©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Modulation of Cholesterol Efflux from Fu5AH Hepatoma Cells by the Apolipoprotein Content of High Density Lipoprotein Particles
PARTICLES CONTAINING VARIOUS PROPORTIONS OF APOLIPOPROTEINS A-I AND A-II (*)

Laurent Lagrost (1)(§), Catherine Dengremont (2), Anne Athias (1), Catherine de Geitere (2), Jean-Charles Fruchart (2), Christian Lallemant (1), Philippe Gambert (1), Graciela Castro (2)

From the (1) Laboratoire de Biochimie des Lipoprotéines, INSERM CJF 93-10, Faculté de Médecine, 21033 Dijon, France, and the (2) Institut Pasteur, Service d'Etude et de Recherche sur les Lipoprotéines et l'Athérosclérose, INSERM Unité 325, 1 rue du Professeur Calmette, F-59019 Lille, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The influence of apolipoproteins (apo) A-I and A-II on the ability of high density lipoproteins (HDL) to remove cholesterol from cultured Fu5AH rat hepatoma cells was studied independently on alterations in the overall structure and lipid composition of the lipoprotein particles. To this end, apoA-I was progressively replaced by apoA-II in ultracentrifugally isolated HDL without inducing changes in other remaining lipoprotein components. As apoA-II was progressively substituted for apoA-I in HDL (A-II:A-I+A-II percentage mass: 29.5, 47.6, 71.5, 97.4, and 98.9%), the rate of cholesterol efflux from Fu5AH hepatoma gradually and significantly decreased after 2 or 4 h of incubation at 37 °C (cholesterol efflux: 30.4 ± 0.8, 24.1 ± 1.0, 19.8 ± 1.2, 15.7 ± 1.4, and 13.4 ± 1.3%/2 h, respectively; 38.4 ± 1.5, 29.2 ± 0.9, 27.0 ± 0.2, 20.4 ± 0.4, and 17.5 ± 1.0%/4 h, respectively) (p < 0.01 with all A-II-enriched HDL fractions as compared with non-enriched homologues). In agreement with data obtained with total HDL, increasing the A-II:A-I+A-II percentage mass in HDL particles containing initially only apoA-I (HDL-A-I) progressively reduced cellular cholesterol efflux. After 2 h of incubation, cholesterol efflux correlated negatively with A-II:A-I+A-II percentage mass (r = -0.86; p < 0.0001; n = 20), but not with either free cholesterol:phospholipid ratio, A-I+A-II:total lipid ratio or mean size of HDL. As determined by using Spearman rank correlation analysis, the A-II:A-I+A-II% mass ratio correlated negatively with the apparent maximal efflux (V) ( = -0.68; p < 0.05, n = 10), but not with the HDL concentration required to obtain 50% of maximal efflux (K) ( = -0.08; not significant, n = 10). It was concluded that the apoA-I and apoA-II content of HDL is one determinant of its ability to promote cholesterol efflux from Fu5AH rat hepatoma cells.


INTRODUCTION

The high density lipoprotein (HDL)() fraction of human plasma has been shown to correlate negatively with the incidence of coronary artery disease (1) , reflecting its putative role in transporting cholesterol from peripheral tissues to the liver, namely the reverse cholesterol transport pathway. It has been now well established that HDLs constitute a collection of particles that vary in size, density, and composition. In particular, the two major HDL apolipoproteins, i.e. apoA-I and apoA-II, define two major populations of particles which contain either both apoA-I and apoA-II, designated LpA-I/A-II, or apoA-I and no apoA-II, designated LpA-I (2) . In addition, small amounts of lipoprotein particles containing apoA-II but no apoA-I, designated LpA-II, have been recently identified in human plasma (3, 4, 5) . Since in vivo studies demonstrated that LpA-I and LpA-IA-II represent distinct metabolic entities (6, 7) , variations in the apolipoprotein content of HDL particles might have some important implications in terms of cholesterol transport. Recent studies in human apoA-I and human apoA-I/apoA-II transgenic mice provided direct evidence for a more pronounced anti-atherogenic potential of LpA-I as compared with LpA-I/A-II, suggesting that LpA-I might be more active in accepting cholesterol from peripheral tissues and promoting its transport to the liver (8, 9) . Whereas a number of in vitro studies investigated the influence of HDL structure and composition on cellular cholesterol efflux, reported roles of apolipoproteins A-I and A-II in the initial step of the reverse cholesterol transport pathway are still controversial (10) . Indeed, cholesterol efflux was shown to be either decreased (11, 12, 13, 14, 15, 16) , unchanged (17-20), or increased (17, 21) in the presence of apoA-II-containing lipoproteins as compared with lipoprotein particles containing only apoA-I. Conflicting observations might relate to the heterogeneity of lipoprotein substrates used, i.e. native HDL or reconstituted particles. Indeed, a number of factors, among them the size (22, 23, 24, 25) , density (26) , and lipid content (25, 26, 27, 28) of lipoprotein particles, were shown to affect independently the cholesterol efflux process. It results therefore that neither native HDL nor reconstituted phospholipid emulsions may constitute ideal models for determining the specific effects of HDL apolipoproteins on cellular cholesterol efflux. In the former case, LpA-I and LpA-I/A-II isolated directly from human plasma do not vary only in their apoA-I and apoA-II content but also in their size and lipid composition (29, 30, 31) . It is possible to circumvent some of the concerns linked to the use of native HDL by preparing phospholipid/apoA-I and phospholipid/apoA-II discoidal complexes with similar size and phospholipid/protein mass ratio (14, 32) . However, in that latter case, recombinant particles do not match exactly the overall structure and composition of native particles, a drawback that may alter the conformational state of apolipoproteins (33, 34, 35) . In addition to alterations in lipoprotein substrates, diversity of selected cell types may account for discrepancies among reported cholesterol efflux measurements (14, 32, 36) , possibly reflecting differences in cholesterol packing within cell membranes (37) .

The present study was focused on the influence of apoA-I and apoA-II on cellular cholesterol efflux independently on the structure and lipid composition of HDL particles. To this end, apoA-I was progressively replaced by apoA-II in ultracentrifugally isolated HDL particles without inducing changes in other remaining lipoprotein components, a strategy that has been used previously in our laboratory to compare the effects of apoA-I and apoA-II on cholesteryl ester transfer protein activity (38) . The cell line used in the present study was the Fu5AH rat hepatoma, which has been shown previously to constitute a convenient and reproducible model for measurement of free cholesterol surface transfer (39) . By using that experimental system, the ability of HDL particles to promote cellular cholesterol efflux was shown to be significantly influenced by their apoA-I and apoA-II content.


MATERIALS AND METHODS

Isolation of HDLParticles

Blood from healthy normolipidemic volunteers was collected into tubes containing EDTA-Na (final concentration, 1 mg/ml), and plasma was immediately separated by centrifugation at 4 °C. Subsequently, HDL were isolated as the plasma fraction of density 1.13-1.21 g/ml by sequential ultracentrifugation as described previously (38) .

Anti-apoA-II Immunoaffinity Chromatography

HDL containing only apoA-I (HDL-A-I) were separated from HDL containing apoA-I and apoA-II (HDL-A-I/A-II) by chromatography on an anti-apoA-II column as described previously (38) . The A-II:A-I+A-II percentage mass in the immunopurified HDL-A-I particles was constantly lower than 5%.

Preparation of ApoA-II-enriched HDL

HDL were progressively enriched with apoA-II according to the general procedure previously described (38). Briefly, apoA-II-enriched HDL was prepared by replacing apoA-I by apoA-II upon the incubation of HDL in the presence of increasing amounts of delipidated HDL apolipoproteins (38). The HDL protein to apoHDL ratio ranged between 1:0 and 1:4, allowing to obtain HDL particles with increasing apoA-II:apoA-I+apoA-II percentage mass (38) . As observed by using SDS-polyacrylamide gradient gel electrophoresis, apoA-I and apoA-II were virtually the only proteins present in the isolated HDL fractions (38) .

Measurement of Cellular Cholesterol Efflux

Cellular cholesterol efflux was determined by using rat Fu5AH hepatoma cells following the procedure previously described by de la Llera Moya and co-workers (16) . Briefly, the cells were maintained in minimal essential medium (MEM) containing 5% calf serum. 20,000 Fu5AH cells/ml were plated on 2.4 cm multiwell plates (Linbro, Polylabo) using 2 ml/well. Two days after plating, cellular cholesterol (approximately 25 µg/well) was labeled during a 48-h incubation with [H]cholesterol (NEN, Dupont de Nemours) (1 µCi/well). To allow equilibration of the label, the cells were rinsed and incubated for 24 h in MEM containing 0.5% bovine serum albumin. For determination of cholesterol efflux, the cells were washed with phosphate-buffered saline and incubated at 37 °C with isolated HDL fractions diluted in MEM without albumin supplementation. At the end of the efflux period, medium was removed and centrifugated. Cell monolayer was washed three times with phosphate-buffered saline and harvested with 0.5 ml of 0.1 mol/liter NaOH. Finally, radioactivity was measured in both medium and cells, allowing to determine the total radioactivity content in each well. Fractional cholesterol efflux, expressed as percent, was calculated as the amount of label recovered in the medium divided by the total label in each well.

Native Polyacrylamide Gradient Gel Electrophoresis

HDL particle size was determined by electrophoresis in 1.5-25.0 g/liter polyacrylamide gradient gels according to the general procedure previously described (40) . Mean apparent diameters of HDL fractions were determined by comparisons with protein standards (high molecular weight protein calibration kit, Pharmacia Biotech Inc.), which were submitted to electrophoresis together with the samples.

Protein and Lipid Analyses

Chemical assays were performed on a Cobas-Fara Centrifugal Analyzer (Roche). Total cholesterol, unesterified cholesterol, triglyceride, and phospholipid concentrations were measured by enzymatic methods using Boehringer Mannheim reagents. Concentrations of apoA-I and apoA-II were determined by immunoturbidimetry (41) with anti-apoA-I and anti-apoA-II antibodies purchased from Behringwerke AG (Marburg, Germany). ApoA-I standard was purchased from Behringwerke AG. ApoA-II standard was purchased from Immuno AG (Vienna, Austria).

Statistical Analyses

One-way analysis of variance was used to determine the significance of the difference between data means. Coefficients of correlation, r and , were calculated by using linear regression and Spearman rank correlation analysis, respectively.


RESULTS

Characterization of ApoA-II-enriched HDLParticles

Incubation of ultracentrifugally isolated HDL in the presence of increasing concentrations of delipidated HDL apolipoproteins (native HDL:delipidated HDL protein ratio ranging between 1:0 and 1:4) induced the progressive replacement of apoA-I by apoA-II (). As reported previously when substituting apoA-II for apoA-I in canine HDL(42) , apoA-I-phosphatidylcholine-cholesterol complexes (43) , and human HDL (38, 44, 45), approximately two molecules of apoA-II displaced one molecule of apoA-I from HDL. In agreement with previous observations from our laboratory (38) , apoA-II enrichment was not accompanied by significant changes in the lipid composition of HDL ().

Time Course of Cholesterol Efflux as Induced by ApoA-II-enriched HDLParticles

In order to set up appropriate experimental conditions to compare the effect of various HDL particles on cholesterol efflux, Fu5AH cells were incubated for up to 24 h in the presence of HDL containing various proportions of apoA-I and apoA-II. As shown in Fig. 1, the rise in cholesterol efflux was approximately linear during the first 4 h of incubation, while kinetic curves tended to reach a plateau for longer incubation times. Interestingly, enrichment of HDL particles with apoA-II seemed to be linked to a substantial reduction of cellular cholesterol efflux during the first part of the incubation period. In contrast, after 24 h of incubation, an inverted tendency was observed and curves obtained with various HDL particles intersected for incubation time longer than 8 h. Since differences in the ability of various HDL substrates to promote cholesterol efflux are susceptible to be more apparent in the linear portion of time-dependent curves, subsequent experiments compared cholesterol efflux rates after relatively short incubation periods, not exceeding 4 h.


Figure 1: Effect of apoA-II-enriched HDL on the time course of cholesterol efflux from Fu5AH hepatoma. [H]Cholesterol-labeled cells were incubated at 37 °C in the presence of HDL (phospholipid concentration, 50 mg/liter) containing various proportions of apoA-I and apoA-II. At the end of the incubation period, cholesterol efflux was calculated as the percentage of total label released (see ``Materials and Methods''). Each point represents the mean of duplicate determinations.



Relations between the Composition of HDLand Cellular Cholesterol Efflux

As compared with non-enriched HDL (A-II:A-I+A-II percentage mass of 29.5%), the rate of cholesterol efflux was gradually and significantly decreased as apoA-II was progressively substituted for apoA-I in apoA-II-enriched homologous particles (A-II:A-I+A-II percentage mass, 47.6-98.9%) (Fig. 2). Consistent data were obtained after 2 and 4 h of incubation (Fig. 2). By using linear regression analysis, cholesterol efflux after 2 h of incubation was shown to correlate negatively with A-II:A-I+A-II percentage mass in HDL particles (r = -0.86; p < 0.0001; n = 20) (Fig. 3). In contrast, no significant relationships were observed between the rate of cholesterol efflux and either free cholesterol:phospholipid ratio (r = -0.04; not significant) or A-I+A-II:total lipid ratio (r = -0.28; not significant) in HDL particles (Fig. 3). In addition, cholesterol efflux did not correlate with the mean size of HDL (r = 0.04; not significant).


Figure 2: Comparative effect of various apoA-II-enriched HDL on cholesterol efflux from Fu5AH hepatoma. [H]Cholesterol-labeled cells were incubated for 2 or 4 h at 37 °C in the presence of HDL (phospholipid concentration, 50 mg/liter; A-II:A-I+A-II%, 29.5) or apoA-II-enriched HDL (A-II:A-I+A-II%, 47.6-98.9). At the end of the incubation period, cholesterol efflux was calculated as the percentage of total label released (see ``Materials and Methods''). Data are mean ± S.D. of triplicate determinations and are representative of four similar experiments. Significance of differences between HDL and apoA-II-enriched HDL: *, p < 0.01;**, p < 0.001;***, p < 0.0001.




Figure 3: Correlation between the composition of HDL and cholesterol efflux from Fu5AH hepatoma. [H]Cholesterol-labeled cells were incubated for 2 h at 37 °C in the presence of HDL (phospholipid concentration, 50 mg/liter) containing various proportions of apoA-I and apoA-II (A-II:A-I+A-II mass ratio ranging from 0.24 to 0.99). Linear regression graphs present efflux values obtained with 20 apoA-II-enriched HDL fractions from four distinct experiments.



Experiments described above were conducted with total HDL, which consist of a mixture of two types of particles containing either only apoA-I (HDL-A-I) or both apoA-I and apoA-II (HDL-A-I/A-II). It results that total HDL do not allow investigation of the effect of apoA-II in the low range values (i.e. A-II:A-I+A-II percentage mass lower than 29%) (see ). In order to circumvent that problem, HDL-A-I were isolated by immunoaffinity chromatography and were progressively enriched with apoA-II (see ``Materials and Methods''). As shown in , and as observed with the total HDL fraction (), the replacement of apoA-I by apoA-II did not induce marked changes in the lipid composition of the particles. In agreement with cholesterol efflux data obtained with total HDL enriched with apoA-II, increasing the A-II:A-I+A-II percentage mass in HDL-A-I progressively reduced cholesterol efflux from the Fu5AH hepatoma cells (Fig. 4).


Figure 4: Effect of enrichment of HDL-A-I particles with apoA-II on cholesterol efflux from Fu5AH hepatoma. [H]Cholesterol-labeled cells were incubated for 2 h at 37 °C in the presence of apoA-II-enriched HDL (phospholipid concentration, 50 mg/liter) with A-II:A-I+A-II percentage mass ranging from 4.4 to 80.8% (see Table II). At the end of the incubation period, cholesterol efflux was calculated as the percentage of total label released (see ``Materials and Methods''). Data are mean ± S.D. of quadruplicate determinations and are representative of two similar experiments.



Concentration-dependent Effect of ApoA-II-enriched HDLon Cholesterol Efflux

In order to gain insight into the mechanism by which apoA-I and apoA-II can modulate cholesterol efflux, Fu5AH cells were incubated for 2 h at 37 °C in the presence of different concentrations of HDL (HDL phospholipid, 12.5-200.0 mg/liter), which varied only in their apoA-I and apoA-II contents. Fig. 5A shows three dose-response curves, which were obtained with either non-enriched HDL (A-II:A-I+A-II percentage mass, 29.4%) or apoA-II-enriched homologous particles (A-II:A-I+A-II percentage mass, 50.6% and 87.0%). As HDL concentration increased, cholesterol efflux increased progressively and tended to reach a maximal value with the highest concentration studied (Fig. 5A).


Figure 5: Concentration-dependent effect of HDL and apoA-II-enriched HDL on cholesterol efflux from Fu5AH hepatoma. [H]Cholesterol-labeled cells were incubated for 2 h at 37 °C in the presence of various concentrations of HDL (A-II:A-I+A-II%, 29.4) or apoA-II-enriched HDL (A-II:A-I+A-II%, 50.6-87.0) (phospholipid concentration, 12.5-200 mg/liter). A, direct plot; B, linear double-reciprocal plot. Values are mean ± S.D. of triplicate determinations.



As proposed by others with various cell models (16, 46, 47) , estimates of the apparent maximal efflux (V) and of the HDL concentration required to obtain 50% of maximal efflux (K) were determined from double-reciprocal plots as the intercepts of the y and x axes, respectively. As illustrated in Fig. 5B, whereas similar K values were obtained with various HDL fractions, V tended to be progressively reduced as the A-II:A-I+A-II percentage mass increased. That tendency was confirmed by calculating V and K values from dose-response curves, which were obtained with 10 distinct HDL preparations containing various proportions of apoA-I and apoA-II (A-II:A-I+A-II percentage mass, 29.4-96.0%). Indeed, as determined by using Spearman rank correlation analysis, the A-II:A-I+A-II mass ratio was shown to correlate negatively and significantly with maximal cholesterol efflux ( = -0.68; p < 0.05). In contrast, no significant correlation was found between A-II:A-I+A-II percentage mass and K values ( = -0.08; not significant).


DISCUSSION

It has been demonstrated clearly that both the structure and concentration of lipoprotein acceptors can influence the transfer of cellular cholesterol toward the extracellular medium (22) . That latter statement raised considerable interest in determining the precise effects of variations in the structure and composition of lipoprotein particles on their ability to remove cholesterol from cells. While the knowledge of the precise role of the two major HDL apolipoproteins, apoA-I and apoA-II, in cellular cholesterol efflux would contribute to a better understanding of the reverse cholesterol transport pathway, previous studies from different laboratories led to inconsistent results. Earlier studies showed that cholesterol efflux from cultured fibroblasts relates mainly to the presence of a specific, dense, minor lipoprotein fraction containing apoA-I but no apoA-II (26, 48) . In fact, subsequent studies demonstrated that a specific, pre--migrating LpA-I fraction, namely LpA-Ipre1, constitutes the preferential recipient of cellular cholesterol (49) , suggesting that LpA-I rather than LpA-I/A-II would mediate cellular cholesterol efflux. In accordance with that latter view, only LpA-I, but not LpA-I/A-II, were shown to promote cholesterol efflux from Ob1771 cultured adipose cells (11, 12, 13) . However, conclusions drawn from experiments with cultured adipocytes were substantially weakened by data obtained with other cell types (19, 20) , suggesting that apparent effects of LpA-I and LpA-I/A-II on cholesterol efflux may well be dependent on the cell type studied (10, 32) . In particular, phosphatidylcholine/apoA-I complexes were more efficient cholesterol acceptors than phosphatidylcholine/apoA-II counterparts when incubated with human fibroblasts, L-cell mouse fibroblasts, and J774 murine macrophages, whereas no differences appeared with Fu5AH hepatoma, aortic smooth muscle cells, or HepG2 human hepatoma (32, 36) . In fact, discrepancies among data previously reported might relate to the diversity of both lipoprotein substrates and experimental conditions used (such as the incubation time which is dependent on the cell type studied). Consequently, we chose to reconsider putative roles of apoA-I and apoA-II in cellular cholesterol efflux by using well characterized lipoprotein substrates and a well known, widely used cell model, i.e. the Fu5AH hepatoma.

In contrast with previous studies, which compared the cholesterol efflux potency of either immunopurified LpA-I and LpA-I/A-II particles or artificial phospholipid-apolipoprotein complexes, we chose to progressively replace apoA-I by apoA-II both in total HDL and in immunopurified HDL particles containing only apoA-I. That latter methological approach presents several advantages over previous approaches. Indeed, as shown in the present report, apoA-II-enriched, spherical HDL present the same lipid composition, in particular the same surface/core lipid ratio, as native particles and differ from each other only in their apolipoprotein composition. In addition, the progressive substitution of apoA-II for apoA-I, which is not accompanied by alterations in either structure or lipid composition of HDL(38) , is more likely to preserve the optimal conformation of HDL apolipoproteins and allows more gradual study of the potential changes in cholesterol efflux than phospholipid complexes containing either only apoA-I or only apoA-II (32) . Furthermore, since observed variations in cholesterol efflux have been explained recently in terms of kinetically distinct cholesterol pools (32), we suspect that experimental conditions, such as incubation time, might also account for some of the discrepancies between the conclusions drawn by several groups (11, 12, 32, 36) . Indeed, in some of the previous studies (32, 36) , the ability of various particles to act as cholesterol acceptors from different cell types was compared after long term incubations (incubation periods ranging between 9 and 12 h). It results therefore that these experimental conditions may well have been optimal for determination of cholesterol efflux from J774 macrophages in which about 90% of cholesterol are transferred from a slow pool with a t of about 20 h, but not from Fu5AH hepatoma cells in which a fast pool with a t of about 2 h predominates (32) . That concern is illustrated by comparison of time course curves of cholesterol efflux from Fu5AH and J774 cells (32) . Indeed, in agreement with data of the present report, cholesterol efflux from Fu5AH hepatoma cells was shown to approximate linear kinetic after 4 h of incubation, and time-dependent curves tended subsequently to be inflected toward a maximal value for longer incubation times (16, 32) . Whereas the inflections of the time-dependent curves may reflect a decrease in apparent cholesterol efflux rates, they might also simply reflect the dilution in the cells of cholesterol specific activity, due to the exchange of unlabeled cholesterol between the lipoproteins and the cells. In contrast to data obtained with Fu5AH hepatoma cells, the overall kinetic observed with J774 macrophages was markedly slower and cholesterol efflux kept to increase gradually after 12 h of incubation (32). Therefore, it is tempting to speculate that long term incubations constitute appropriate conditions to compare the ability of various lipoprotein substrates to act as cholesterol acceptors with J774, but not with Fu5AH cells. Consequently, in accordance with a recent report, which confirmed that short incubation periods are the most suitable experimental conditions to compare the ability of various human serum samples to promote cholesterol efflux from Fu5AH cells (16) , we selected incubation times not exceeding 4 h to compare the cholesterol efflux efficiency of HDL and A-II-enriched HDL.

In contrast to previous results obtained with the same cell type by using artificial phospholipid/apolipoprotein complexes and longer incubation times (32) , the present study revealed that cholesterol efflux is significantly lower with apoA-II-enriched HDL than with non-enriched homologous particles. Since the rate of cholesterol efflux correlated significantly with A-II:A-I+A-II ratio of HDL but not with their size or lipid content, it was concluded that the apoA-I and apoA-II content of HDL is one determinant of their cholesterol efflux potency.

One can distinguish at least two general models among various mechanisms that might account for cellular cholesterol efflux: the ``receptor-mediated model'' and the ``aqueous diffusion model.'' According to the former hypothesis, the initial interaction of HDL with a specific binding site would regulate several related events, including cellular cholesterol efflux, low density lipoprotein catabolism, and sterol synthesis (50, 51, 52) . According to the second hypothesis, the transfer of free cholesterol from cells to lipoprotein particles does not necessarily involve specific cell surface binding of HDL. Plasma membrane cholesterol would desorb into the unstirred water layer surrounding the cells, prior to be taken up by acceptor lipoprotein substrates (39, 53) . In that latter case, apolipoproteins could also affect the rate of cholesterol desorption by modulating packing at the lipid-water interface, as shown previously with donor phospholipid/cholesterol complexes (54) . In the present study, in order to investigate whether the apolipoprotein content of HDL might affect the rate of cholesterol desorption, cholesterol efflux from Fu5AH hepatoma cells was measured in the presence of increasing concentrations of HDL. Indeed in the presence of high levels of lipoprotein acceptors, the rate of desorption of cholesterol molecules from the cell plasma membrane has been shown to constitute the rate-limiting step for efflux (10) . Concentration-dependent curves of the present study revealed that the apoA-II enrichment of HDL reduces progressively and significantly apparent V without affecting apparent K values. These latter observations are in good agreement with data of Hara and Yokoyama (46) , who observed, by studying the effect of free apolipoproteins on cholesterol release from macrophages, that estimates of V values with apoA-II were about 3 times lower than with apoA-I, while estimates of K values were similar. Taken together, these observations would be compatible with putative, differential roles of apoA-I and apoA-II in promoting the desorption of plasma membrane cholesterol. This hypothesis is compatible with observations of Mahlberg and Rothblat (32) , who demonstrated that differential effects of various apolipoprotein/phospholipid complexes on cholesterol efflux from J774 macrophages were largely explained by processes modulating the kinetic characteristics of cholesterol pools at the plasma membrane level.

Whereas results of the present study demonstrated that the ability of HDL particles to promote cholesterol efflux is dependent on the nature of their apolipoprotein content, the structural elements of apoA-I and apoA-II that could account for their differential effects remain to be identified. As suggested by membrane/lipoprotein binding experiments (44, 55-57) and by studies with genetic variants (58) , proteolytic fragments (59) , and specific monoclonal antibodies (60, 61, 62, 63) , HDL apolipoproteins might contain one or several specific structural domains implicated in the interaction with cell membranes. However, another hypothesis holds that binding of HDL apolipoproteins to plasma membranes (64) as well as cellular lipid efflux (46, 65) would more likely involve amphipathic -helical segments of apolipoproteins rather than one specific domain. If this latter hypothesis was proven to be true, differences in the ability of apoA-I and apoA-II to favor cholesterol desorption might relate to the heterogeneity of their -helical content (66) rather than to specificities of their amino acid sequences.

The physiological significance of the role of HDL apolipoproteins in cellular cholesterol efflux was not addressed in the present study. However, recent observations suggest that in vivo alterations in the apoA-I and apoA-II content of HDL may alter markedly the efficiency of reverse cholesterol transport, and in particular cellular cholesterol efflux. When a large number of human serum samples were tested for their ability to promote cholesterol efflux from Fu5AH hepatoma cells, partial correlation analysis revealed a greater association with LpA-I than with LpA-I/A-II, suggesting that LpA-I are better cholesterol acceptors than LpA-I/A-II (16) . The data of the present report, obtained under similar experimental conditions, brought direct evidence in favor of that latter hypothesis. Studies in apoA-I and apoA-I/apoA-II transgenic mice provided other strong arguments in favor of a differential role of apoA-I and apoA-II in reverse cholesterol transport (8, 9) . Indeed, susceptibility to atherogenesis was shown to be significantly decreased in apoA-I compared to apoA-I/apoA-II transgenic mice (8) , reflecting the higher ability of LpA-I to remove cholesterol excess from peripheral tissues (9). In fact, data from the past few years revealed that LpA-I/A-II can be significantly less efficient than LpA-I in acting as a lipoprotein substrate at several other stages of the reverse cholesterol transport pathway, including plasma cholesterol esterification (67, 68) , plasma cholesteryl ester transfer (38) , and bile acid synthesis (69) . Taken together, these observations might account for the slower catabolism of LpA-I/A-II compared to LpA-I (6) and might explain why the incidence of coronary artery disease in humans can be predicted by significant variations in plasma levels of LpA-I but not LpA-I/A-II (7) .

  
Table: Composition (mass percent) of HDL after enrichment with apoA-II

Ultracentrifugally isolated HDL particles were progressively enriched with apoA-II by incubation with increasing concentrations of delipidated HDL apolipoproteins as described under ``Materials and Methods.'' Values are mean ± S.D. of three distinct experiments.


  
Table: Composition (mass percent) of HDL after enrichment of HDL -A-I with apoA-II

HDL particles containing only apoA-I (HDL-A-I) were isolated from human plasma by a sequential procedure, which combined ultracentrifugation and anti-apoA-II immunoaffinity chromatography. Subsequently, HDL-A-I were progressively enriched with apoA-II by incubation with increasing concentrations of delipidated HDL apolipoproteins as described under ``Materials and Methods.'' Values are representative of two distinct experiments.



FOOTNOTES

*
This research was supported by grants from the Université de Bourgogne, the Conseil Régional de Bourgogne, INSERM, the Fondation pour la Recherche Médicale, and the Bioavenir Program (Rhône Poulenc Rorer and the French government). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratoire Central de Biochimie Médicale, Hôpital du Bocage, 21034 Dijon, France. Tel.: 80-29-38-25; Fax: 80-29-36-61.

The abbreviations used are: HDL, high density lipoprotein; HDL, high density lipoprotein subfraction 3; apo, apolipoprotein; LpA-I, lipoprotein particle containing apoA-I but no apoA-II; LpA-I/A-II, lipoprotein particle containing apoA-I and apoA-II; LpA-II, lipoprotein particle containing apoA-II but no apoA-I; HDL-A-I, HDL particle containing apoA-I but no apoA-II; HDL-A-I/A-II, HDL particle containing apoA-I and apoA-II; MEM, minimal essential medium.


ACKNOWLEDGEMENTS

Fu5AH hepatoma cells were a kind gift from Dr. George Rothblat.


REFERENCES
  1. Miller, G. J., and Miller, N. E.(1975) Lancet i, 16-19
  2. Cheung, M. C., and Albers, J. J.(1984) J. Biol. Chem. 259, 12201-12209 [Abstract/Free Full Text]
  3. März, W., and Gross, W.(1988) Biochim. Biophys. Acta 962, 155-158 [Medline] [Order article via Infotrieve]
  4. Bekaert, E. D., Alaupovic, P., Knight-Gibson, C. S., Laux, M., Pelachyk, J. M., and Norum, R. A.(1991) J. Lipid Res. 32, 1587-1599 [Abstract]
  5. Bekaert, E. D., Alaupovic, P., Knight-Gibson, C., Norum, R., Laux, M. J., and Ayrault-Jarrier, M.(1992) Biochim. Biophys. Acta 1126, 105-113 [Medline] [Order article via Infotrieve]
  6. Rader, D. J., Castro, G., Zech, L. A., Fruchart, J.-C., and Brewer, H. B., Jr.(1991) J. Lipid Res. 32, 1849-1859 [Abstract]
  7. Fruchart, J.-C., and Ailhaud, G.(1992) Clin. Chem. 38, 793-797 [Abstract/Free Full Text]
  8. Schultz, J. R., Verstuyft, J. G., Gong, E. L., Nichols, A. V., and Rubin, E. M.(1993) Nature 365, 762-764 [CrossRef][Medline] [Order article via Infotrieve]
  9. Castro, G. R., Nihoul, L., Dengremont, C., Tailleux, A., Duverger, N., Denefle, P., Rubin, E. M., and Fruchart, J.-C.(1994) Circulation 90, I-240 (abstr.)
  10. Johnson, W. J., Mahlberg, F. H., Rothblat, G. H., and Phillips, M. C. (1991) Biochim. Biophys. Acta 1085, 273-298 [Medline] [Order article via Infotrieve]
  11. Barbaras, R., Grimaldi, P., Negrel, R., and Ailhaud, G.(1986) Biochim. Biophys. Acta 888, 143-156 [Medline] [Order article via Infotrieve]
  12. Barbaras, R., Puchois, P., Fruchart, J.-C., and Ailhaud, G.(1987) Biochem. Biophys. Res. Commun. 142, 63-69 [Medline] [Order article via Infotrieve]
  13. Barkia, A., Puchois, P., Ghalim, N., Torpier, G., Barbaras, R., Ailhaud, G., and Fruchart, J.-C.(1991) Atherosclerosis 87, 135-146 [Medline] [Order article via Infotrieve]
  14. Mahlberg, F. H., Glick, J. M., Lund-Katz, S., and Rothblat, G. H. (1991) J. Biol. Chem. 266, 19930-19937 [Abstract/Free Full Text]
  15. Nakamura, R., Ohta, T., Ikeda, Y., and Matsuda, I.(1993) Arterioscler. Thromb. 13, 1307-1316 [Abstract]
  16. de la Llera Moya, M., Atger, V., Paul, J. L., Fournier, N., Moatti, N., Giral, P., Friday, K. E., and Rothblat, G.(1994) Arterioscler. Thromb. 14, 1056-1065 [Abstract]
  17. Johnson, W. J., Kilsdonk, E. P. C., van Tol, A., Phillips, M. C., and Rothblat, G.(1993) J. Lipid Res. 32, 1993-2000 [Abstract]
  18. von Hodenberg, E., Heinen, S., Howell, K. E., Luley, C., Kübler, W., and Bond, H. M.(1991) Biochim. Biophys. Acta 1086, 173-184 [Medline] [Order article via Infotrieve]
  19. Oikawa, S., Mendez, A. J., Oram, J. F., Bierman, E. L., and Cheung, M. C.(1993) Biochim. Biophys. Acta 1165, 327-334 [Medline] [Order article via Infotrieve]
  20. Cheung, M. C., Mendez, A. J., Wolf, A. C., and Knopp, R. H.(1993) J. Clin. Invest. 91, 522-529 [Medline] [Order article via Infotrieve]
  21. Jackson, R. L., and Gotto, A. M.(1975) J. Biol. Chem. 250, 7204-7209 [Abstract]
  22. Rothblat, G. H., and Phillips, M. C.(1982) J. Biol. Chem. 257, 4775-4782 [Free Full Text]
  23. Duverger, N., Rader, D., Duchateau, P., Fruchart, J.-C., Castro, G., and Brewer, H. B.(1993) Biochemistry 32, 12372-12379 [Medline] [Order article via Infotrieve]
  24. Agnani, G., and Marcel, Y. L.(1993) Biochemistry 32, 2643-2649 [Medline] [Order article via Infotrieve]
  25. Jonas, A., Bottum, K., Theret, N., Duchateau, P., and Castro, G. (1994) J. Lipid Res. 35, 860-870 [Abstract]
  26. Oram, J. F., Albers, J. J., Cheung, M. C., and Bierman, E. L.(1981) J. Biol. Chem. 256, 8348-8356 [Free Full Text]
  27. Johnson, W. J., Bamberger, M. J., Latta, R. A., Rapp, P. E., Phillips, M. C., and Rothblat, G. H.(1986) J. Biol. Chem. 261, 5766-5776 [Abstract/Free Full Text]
  28. Sola, R., Motta, C., Maille, M., Bargallo, M. T., Boisnier, C., Richard, J. L., and Jacotot, B.(1993) Arterioscler. Thromb. 13, 958-966 [Abstract]
  29. James, R. W., Hochstrasser, D., Tissot, J.-D., Funk, M., Appel, R., Barja, F., Pellegrini, C., Muller, A. F., and Pometta, D.(1988) J. Lipid Res. 29, 1557-1571 [Abstract]
  30. Ohta, T., Nakamura, R., Ikeda, Y., Shinohara, M., Miyazaki, A., Horiuchi, S., and Matsuda, I.(1992) Biochim. Biophys. Acta 1165, 119-128 [Medline] [Order article via Infotrieve]
  31. Kilsdonk, E. P. C., van Gent, T., and van Tol, A.(1990) Biochim. Biophys. Acta 1045, 205-212 [Medline] [Order article via Infotrieve]
  32. Mahlberg, F. H., and Rothblat, G. H.(1992) J. Biol. Chem. 267, 4541-4550 [Abstract/Free Full Text]
  33. Curtiss, L. K., and Edgington, T. S.(1985) J. Biol. Chem. 260, 2982-2993 [Abstract]
  34. Collet, X., Perret, B., Simard, G., Raffa, E., and Marcel, Y. L.(1991) J. Biol. Chem. 266, 9145-9152 [Abstract/Free Full Text]
  35. Davidson, W. S., Sparks, D. L., Lund-Katz, S., and Phillips, M. C. (1994) J. Biol. Chem. 269, 8959-8965 [Abstract/Free Full Text]
  36. DeLamatre, J., Wolfbauer, G., Phillips, M. C., and Rothblat, G. H. (1986) Biochim. Biophys. Acta 875, 419-428 [Medline] [Order article via Infotrieve]
  37. Rothblat, G. H., Mahlberg, F. H., Johnson, W. J., and Phillips, M. C. (1992) J. Lipid Res. 33, 1091-1097 [Abstract]
  38. Lagrost, L., Perségol, L., Lallemant, C., and Gambert, P.(1994) J. Biol. Chem. 269, 3189-3197 [Abstract/Free Full Text]
  39. Phillips, M. C., McLean, L. R., Stoudt, G. W., and Rothblat, G. H. (1980) Atherosclerosis 36, 409-422
  40. Blanche, P. J., Gong, E. L., Forte, T. M., and Nichols, A. V.(1981) Biochim. Biophys. Acta 1085, 209-216
  41. Rifai, N., and King, M. E.(1986) Clin. Chem. 32, 957-961 [Abstract/Free Full Text]
  42. Lagocki, P. A., and Scanu, A. M.(1980) J. Biol. Chem. 255, 3701-3706 [Free Full Text]
  43. Rosseneu, M., Van Tornout, P., Lievens, M.-J., and Assman, G.(1981) Eur. J. Biochem. 117, 347-352 [Abstract]
  44. Vadiveloo, P. K., and Fidge, N. H.(1990) Biochim. Biophys. Acta 1045,135-141 [Medline] [Order article via Infotrieve]
  45. Van Tornout, P., Caster, H., Lievens, M.-J., Rosseneu, M., and Assman, G.(1983) Biochim. Biophys. Acta 751, 175-188 [Medline] [Order article via Infotrieve]
  46. Hara, H., and Yokoyama, S.(1991) J. Biol. Chem. 266, 3080-3086 [Abstract/Free Full Text]
  47. Li, Q., Komaba, A., and Yokoyama, S.(1993) Biochemistry 32, 4597-4603 [Medline] [Order article via Infotrieve]
  48. Fielding, C. J., and Fielding, P. E.(1981) J. Biol. Chem. 78, 3911-3914
  49. Castro, G. R., and Fielding, C. J.(1988) Biochemistry 27, 25-29 [Medline] [Order article via Infotrieve]
  50. Oram, J. F.(1983) Arterioscler. Thromb. 3, 420-432 [Abstract]
  51. Biesbroeck, R., Oram, J. F., Albers, J. J., and Bierman, E. L.(1983) J. Clin. Invest. 71, 525-539 [Medline] [Order article via Infotrieve]
  52. Brinton, E. A., Oram, J. F., and Bierman, E. L.(1987) Biochim. Biophys. Acta 920, 68-75 [Medline] [Order article via Infotrieve]
  53. Johnson, W. J., Mahlberg, F. H., Chacko, G. K., Phillips, M. C., and Rothblat, G. H.(1988) J. Biol. Chem. 263, 14099-14106 [Abstract/Free Full Text]
  54. Letizia, J. Y., and Phillips, M. C.(1991) Biochemistry 30, 866-873 [Medline] [Order article via Infotrieve]
  55. Fidge, N. H., and Nestel, P. J.(1985) J. Biol. Chem. 260, 3570-3575 [Abstract]
  56. Steinmetz, A., Barbaras, R., Ghalim, N., Clavey, V., Fruchart, J.-C., and Ailhaud, G.(1990) J. Biol. Chem. 265, 7859-7863 [Abstract/Free Full Text]
  57. Vadiveloo, P. K., and Fidge, N. H.(1992) Biochem. J. 284, 145-151 [Medline] [Order article via Infotrieve]
  58. von Eckardstein, A., Castro, G., Wybranska, I., Theret, N., Duchateau, P., Duverger, N., Fruchart, J.-C., Ailhaud, G., and Assman, G.(1993) J. Biol. Chem. 268, 2616-2622 [Abstract/Free Full Text]
  59. Morrison, J. R., McPherson, G. A., and Fidge, N. H.(1992) J. Biol. Chem. 267, 13205-13209 [Abstract/Free Full Text]
  60. Allan, C. M., Fidge, N. H., and Kanellos, J.(1992) J. Biol. Chem. 267, 13257-13261 [Abstract/Free Full Text]
  61. Allan, C. M., Fidge, N. H., Morrison, J. R., and Kanellos, J.(1993) Biochem. J. 290, 449-455 [Medline] [Order article via Infotrieve]
  62. Luchoomun, J., Theret, N., Clavey, V., Duchateau, P., Rosseneu, M., Brasseur, R., Denefle, P., Fruchart, J.-C., and Castro, G. R.(1994) Biochim. Biophys. Acta 1212, 319-326 [Medline] [Order article via Infotrieve]
  63. Fielding, P. E., Kawano, M., Catapano, A. L., Zoppo, A., Marcovina, S., and Fielding, C. J.(1994) Biochemistry 33, 6981-6985 [Medline] [Order article via Infotrieve]
  64. Leblond, L., and Marcel, Y. L.(1991) J. Biol. Chem. 266, 6058-6067 [Abstract/Free Full Text]
  65. Hara, H., Hara, H., Komaba, A., and Yokoyama, S.(1992) Lipids 27, 302-304 [Medline] [Order article via Infotrieve]
  66. Segrest, J. P., Jones, M. K., De Loof, H., Brouillette, C. G., Venkatachalapathi, Y. V., and Anantharamaiah, G. M.(1992) J. Lipid Res. 33, 141-166 [Abstract]
  67. Fielding, C. J., Shore, V. G., and Fielding, P. E.(1972) Biochem. Biophys. Res. Commun. 46, 1493-1498 [Medline] [Order article via Infotrieve]
  68. Chung, J., Abano, D. A., Fless, G. M., and Scanu, A. M.(1979) J. Biol. Chem. 254, 7456-7464 [Medline] [Order article via Infotrieve]
  69. Pieters, M. N., Castro, G. R., Schouten, D., Duchateau, P., Fruchart, J.-C., and van Berkel, T. J. C.(1993) Biochem. J. 292, 819-823 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.