HDL involves plasma membrane microsolubilization.

High density lipoprotein (HDL) is able to remove unesterified cholesterol from peripheral cells in the process of reverse cholesterol transport by an aqueous diffusion mechanism as well as by an apolipoprotein (apo)-mediated process. The aqueous diffusion mechanism is understood but the molecular mechanism of lipid-poor pre-beta-HDL-(apo-) mediated cholesterol removal is not known. Measurements of the initial rates of efflux of unesterified cholesterol and phospholipid from human fibroblasts to lipid-free, human apoA-I showed that both lipids are released from the cells during a 10-min incubation with apoA-I. The concentration-dependence of efflux of the lipids is the same (Km = 0.4 and 0.6 microg apoA-I/ml for cholesterol and phospholipid flux, respectively), suggesting a membrane microsolubilization process. A finite pool of about 1% of the plasma membrane cholesterol is accessible for release by solubilization; the limited size of this cholesterol pool is not due to a lack of availability of apoA-I, but rather to the restricted amount of phospholipid that is removed from the plasma membrane. Plasma membrane domains may be involved in membrane microsolubilization, but caveolar cholesterol seems not to be specifically accessed in this process. Membrane microsolubilization is the process by which pre-beta1-HDL removes cell cholesterol in the first step of reverse cholesterol transport. When apoA-I is present in the extracellular space, the relative contributions of cholesterol efflux by membrane microsolubilization and by aqueous diffusion are determined by the degree of lipidation of the apoA-I molecules.

Dietary modification of high density lipoprotein phospholipid and influence on cellular cholesterol efflux.

African green monkeys fed fat-specific diets served as a model to investigate the effect of phospholipid acyl chain modification on high density lipoprotein (HDL)-mediated cellular cholesterol efflux. Diets enriched in saturated, monounsaturated, n-6 polyunsaturated, or n-3 polyunsaturated fats were provided during both low cholesterol and cholesterol-enriched stages; sera and HDL3 samples were obtained at specific points during the treatment period. Analysis of the HDL phospholipid composition revealed significant acyl chain modification, consistent with the respective fat-specific diet. Cholesterol efflux from mouse L-cell fibroblasts to HDL3 isolated from the specific diet groups was measured and revealed no differences in the abilities of the particles to accept cellular cholesterol; determination of the bidirectional flux of cholesterol between the cells and HDL3 species further demonstrated no effect of phospholipid acyl chain modification on this process. The effects of dietary modification of phospholipid acyl chains on cellular cholesterol efflux were directly examined by isolating the HDL phospholipid and combining it with human apolipoprotein A-I to form well-defined reconstituted HDL particles. These complexes did not display any differences with respect to their ability to stimulate cellular cholesterol efflux. Incubations with 5% sera further confirmed that the fat-specific diets do not influence cholesterol efflux. These results suggest that the established influences of specific dietary fats on the progression of atherosclerosis are due to effects on cholesterol metabolism other than the efflux of cellular cholesterol in the first step of reverse cholesterol transport.

Cholesterol metabolism and efflux in human THP-1 macrophages.

This study has investigated in detail factors regulating accumulation, esterification, and mobilization of cholesterol in human THP-1 macrophages. Human THP-1 monocytes were differentiated into macrophages and then cholesterol enriched by exposure to acetylated LDL (AcLDL), together with [3H]free cholesterol (FC). Although THP-1 macrophages accumulated FC and esterified cholesterol (EC), assessed by both mass and radioactivity, cellular EC always demonstrated a much lower specific activity (cpm/ microg) than did cellular FC, and several potential causes of this finding were investigated. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) during loading decreased cell [3H]EC by 95+/-1.4% but decreased cell EC mass by only 66.0+/-4.0%, indicating that some intracellular undegraded AcLDL-derived EC was present in these cells. Esterification of [3H]oleate to EC in THP-1 cells loaded with AcLDL was 2.0 nmol x mg-1 x h-1, consistent with previous literature. However, EC, triglyceride, and phospholipid fractions respectively contained 1.0+/-0.07%, 80.0+/-0.5%, and 18.9+/-0.3% of cell [3H]oleate, indicating triglycerides were much more metabolically active than EC. In addition, the mass of triglyceride in THP-1 macrophages exceeded that of EC both before and after exposure to AcLDL. Esterification of nonlipoprotein-derived cholesterol was compared in THP-1 cells and nonhuman Fu5AH, CHO, and RAW macrophage cells. Whereas the nonhuman cell lines all esterified over 30% of 2-hydroxypropyl-beta-cyclodextrin (hp-ss-CD)-delivered cholesterol within 6 hours, THP-1 cells esterified <8.0% of incorporated cholesterol. Kinetics of cholesterol efflux from AcLDL-loaded THP-1 cells were first investigated after loading with only FC, and interactions between efflux and EC hydrolysis were further assessed after loading cells with both EC and FC. Over 24 hours, human apolipoprotein (apo) A-I, apoHDL reconstituted with phosphatidylcholine, and HDL3 respectively removed 46.6+/-3.7%, 61. 3+/-3.4%, and 76.4+/-10.1% of [3H]FC from FC-enriched THP-1 cells. Cholesterol efflux to apoA-I was saturated by 24 hours and was enhanced by using apoA-I-phospholipid instead of pure apoA-I. Kinetic modeling identified that 97% of effluxed FC derived from a slow pool, with a T1/2 ranging from 27.7 hours for HDL to 69.3 hours for apoA-I. Although efflux enhanced net clearance of EC, hydrolysis of EC during concurrent inhibition of ACAT was unaffected by cholesterol efflux. Supplementation of THP-1 cultures with cAMP to stimulate hormone-sensitive lipase did not significantly enhance net hydrolysis of EC or cholesterol efflux. In conclusion, human THP-1 macrophages contain a large and metabolically active pool of triglyceride and a relatively inactive pool of EC. The low specific activity of EC relative to FC is contributed to by reduced esterification of FC, slow hydrolysis of EC, and accumulated lipoprotein EC. The relative inactivity of the EC pool may further contribute to already impaired cholesterol efflux from these cells. Net cholesterol efflux from human macrophages is achieved by pure apoA-I and is substantially further enhanced by the presence of phospholipid in acceptor particles.