From the
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
The high density lipoprotein (HDL)
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
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-
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
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
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
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
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) .
Ultracentrifugally isolated
HDL
HDL
Fu5AH hepatoma cells were a kind gift from Dr. George
Rothblat.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
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) .
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.
Isolation of HDL
Blood from healthy normolipidemic volunteers
was collected into tubes containing EDTA-NaParticles
(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.
Characterization of ApoA-II-enriched HDL
Incubation of ultracentrifugally isolated
HDLParticles
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 HDL
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 HDLParticles
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 HDL
As compared with
non-enriched HDLand Cellular Cholesterol Efflux
(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
HDL
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 HDLon Cholesterol Efflux
(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).
-migrating LpA-I fraction,
namely LpA-Ipre
1, 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.
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
.
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.
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.
-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.
Table:
Composition (mass percent) of HDL after enrichment with apoA-II
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
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.
, 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.