©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Effect of High Density Lipoprotein Phospholipid Acyl Chain Composition on the Efflux of Cellular Free Cholesterol (*)

(Received for publication, November 7, 1994; and in revised form, December 9, 1994)

W. Sean Davidson Kristin L. Gillotte Sissel Lund-Katz William J. Johnson George H. Rothblat Michael C. Phillips (§)

From the Medical College of Pennsylvania, Department of Biochemistry, Philadelphia, Pennsylvania 19129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

High density lipoprotein (HDL) phospholipid (PL) fatty acyl chain composition has been proposed to affect the ability of HDL to participate in the first step of reverse cholesterol transport. To examine the effects of PL fatty acid chain length and degree of unsaturation in this process, reconstituted HDL (rHDL) particles were made with human apolipoprotein (apo) A-I and PL containing fatty acid chains from 14 to 18 carbons in length, which were either fully saturated or unsaturated in one or both chains. These particles were characterized structurally and for their ability to promote free (unesterified) cholesterol (FC) efflux from cells growing in culture. The discoidal rHDL particles were homogeneous and exhibited similar hydrodynamic diameters (10.4 ± 1.0 nm) indicating that apoA-I forms similarly sized discs with a variety of PL. Measurements of particle surface charge, apoA-I alpha-helix content, and conformational stability indicated that the conformation of apoA-I varies among the particles. These conformational effects on apoA-I are consistent with the PL fluidity influencing the interaction between the amphipathic alpha-helical segments and PL acyl chains. Differential scanning calorimetry demonstrated that the physical state of the rHDL PL at 37 °C varied according to acyl chain length and degree of unsaturation; the FC efflux efficiencies for particles with PL in either the gel or liquid crystal states were determined. The ability of the rHDL to accept cellular FC depended on the physical state of the PL in the rHDL. Liquid crystal PL formed the most efficient FC acceptor particles exhibiting a maximal efflux velocity (V(max)) of 12-14% release of total cellular FC per h. Gel-phase PL formed inefficient rHDL acceptors with a V(max) of about 3%/h. A similar hierarchy of FC efflux efficiency was noted when either mouse L-cells or rat Fu5AH hepatoma cells were used as the FC donors. Furthermore, this hierarchy was found to be due to the characteristics of the PL and not due to variable apoA-I conformation because protein-free, small unilamellar vesicles made with the same PL exhibited similar relative efflux capabilities. Generally, the ability of a given rHDL particle to accept cellular FC was related to rHDL PL acyl chain length and degree of unsaturation; decreases in PL acyl chain length and increases in chain unsaturation tended to result in more efficient FC acceptor particles. These results suggest that rHDL acceptor particles that contain highly fluid surfaces sequester FC molecules that have diffused from the cell plasma membrane at a significantly faster rate than those containing highly organized lipid surfaces with restricted PL acyl chain mobility. This information forms a basis for understanding the role of lipid content in the structural and functional diversity of HDL.


INTRODUCTION

High density lipoprotein (HDL) (^1)exhibits substantial structural heterogeneity (1) and recent evidence indicates that this lipoprotein class may be functionally heterogeneous as well. Distinct HDL subspecies may be involved in different aspects of reverse cholesterol transport, the process by which excess peripheral cell cholesterol (FC) is returned to the liver for catabolism(2) . Castro and Fielding (3) have demonstrated that a minor HDL charge subspecies, pre-beta HDL, may be an important mediator of the first step of reverse cholesterol transport, the transfer of FC from cells to HDL. It has been proposed (3) that this particle initially accepts peripheral cell FC and subsequently transports it to larger HDL species that contain lecithin-cholesterol acyltransferase where it can be esterified. Rader et al.(4) have shown that other HDL particles exhibit an increased rate of clearance from the plasma by the liver. Still other HDL particles appear to be able to interact with plasma enzymes such as lecithin-cholesterol acyltransferase(5) . Although the various HDL subspecies are known to have variable lipid and protein compositions, little is known about the structural features of the HDL subspecies that give rise to functional diversity.

Previously, we have investigated the role of HDL apolipoprotein structure in the transfer of FC from cells to HDL(6) . The current study focuses on the PL component of HDL. Several studies have shown that the ability of a given particle to accept cellular FC is related to the amount of PL present in the particle(6, 7) . In addition, there is evidence that the types of phospholipids present in HDL may be important. Sola et al.(8) demonstrated that diet-induced changes in the fatty acyl chain composition of HDL(3) lead to differences in the ability of HDL(3) to remove FC from cultured fibroblasts. Increases in the percentage of saturated acyl chains in the HDL(3) reduced its ability to accept cellular FC relative to HDL(3) that was enriched in unsaturated acyl chains. This effect was attributed to decreases in the lipid fluidity of the HDL(3) surface as a result of the increased percentage of saturated acyl chains(9) . Dietary modifications such as these have been suggested to have an effect on the FC transfer to HDL in vivo(10) , although such studies cannot distinguish between the lipid modification of HDL and modifications of peripheral cell membranes which can also affect FC transfer to HDL(11) .

Our objective in this study was to test the hypothesis that the PL acyl chain content can affect the ability of HDL particles to remove FC from cells. A second objective was to examine the effect of PL acyl chain content on the structure of associated apoA-I. In order to avoid complicating factors such as variable PL:protein ratios, the presence of lipids other than PL, variable protein contents, and large changes in particle size that can occur in studies using in vivo modified HDL, we reconstituted well-defined rHDL particles that were similar in size as well as composition. A variety of synthetic PL containing saturated acyl chains from 14 to 18 carbons in length as well as length-matched PL containing unsaturated acyl chains were complexed to human apoA-I using a sodium cholate removal technique. The resulting particles were characterized with respect to composition, size, charge, lipid physical state, and apoA-I conformational stability. The relative efficiencies of the rHDL in removing FC from cultured cells were determined over a range of concentrations. The results show that the physical state of the PL molecules in the rHDL particles significantly affects the ability of the particles to remove cell FC. The information derived from this study has implications for understanding the role of HDL lipids in the first step of reverse cholesterol transport.


EXPERIMENTAL PROCEDURES

Materials

Bovine brain sphingomyelin (SM), sodium cholate, and bovine serum albumin were purchased from Sigma. 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-oleoyl-2-palmitoyl phosphatidylcholine (OPPC), 1,2-dimyristoyl phosphatidylcholine (DMPC), 1,2-dioleoyl phosphatidylcholine (DOPC), 1,2-distearoyl phosphatidylcholine (DSPC), and 1-palmitoyl 2-stearoyl phosphatidylcholine (PSPC) were purchased from Avanti Polar Lipids (Birmingham, AL) (+99% grade). 1,2-Dipalmitoyl phosphatidylcholine (DPPC) was from Calbiochem (La Jolla, CA). [1,2-^3H]Cholesterol was obtained from DuPont NEN (Boston, MA). The PC samples contained no lysophosphatidylcholine or free fatty acid that could be measured by TLC on Silica Gel G in petroleum ether/diethyl ether/acidic acid (75:24:1, v/v/v). Minimal essential medium and bovine calf and fetal serum were obtained from Life Technologies, Inc. (Grand Island, NY). Media were supplemented with 50 µg/ml gentamycin (Sigma). All other reagents were analytical grade.

Methods

Purification of Apolipoprotein A-I

Human HDL was isolated from the fresh plasma of normolipidemic subjects by sequential ultracentrifugation as described previously(12) . HDL was delipidated in ethanol/diethyl ether as described by Scanu and Edelstein(13) , and purified apoA-I was isolated by anion exchange chromatography on Q-Sepharose (14) and stored in lyophilized form at -70 °C. Prior to use, purified apoA-I was resolubilized in 6 M guanidine HCl and dialyzed extensively against Tris buffer (10 mM Tris, 150 mM NaCl, 1.0 mM EDTA, pH 8.2).

Preparation and Characterization of Reconstituted HDL Particles

All discoidal particles were reconstituted using the sodium cholate removal method described in detail elsewhere(15) . Initial mass ratios of PL to apoA-I are shown in Table 1. Due to the high gel to liquid-crystal phase transition temperatures of saturated long acyl chain PL, reconstitutions with DSPC and PSPC required that the incubation temperature be increased from 37 to 52 °C during the steps prior to the addition of apoA-I. Once apoA-I was added, all complexes were incubated at 37 °C to avoid denaturation of the protein. Most of the PL used in this study formed homogeneous rHDL that were in a common size range (9.7-10.0 nm, see ``Results''). However, certain PL formed particles that were substantially larger in size (DSPC (11.5 nm), PSPC (10.7 nm), and SM (10.9 nm)) and the DOPC preparation was significantly heterogeneous; it contained a major band at 10.2 nm and four additional bands at 12.5, 11.2, 8.4, and 7.2 nm as observed by polyacrylamide gradient gel electrophoresis (PAGGE). To optimize the amount of protein in the major band of the DOPC preparation and to decrease the size of the DSPC, PSPC, and SM complexes, additional reconstitutions were performed with varying initial PL:apoA-I stoichiometries. Such changes had no effect on the degree of heterogeneity of the DOPC product but had the deleterious effect of reducing the percentage of protein in the major band. The DSPC preparation produced a moderately smaller particle (10.8 nm) at an initial PL:apoA-I ratio of 1.5:1 and subsequent decreases failed to produce a smaller particle (only increases in free apoA-I). No further reductions in particle size could be obtained for the PSPC and SM preparations. Once the best initial stoichiometry was determined for each lipid, rHDL discs were prepared and separated from unreacted lipid and protein by gel filtration chromatography on a (2.5 times 100 cm) Superose 6 column eluted with Tris buffer (0.7 ml/min at 25 °C). For the heterogeneous DOPC preparation, fractions that contained the highest percentage of apoA-I in the major band were collected and combined. This resulted in a preparation with 80-85% of the apoA-I in the major band with the remainder present in two smaller particles. The size and homogeneity of the resulting particles were estimated by electron microscopy and nondenaturing PAGGE. Negative stain electron microscopy was performed at 80,000 magnification as described by Forte and Nordhausen(16) . Mean particle dimensions of 100 particles were determined from each negative. The hydrodynamic diameters of rHDL particles were estimated by Phast electrophoresis using pre-cast 8-25% polyacrylamide gels (Pharmacia Biotech Inc.)(15) . The particle migrations into the gel were determined to within 0.1 mm by digitizing the gel with a Mustek 105 hand scanner followed by computer image analysis (Sigma Scan/Image, Jandel Scientific, San Rafael, CA). Intact rHDL particles were chemically analyzed where appropriate using the Markwell modification of the Lowry protein assay (17) while phospholipids were determined as inorganic phosphorus by the method of Sokoloff and Rothblat(18) . The number of apoA-I molecules per particle was determined by cross-linking the apoA-I with dimethyl suberimidate (19) . The electrophoretic mobilities of native and reconstituted HDL were measured on Beckman Paragon preformed 0.5% agarose gels and the net number of negative charges per complex (valence, V) and the potential at the particle surface of shear (surface potential, S) were calculated as described by Sparks and Phillips(20) .



Circular Dichroism and Isothermal Denaturation Studies

The average alpha-helix content of apoA-I when complexed to various PL was determined by circular dichroism (CD) spectroscopy using a Jasco J41A spectropolarimeter. Spectra were measured at 25 °C in a 0.1-cm path length quartz cuvette as described previously(15) . The percent alpha-helix was determined from the molar ellipticities at 222 nm. The effect of guanidine HCl (GdnHCl) on the alpha-helix content of the rHDL particles (15) was used to obtain the free energy of unfolding of apoA-I as proposed by Aune and Tanford(21) .

Differential Scanning Calorimetry

The gel to liquid crystal (LC) phase transition temperatures (T(m)) which were above 0 °C were determined for the PL in both liposomes and rHDL discs in a MicroCal MC-2 differential scanning calorimeter (MicroCal Inc., Northampton, MA). Samples were suspended in 50 mM phosphate buffer (pH 7.2) in matched 1-ml sample cells and scanned at 1 °C/min over the range of 10-60 °C. The temperature at the peak of the change in heat capacity was taken as the T(m).

Efflux of Plasma Membrane Cholesterol

Mouse L-cell fibroblast or rat Fu5AH hepatoma cells were used to monitor the FC efflux efficiencies of the various rHDL. The methods for the efflux assay as well as the post-experiment work-up were as previously reported (6) with the following exceptions. The L-cells were plated 6 days prior to the experiment in 12-well cell plates (22 mm) at 75-100 times 10^3 cells per well rather than in 6-well plates. The cells were labeled with 1.0-1.5 µCi/ml [^3H]FC in bicarbonate-buffered minimal essential medium with 3% fetal calf serum for 24 h followed by a 12-h incubation in minimal essential medium/bicarbonate containing 1% bovine serum albumin to equilibrate the label between the various cellular sterol pools. After a brief wash, the efflux measurements were initiated by the application of 1.0 ml/well of the test medium containing 0.5% bovine serum albumin and the rHDL complex present at the indicated concentrations. Typical cell protein values were between 250 and 300 µg of protein/well. Efflux experiments that used rat Fu5AH hepatoma cells were done exactly the same as the L-cell efflux experiments except that only 0.5-1.0 µCi of labeled FC was used to label the cells.

Data Analysis

The fractional release of cellular FC determined experimentally was analyzed as originally proposed by Johnson et al.(22) and as described in detail for this system previously(6) . Briefly, the kinetic analysis assumes a closed system in which FC exists in one of two kinetic pools, either the cellular FC pool or the acceptor FC pool. The equilibration of FC between these pools is fit to the single exponential equation: Y = H(1)e + H(2) which has been shown to fit the data better than a double exponential equation by comparison of the F statistic (6) . Y represents the fraction of radiolabeled FC remaining in the cells, t is the incubation time in hours, H(1) is a pre-exponential term that reflects the fraction of cellular FC that exists in the medium at equilibrium, g is the sum of the rate constants for efflux (k(e)) and influx (k(i)), and H(2) is a constant that represents the fraction of labeled cell FC that remains associated with the cells at equilibrium due to a constant retrograde flux of FC from the extracellular acceptor to the cell. H(1), g, and H(2) are variables that can be fit to the experimental data by computer (Sigma Plot, Jandel Scientific, San Rafael, CA). The apparent rate constant for the efflux process (k(e)) is the product of H(1) and g. This value should be considered ``apparent'' because it is dependent on acceptor particle concentration under some conditions. The apparent t value in hours is then calculated as follows: t = ln2/k(e).


RESULTS

The Effect of PL Fatty Acid Content on rHDL Structure

Composition and Dimensions of rHDL Particles

To study the effects of acyl chain length on the structure and function of rHDL particles, complexes were made with DMPC (14:0, 14:0), DPPC (16:0, 16:0), DSPC (18:0, 18:0), and PSPC (16:0, 18:0). POPC (16:0, 18:1) and DOPC (18:1, 18:1) were used to study the effect of acyl chain unsaturation. Comparison of either PSPC versus POPC or DSPC versus DOPC allowed comparison of otherwise equal length acyl chains that differ only in chain saturation. To determine the effect of the position of unsaturated acyl chains with respect to the PL glycerol backbone, reconstitutions were carried out with OPPC (18:1, 16:0) and the rHDL were compared to those made with POPC. To simplify the comparison of cell FC efflux to the various rHDL, the particles were reconstituted to be as similar in size and composition as possible. Preliminary experiments using POPC showed that reconstitutions at an initial PL:apoA-I mass ratio of 2.4:1 resulted in homogeneous particles with hydrodynamic diameters in the range 9.6-9.8 nm. No post-reconstitution purification was required because over 95% of the protein was associated with the major band visualized on native PAGGE. Preliminary reconstitutions with the other PL yielded similarly sized particles by native PAGGE for DMPC (9.7 nm), DPPC (9.9 nm), OPPC (9.8 nm), and DOPC (10.2 nm). These results were in good agreement with the studies of Zorich et al.(23) who reported a size range of 9.0-10.0 nm for particles made with a variety of phosphatidylcholines at a similar initial stoichiometry. The particles in this study appeared to be more homogeneous than those of Zorich et al.(23) using the same PL but the particles in their study contained up to 10 mol % FC. FC incorporation into rHDL such as these results in increased particle heterogeneity (24) and likely explains these differences.

Table 1shows the compositions and diameters of particles from four independent sets of reconstitutions. On a molar basis, the final PL:protein molar ratios were in the range from 75 to 100:1 with each particle containing two molecules of apoA-I per particle. The exceptions were the particles containing DSPC and PSPC which ranged from 55 to 60:1 (PL:apoA-I) and contained 3 molecules of apoA-I. The particle diameters fell generally within a 1.5-nm range with an overall average of 10.4 nm by PAGGE and 10.1 nm by electron microscopy. POPC/apoA-I and OPPC/apoA-I particles exhibited identical sizes and compositions suggesting that the position of unsaturated acyl chains did not affect the binding of apoA-I to PL. DSPC/apoA-I and PSPC/apoA-I particles had the largest hydrodynamic diameters despite their reduced PL:apoA-I ratio; the increased disc diameters were most likely due to the presence of the third molecule of apoA-I.

Physical State of the PL Composing the rHDL Particles

To determine the physical state of the PL at 25 and 37 °C, differential scanning calorimetry was used to determine the main transition temperature (T(m)) of each phospholipid in protein-free liposomes and in apoA-I-containing rHDL (Table 1). The T(m) is the temperature at which lipids existing in the gel-phase melt to the more fluid liquid crystalline state (LC). The T(m) values measured for the PL that melt above 0 °C when present as hydrated liposomes agreed well with the published values for such systems(25) . When the lipids were complexed to apoA-I, the temperature range at which the lipid phase transition occurs was broadened and shifted to a slightly higher temperature(26) . This indicates that discoidal rHDL particles containing DOPC, OPPC, and POPC exist in the LC state at 25 and 37 °C, whereas the PSPC and DSPC particles exist in the gel state at both of these temperatures. The DMPC particle was found to exist in a partially melted state at room temperature with PL acyl chains in both the LC and gel states, but at 37 °C the lipid was completely in the LC state. The DPPC particle was in the gel state at room temperature but was close to its T(m) at 37 °C so that gel and LC states coexist (Table 1).

Effect of Acyl Chain Composition on the Charge Characteristics of rHDL Particles

Agarose gel electrophoresis was used to determine the effect of the various PL acyl chain combinations on the charge of the rHDL particles. All rHDL exhibited surface potential values in the pre-beta migration range (Table 2) according to the classification scheme proposed by Sparks and Phillips (-7.0 to -10.5 mV)(20) . Lipid-free apoA-I also migrates in this range(27) . The particle valences (V, the number of excess negative charges per particle) varied between -6.6 and -8.7e indicating that all particles were negatively charged at pH 8.6. Although significant differences existed among the particle charges, these differences did not correlate with the physical state of the rHDL PL (compare PSPC/apoA-I and POPC/apoA-I). Previous studies have shown that variations in rHDL particle valence of up to 2e can be caused by addition of FC(24) , or change in particle shape(27) . The difference in charges in Table 2did not arise from changes in particle shape because electron microscopy showed a discoidal morphology for all particles. Therefore, the charge differences likely occurred in response to the different chemical natures of the various PL acyl chains. The charge density relates the overall charge to the particle surface area. The complexes with the saturated PL exhibited a lower charge density which accounts for their lower S. The charge characteristics of OPPC/AI and POPC/AI discs were identical, indicating that the relative positions of saturated and unsaturated acyl chains did not influence the particle charge. Apparently, different PL acyl chains can induce a change in the charge of associated apoA-I molecules. As discussed later, this most likely occurs via a conformational change in apoA-I.



ApoA-I Secondary Structure and Conformational Stability

CD was used to determine the effect of the various PL acyl chains on the average alpha-helix content of the apoA-I molecules in HDL at 25 °C. In the case of the particles that contain PL with the fully saturated acyl chains, the alpha-helix content decreased with increasing acyl chain length (Table 3). This suggests that bilayers that contain PL in the LC state (DMPC) allow the formation of more alpha-helix in the associated apoA-I than bilayers in the gel state. In agreement with this, the most fluid PL (DOPC and POPC) formed rHDL particles with high alpha-helicity. The stability of apoA-I alpha-helices was monitored in the presence of increasing concentrations of GdnHCl. The particles containing LC PL were progressively denatured by GdnHCl and the process was complete at about 4.5 M (data not shown). The DPPC/apoA-I particle required 6 M GdnHCl to fully denature whereas rHDL made with DSPC, PSPC, or SM were not fully denatured over the GdnHCl concentrations studied. SMbulletapoA-I complexes have been shown previously to contain measurable alpha-helix content at concentrations of up to 7 M GdnHCl(28) . The concentration of GdnHCl required to 50% denature apoA-I (D) was lowest for lipid-unassociated (free) apoA-I and increased when apoA-I was complexed to lipid (Table 3). Furthermore, complexes containing gel-phase PL required more denaturant than those containing LC PL. The D values for the POPC/apoA-I and the DPPC/apoA-I particles agreed with published values of about 2.2 (29) and 3.2 M GdnHCl(30) , respectively.



The D term must be interpreted with caution when comparing apoA-I conformational stabilities in different rHDL particles. Previous reports have suggested that PL complexed to apoA-I may inhibit the binding of GdnHCl molecules to the target protein(15, 31) . Therefore, apparent increases in D when studying rHDL containing various PL may be artifactual because of the interference of GdnHCl binding by the PL. The analysis used here accounts for the variable activities of GdnHCl to give a standard free energy of denaturation (DeltaG) to describe the stability of the alpha-helical segments of apoA-I. The Deltan term in Table 3is the number of GdnHCl molecules that bind to each molecule of apoA-I during the denaturation. The denaturation of free apoA-I exhibited a high Deltan, whereas apoA-I complexed to LC PL exhibited a 2-fold lower value; an even lower value was evident when apoA-I was complexed to gel-phase PL (DPPC and PSPC). These results are consistent with the concept that PL interferes with GdnHCl binding to apoA-I and that gel-phase PL acyl chains perturb this interaction more than fluid PL. When these factors are taken into account, apoA-I in rHDL containing LC PL exhibited similar DeltaG values to free apoA-I, whereas the apoA-I in the gel-phase rHDL particles was less stable.

The Effect of PL Fatty Acid Content on the Ability of Discoidal rHDL to Remove Cell FC

The abilities of the particles shown in Table 1to act as acceptors of cellular FC were compared using mouse L-cells that had been labeled with [^3H]FC. The rHDL were present at a phospholipid concentration of 100 µg of PL/ml, and for the experiments of Fig. 1, the relative particle numbers (normalized to DMPC/apoA-I) for each rHDL were: DOPC/apoA-I, 1.0; POPC/apoA-I, 0.9; PSPC/apoA-I, 1.0; DMPC/apoA-I, 1.0; DPPC/apoA-I, 1.1; DSPC/apoA-I, 1.0; and SM/apoA-I, 1.2. This demonstrates that a similar number of particles was present per ml of extracellular medium for all samples. We have reported previously (6) that gas liquid chromatography analysis confirms that decreases in cellular FC mass closely parallel decreases in cellular FC radioactivity upon exposure to acceptor-containing medium, indicating that the radiolabel correctly reflects movement of FC mass. The PL concentration of the extracellular medium was found to be constant over the 6-h time course for rHDL containing both gel- and LC-phase PL, indicating that the particles did not precipitate out of solution during the experiment. Gel filtration chromatography of test medium recovered after a 6-h cell efflux incubation showed that >95% of the FC radioactivity that appeared in the medium eluted at a volume which was characteristic of the discoidal rHDL particle that was present initially in the medium (data not shown). This indicated the following: (a) the acceptor particles sequestered cellular FC, and (b) the acceptor particles did not significantly change size during the efflux assay (i.e. there was no fusion or aggregation of rHDL).


Figure 1: Time course of [^3H]free cholesterol efflux from mouse L-cell fibroblasts to discoidal rHDL containing various PL acyl chains. Mouse L-cell fibroblasts that had been trace labeled with [^3H]free cholesterol in 22-mm tissue culture wells were incubated for 6 h at 37 °C in a humidified incubator (5% CO(2)) with 1.0 ml of test medium containing 0.5% bovine serum albumin, 1.0 µg/ml Sandoz compound 58035, and the rHDL containing the indicated PL at 100 µg of PL/ml. The rHDL particles were: POPC/apoA-I (bullet), DOPC/apoA-I (), DMPC/apoA-I (), DSPC/apoA-I (circle), PSPC/apoA-I (box), DPPC/apoA-I (Delta), and SM/apoA-I (). The vertical axis indicates the fraction of initial labeled free cholesterol that remained in the cell at the designated times. Each point represents the mean of six cell wells. The error bars represent 1 S.D. All curves were obtained by fitting the entire time course to the model for tracer equilibration between two pools (see ``Methods'').



Fig. 1shows clear differences between the various rHDL with the POPC/apoA-I and DOPC/apoA-I particles being the most effective acceptors and DSPC/apoA-I and PSPC/apoA-I particles being the least effective. The half-times (t, corrected for FC efflux to control medium) for FC efflux from L-cells to the rHDL made with DOPC, POPC, PSPC, DMPC, DPPC, DSPC, and SM were 12.8 ± 1.4, 10.6 ± 0.9, 32.4 ± 3.8, 25.0 ± 3.6, 22.2 ± 1.0, 32.2 ± 6.0, and 13.8 ± 3.0 h, respectively. OPPC/apoA-I and POPC/apoA-I particles showed identical kinetics indicating that the position of unsaturated acyl chains was not important in determining FC efflux efficiency (data not shown). The PL that were in the LC state at 37 °C promoted faster FC efflux than those in the gel-phase. Furthermore, the relative efflux efficiency was inversely related to the T(m) (Table 1) of the PL composing the particle. For example, the DMPC/apoA-I disc, although in a LC state at 37 °C, did not promote as much FC transfer as DOPC/apoA-I and POPC/apoA-I discs. A similar relationship was noted among the gel-phase rHDL. To determine the efflux over a range of concentrations, FC efflux rate constants were derived for each particle at each of five concentrations ranging from 20 to 500 µg of PL/ml of medium (Fig. 2). Kinetic analysis of the time courses at the highest acceptor particle concentrations indicated that the predicted equilibrium distribution of cellular FC was generally similar for both LC- and gel-phase rHDL (about 70% of cell FC was present in the acceptor pool at equilibrium). The relative FC efflux rate differences observed at 100 µg of PL/ml (Fig. 1) persisted at 5-fold higher acceptor particle concentrations. To estimate the maximal efflux velocity (V(max)) that was attained by these complexes, the initial rates of cellular FC efflux (occurring during the first 2 h) were plotted as a function of particle concentration as proposed by Hofstee(32) . This treatment linearized the data so that a V(max) could be estimated by extrapolating the data to the condition of infinitely high acceptor concentration (for a detailed discussion of the application of this analysis to FC efflux, see (6) and the Appendix). The average (n = 6) V(max) values (percent of cellular FC released per h) for the rHDL made with DOPC, POPC, PSPC, DMPC, DPPC, DSPC, and SM were 14.0 ± 1.7, 12.0 ± 2.2, 2.5 ± 0.2, 6.2 ± 1.1, 4.9 ± 0.7, 3.2 ± 0.6, and 8.8 ± 2.4, respectively. The FC efflux rates were slightly increased in separate incubations in which the cell wells were stirred at 500 rpm on an orbital shaker (2 mm radius), but the differences in FC efficiency that are shown in Fig. 2were found to persist whether the cell plates were stirred or left quiescent (data not shown). These data show that the V(max) values for the various rHDL did not converge to a common value.


Figure 2: The concentration dependence of the FC efflux rate constants (k(e)) from mouse L-cell fibroblasts to discoidal rHDL containing various PL acyl chains. The incubation conditions symbols were the same as described in the legend to Fig. 1. The vertical axis shows the average FC efflux rate constant (k) derived by fitting an equation that describes tracer equilibration between two pools (see ``Methods'') to an experimental time course. Thus each data point is derived from an entire 6-h time course. Each point represents the mean of six cell wells from two separate experiments. The error bars represent 1 S.D. The values shown are corrected for the FC efflux to control medium.



To determine if the relative FC efflux efficiencies are a function of the cell type used, similar FC efflux experiments were performed using rat Fu5AH hepatoma cells which are known to release FC at a faster rate than L-cells in response to PL-containing particles (33) (Fig. 3). In this case, the corrected t values for the efflux of Fu5AH FC to the rHDL made with DOPC, POPC, PSPC, DMPC, DPPC, DSPC, and SM were 7.3 ± 0.3, 5.5 ± 0.3, 33 ± 6.0, 11.2 ± 1.0, 12.2 ± 0.7, 63.4 ± 9.3, and 8.6 ± 0.3 h, respectively. Relative to L-cells, the Fu5AH cells released FC more rapidly to the LC rHDL at a concentration of 100 µg of PL/ml ( Fig. 1and Fig. 3). However, the FC transfer to gel-phase (DSPC/apoA-I and PSPC/apoA-I) particles was the same or slower than from L-cells. The same ranking of the abilities of the various rHDL particles to remove FC was observed with the two cell types. This indicates that the relative abilities of these complexes to remove cellular FC was a particle-dependent phenomenon and was not related to the particular cell type used.


Figure 3: Time courses of [^3H]free cholesterol efflux from rat Fu5AH hepatoma cells to discoidal rHDL containing various PL acyl chains. The incubation conditions and symbols were the same as described in the legend to Fig. 1. The vertical axis indicates the fraction of initial labeled free cholesterol that remained in the cell at the designated times. Each point represents the mean of three cell wells. The error bars represent 1 S.D. All curves were obtained as described in Fig. 2.



It was possible that the nature of the PL in the rHDL determined the particle's ability to accept cellular FC although it was also possible that the conformation of the apoA-I on the surface of the particle could be a determinant. To distinguish between these possibilities, two of the PL types were selected to make protein-free, small unilamellar vesicles (SUV). POPC and DMPC were used because they exhibited large differences in efflux efficiency when complexed to apoA-I (Fig. 2) and preliminary work has established that both PL form well-defined SUV that do not degrade or fuse under the conditions of the FC efflux assay (data not shown). Fig. 4compares the concentration dependence of FC efflux from L-cells to apoA-I-containing rHDL and to protein-free SUV made with POPC and DMPC. At 500 µg of PL/ml, the POPC/apoA-I disc had a k(e) of about 12% cell FC/h and the DMPC/AI had a k(e) of about 6% cell FC/h, a 2-fold difference. At the same concentration, the POPC SUV exhibited a lower rate of 2.5%/h and the DMPC SUV had a k(e) of about 1.1%/h, about a 2-fold difference. Since the 2-fold difference in efflux efficiency between the apoA-I-containing discs is also observed between the SUV made with the same PL, it follows that the relative order of efflux efficiency seen in Fig. 2was due to the nature of the PL present in the rHDL and not to the conformation of the apoA-I on the surface of the particle.


Figure 4: Comparison of the concentration dependence of the FC efflux rate constants (k) from mouse L-cell fibroblasts to apoA-I-containing discoidal rHDL and SUV made with either POPC or DMPC. Panel A shows the data for rHDL discs (replotted from Fig. 2) and panel B shows the data for SUV. Incubation conditions were the same as those for Fig. 1. Each point represents three cell wells and the error bars represent 1 S.D. The values shown are corrected for the FC efflux to control medium.



The rHDL FC efflux efficiency can be related to the length and degree of saturation of the acyl chains of the rHDL PL. Increases in the rHDL PL acyl chain length and degree of saturation apparently decrease the FC efflux efficiency of rHDL. In terms of acyl chain length of rHDL PL, the FC efflux hierarchy is DMPC (2 times 14:0) > DPPC (2 times 16:0) > DSPC (2 times 18:0) (Fig. 1Fig. 2Fig. 3). In the case of degree of unsaturation, the FC efflux ranking is DOPC (2 times 18:1) > DSPC (2 times 18:0), and PSPC (16:0 18:0) <POPC (16:0 18:1).


DISCUSSION

The results of this study show that both the structure and function of discoidal rHDL are significantly affected by PL acyl chain length and degree of unsaturation because these parameters directly determine the physical state of the lipid that composes the particle. The physical state and hence fluidity of the PL at 37 °C affects the conformation of associated apoA-I molecules and the ability of an rHDL particle to remove FC from cells. These two effects are discussed in turn below.

The Effect of PL Physical State on the Structure of Discoidal ApoA-I-containing rHDL

It is known that amphipathic alpha-helical segments are the main lipid binding domains in apoA-I (34) and there is strong evidence that these domains are arranged around the edge of an rHDL disc such that the long axes of the alpha-helices are parallel to the PL acyl chains(35) . Zorich et al.(23) have proposed that amphipathic alpha-helical segments determine the size of discoidal particles obtained when apoA-I is combined with various PL. The present data are consistent with this concept in that most of the rHDL have a diameter of 9.7-10.2 nm despite the variety of PL acyl chains present. The exceptions are the DSPC/apoA-I, PSPC/apoA-I, and SM/apoA-I particles which are slightly larger as a consequence of the incorporation of a third molecule of apoA-I. The conformation of the helical segments appears to be affected by the various PL in that the apoA-I molecules are less helical and relatively unstable when the protein is associated with gel-phase PL rather than LC PL (Table 3). It is thought that the hydrophobic faces of amphipathic helices penetrate into the fatty acyl chain milieu when apoA-I molecules are located at a lipid/water interface such as the edge of a discoidal rHDL particle(36) . The resultant interaction of the residues on the nonpolar face of the alpha-helix with lipid stabilizes the alpha-helix. The current data suggest that the nonpolar faces of the helices are unable to penetrate as deeply into the disc edge when the PL acyl chains are in a quasi-crystalline state rather than in a fluid and relatively compressible LC state. This may result in exposure of the some nonpolar residues to the aqueous environment and formation of short, unstable alpha-helices. In addition, the increased bilayer thickness that occurs with gel-phase PL may lead to a mismatch with the length of the 22-residue apoA-I helical segments around the disc edge(23, 34) . Both effects would contribute to destabilization of the helical segments of apoA-I molecules in discoidal rHDL containing gel-phase PL.

Although the various PL affect the surface charge of the rHDL particles, this parameter does not appear to correlate with the physical state of the associated PL. Since the various PC molecules in the rHDL particles have the same net charge at a given pH, any changes in particle valence must arise from conformational changes in the resident apoA-I molecules that lead to alterations in the ionization states of charged amino acid residues(27) . Although the exact molecular nature of these changes cannot be determined, the variations in the rHDL particle charges reported in Table 2are consistent with the types of conformational effects discussed in the preceding paragraph.

The Effect of the PL Physical State on the Efflux of Cell Membrane FC

The transfer of cell plasma membrane (PM) FC to extracellular particles occurs by the aqueous diffusion mechanism(37, 38) . This study and others have demonstrated that the rates of FC transfer between donor and acceptor particles exhibit a hyperbolic dependence on acceptor concentration (Fig. 2, and (38, 39, 40) ). The form of this curve can be compared to a Michaelis-Menten plot of enzyme velocity against substrate concentration. The cases of FC transfer and enzyme kinetics are similar in that they involve two sequential rate processes. In light of the similar hyperbolic concentration dependence, we have used the well-known methods of analyzing enzyme kinetics to generate maximal velocities (V(max)) for FC efflux (6) . Since the molecular events differ between an enzyme reaction and FC transfer, an analysis of the kinetic steps involved in FC transfer is required. The two-step process of FC transfer involves: 1) the first-order desorption of a FC molecule from the hydrophobic environment of the PM followed by, 2) a second-order collision process involving FC incorporation into the acceptor particle(38) . As outlined under ``Appendix,'' the relative contributions of these steps to the overall rate (v) of FC efflux depends on the concentration of extracellular acceptor particles [A]. At low acceptor concentrations, v = k[A] so that the rate is first-order with respect to [A]. The k term includes the rate constant characterizing the tendency of FC molecules to desorb from the cell PM (k(1)) and a second-order rate constant (k(2)) that describes the subsequent diffusion and incorporation into an acceptor particle; at low acceptor concentrations, both the PM desorption and diffusion steps contribute to the overall efflux rate. However, at high acceptor concentrations, the rate of collision/incorporation into the acceptor particles is fast relative to the PM desorption rate due to the large excess of acceptor particles in the medium. Thus, the kinetics are zero order with respect to [A] because v = k(1)[DC]; [DC] is the concentration of FC in the PM that is available for desorption. Since the desorption step becomes rate-limiting for FC efflux under these conditions, a common maximal efflux rate (V(max)) should be attained for all types of acceptor particles at a sufficiently high [A]. The current efflux data for low and high acceptor particle concentrations are considered in turn below.

Fig. 2shows that, at low rHDL concentrations (<100 µg of PL/ml), rHDL containing LC PL appear to be more efficient than those containing gel-phase PL. Since the diffusion coefficients of the various rHDL particles in the extracellular medium are expected to be similar because of their similar sizes (Table 1) and the diffusion coefficient of FC is the same in all cases, the aqueous diffusion model argues that the number of FC-rHDL collisions should be similar for the various rHDL. Thus, the observed differences must be due to a difference in the fraction of FC-rHDL collisions that result in incorporation of the FC molecules into the rHDL PL surface (effective collisions). It is likely that the percentage of effective FC collisions is higher for rHDL containing the LC PL than those containing the gel-phase PL. This seems reasonable because the incoming FC molecule must overcome the stronger cohesive interactions between the gel-phase PL acyl chains in order to incorporate into the acceptor PL surface.

At high acceptor concentrations (>500 µg of PL/ml), the FC efflux differences between the rHDL particles persist and the V(max) values indicate substantially different maximal FC efflux velocities among the rHDL of different PL content. This result is surprising because, according to the aqueous diffusion mechanism, different acceptor particles should exhibit similar V(max) values that are related to the tendency of FC to desorb from the PM (k(1)). Since the relative FC efflux differences are similar at high and low acceptor concentrations, it is reasonable to infer that the rationale described for the case of low acceptor concentrations can explain the differences between rHDL containing the various PL at all rHDL concentrations. If this is correct, it follows that the reported V(max) k(1)[DC], because no terms are included that describe the interaction of FC molecules with the acceptor rHDL particles. This suggests that the V(max) values that are measured in this study are apparent values that may not represent the true maximal rate of FC efflux at high concentrations of acceptor. The simplest explanation for this discrepancy is that the concentration of the rHDL particles at the surface of the cells (where most of the FC transfer is expected to occur) is limited to a concentration that is: (a) distinct from the bulk medium concentration and (b) below that required to saturate the cellular FC efflux. This difference between the bulk medium and cell surface concentrations is expected to be the same for all of the rHDL particles used in this study because of their comparable size. Under these conditions, the rate constant (k(2)) for FC diffusion to and incorporation into the acceptor particle is expected to affect the FC efflux rate as it does in the case of low acceptor concentrations. Consequently, the differences in FC efflux noted at high concentrations can be explained by the different fraction of effective collisions between desorbed FC and the various rHDL (i.e. the effective FC collision rate is lower with gel-phase rHDL). It is interesting that the equilibrium distribution of FC among the cell and acceptor pools is generally similar for rHDL made with both LC and gel-phase lipids, suggesting that those particles near the cell surface can exchange with those in the bulk medium so that the entire acceptor pool is available to accept cell FC regardless of any limitations in the concentration close to the cell surface. Apparently, the nature of the rHDL PL only affects the rate at which a given rHDL can accept FC and not its capacity to solubilize FC.

It is likely that the complexity of the PM of living cells accounts for the lower concentration of rHDL particles at the cell surface. The most obvious complications inherent in cellular FC transfer systems include the unstirred water layer at the cell surface(41) , the cell glycocalyx, and the convoluted nature of the PM(42) . The thickness of the unstirred water layer over most planar cells is between 300 and 800 µm(41) . Westergaard and Dietschy (43) have demonstrated that this value can be decreased to a minimum of 100 µm with vigorous stirring. Although the effect of stirring on the thickness of the unstirred water layer was not determined in this study, the increased V(max) values observed with stirring of the cell plates support the assertion that the values that are obtained with quiescent cell plates are apparent values. At this point, the cell characteristic(s) that shelters the region proximal to the PM from further increases in acceptor particle concentration remain to be elucidated. Experiments using acceptor particles of varying size are needed to further investigate this phenomenon. In addition, the nature of the donor PM can be changed. For example, the cells can be grown in suspension culture or PM vesicles can be used as FC donors to address the effects of cell surface organization on FC efflux.

Alternatively, if the physical basis for the differences between the gel- and LC-phase rHDL FC efflux efficiencies is not due to differences in the effective collision rates (i.e.V(max) = k(1)[DC]), then one is forced to conclude that the different V(max) values in this study arise from differential effects of the various rHDL on either k(1) or [DC] (or both). A change in the FC desorption rate (k(1)) could be caused by differential interactions of the various rHDL with the cell PM. Such an interaction could be mediated by the apolipoprotein component of the rHDL (6) or by exchange of the PL component of the rHDL into the cell PM. The observation that SUV exhibit similar differences in FC efflux efficiency as apoA-I-containing rHDL (Fig. 4) indicates that the observed differences are due to a property of the acceptor PL. While rHDL PL undoubtedly exchanges into the cells to some extent over the course of the efflux incubation(44, 45) , the rate of transfer of PL is generally slow compared to the transfer of FC(2) ; cells that have been incubated with DOPC (46) and SM (47) liposomes for much longer than the 6-h incubations employed here show no significant change in the fatty acid content of the PM. Thus, during the time frame of our studies, it is unlikely that cell-accumulated PL could sufficiently modify the PM to an extent that has been shown to have noticeable effects on the rate of FC efflux(11) . It could be argued that the various rHDL have differential access to regions of the PM, altering the size of the PM FC pool ([DC]) that is participating in efflux. However, the similar diffusional properties of the various rHDL of comparable size suggests that all of the rHDL particles have equal access to the various regions of the cell PM. Again, more information from experiments where acceptor particle size is systematically changed is required to distinguish between these possibilities.

A full understanding of the different V(max) values reported for the various rHDL awaits further study. Nevertheless, it appears that rHDL which contain gel-phase PL allow a lower fraction of colliding FC molecules to incorporate into the PL surface than those containing LC-phase PL. It is interesting that the SM/apoA-I discoidal rHDL exhibits a faster rate than predicted from its physical state. Since the SM used in this study was isolated from bovine brain, this may be due to the heterogeneous nature of the various acyl chains causing defects that may facilitate the incorporation of incoming FC molecules into the bilayer. It is unlikely that HDL particles exhibiting the large differences in PL fluidity observed with the particles reconstituted with purified PL (Table 1) occur in vivo. However, several lipids that are common constituents of HDL are known to significantly affect the fluidity of lipid surfaces (e.g. FC and SM)(2) . In addition, the PL fatty acyl composition of lipoproteins and cell membranes reflect the dietary intake of saturated fat (8, 44) and such changes are known to have subtle but measurable effects on the fluidity of plasma lipoproteins(9) . These changes may affect the ability of HDL particles to accommodate FC molecules that have desorbed from peripheral cells. The current study using simplified systems forms a basis for understanding the impact of HDL lipid composition on the process of reverse cholesterol transport and offers insight into the mechanism of FC efflux from cells.


APPENDIX

The transfer of lipid molecules between donor and acceptor particles by diffusion has been studied extensively (for reviews, see (38) and (49) ). It is generally agreed that, depending upon the concentrations and nature of the donor and acceptor particles, and the nature of the transferring molecule, complex kinetics with different reaction orders can be observed. For instance, it has been shown that spontaneous transfer of long-chain phospholipids can occur by both a first-order, monomer desorption process and a parallel, concentration-dependent, transfer process in which the rate of lipid monomer desorption from the donor particle is enhanced via transient donor-acceptor collisions(39, 50) ; the contribution of the donor-acceptor collision-mediated process to phospholipid transfer is enhanced at higher concentrations of particles. Interestingly, the transient donor-acceptor collisional contribution is not observed for FC transfer in vesicle systems under conditions where the phospholipid transfer rate is significantly increased by formation of transient vesicle-vesicle complexes(50) . In light of this, the following kinetic model for FC transfer does not include the latter effect; also, such a process is not consistent with the zero-order kinetics seen for FC transfer from cells at high acceptor concentrations. The hyperbolic dependence of FC transfer rate on acceptor concentration is explained in terms of a kinetic scheme in which a first-order reaction to form a FC intermediate state is followed by a second-order interaction with the acceptor particle to transfer the FC molecule from the intermediate state to the acceptor particle.

The scheme for donor particles D and acceptor particles (A) can be written in terms of the two steps (see (39) and (50) -52).

where DC and AC represent FC in the donor and acceptor particles, respectively. C(i) is FC in the intermediate state and the k values are the rate constants for the steps indicated. Under initial velocity conditions where k approx 0, the rate of formation of C(i) from mass action kinetics is given by .

At the steady state equilibrium, d[C(i)]/dt = 0 so that

From , the initial velocity (v) of the transfer reaction

where the terms in square brackets are concentrations at time t approx 0.

describes the hyperbolic dependence of v on the acceptor particle concentration. When k[D] k(2)[A], there is first-order dependence of v on acceptor concentration because v = k[A] at constant donor concentration; the apparent rate constant k = k(1)k(2)[DC]/k[D] contains collisional rate constants k and k(2). When k(2)[A] k [D], or simply [A] [D] when both donor and acceptor species are similar so that similar on-rates for FC are expected (i.e.k(2) approx k), v = k(1)[DC] which indicates that v is independent of the acceptor concentration [A] and zero-order kinetics occur at constant donor concentration. The rate constant k(1) describes the diffusion of FC from the donor particle into the intermediate state C(i); any factors that modulate the packing of FC molecules in the surface of the donor particle can influence k(1) (cf. (38) ). Mixed kinetics occur when k(1)[D] approx k(2)[A].

The molecular events involved in FC transfer cannot be determined unambiguously from the kinetics but some mention of the physical process is worthwhile. FC transfer has been explained in terms of two limiting situations: either an aqueous diffusion mechanism where the desorption rate k(1) is limiting or a collision-mediated mechanism where collisions between donor and acceptor particles are involved. In fact, both models can fit the same kinetic scheme of a first-order formation of an intermediate followed by a second-order transfer of FC from the intermediate state to the acceptor particle so that there is a hyperbolic dependence on [A] (cf. Fig. 2). The nature of C(i) has been the subject of much debate, it has been considered variously as FC monomers in the aqueous phase (for a review, see (38) ) or FC molecules partially dissociated from the donor particle(52) . When [A] is low, the rate of FC transfer is proportional to the number of collisions between FC molecules in the intermediate state and acceptor particles so that v is proportional to [A]. The collision event to form the acceptor-FC complex AC could occur in either of the above loci, with this being influenced by the nature of the donor and acceptor particles and the interaction between them. It should be noted that direct contact between the donor and acceptor particles is not obligatory because FC can transfer between donors and acceptors separated by a semipermeable membrane (for a review, see (38) ). At high [A], v is zero-order with respect to [A] and the transfer rate is limited by k(1) at a constant donor concentration. This rate is affected by the structure of the donor particle and also by donor-acceptor interactions such as might occur in apolipoprotein-containing systems(53) .


FOOTNOTES

*
This work was supported by National Institutes of Health Program Project Grant HL22633 and a predoctoral fellowship (to W. S. D.) from the American Heart Association, Southeastern Pennsylvania Affiliate. 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.

(^1)
The abbreviations used are: HDL, high density lipoprotein; apoA-I, apolipoprotein A-I; DOPC, 1,2-dioleoyl phosphatidylcholine; DMPC, 1,2-dimyristoyl phosphatidylcholine; DSPC, 1,2-distearoyl phosphatidylcholine; FC, free (unesterified) cholesterol; GdnHCl, guanidine hydrochloride; rHDL, reconstituted HDL; LC, liquid crystal state; OPPC, 1-oleoyl-2-palmitoyl phosphatidylcholine; PAGGE, polyacrylamide gradient gel electrophoresis; PL, phospholipid; PM, plasma membrane; POPC, 1-palmitoyl 2-oleoyl phosphatidylcholine; PSPC, 1-palmitoyl-2-stearoyl phosphatidylcholine; S, surface potential in millivolts (mV); SM, sphingomyelin; SUV, small unilamellar vesicle; V, valence in electronic units (e).


ACKNOWLEDGEMENTS

We thank Dr. Julian Snow (Philadelphia College of Pharmacy and Science, Philadelphia, PA) for helpful discussion and Faye Baldwin, Sheila Benowitz, and Margaret Nickel for expert technical assistance.


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