© 1995 American Heart Association, Inc.
Articles |
Lipoproteins Containing Apolipoprotein A-IV but Not Apolipoprotein A-I Take Up and Esterify Cell-Derived Cholesterol in Plasma
From the Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität Münster (A.von E., A.S.S., G.A.), FRG; the Institut für Arterioskleroseforschung an der Universität Münster (Y.H., S.W., G.A.); and the Abteilung für Endokrinologie und Stoffwechsel, Zentrum Innere Medizin, Philipps-Universität Marburg (S.S., A.S.).
Correspondence to Arnold von Eckardstein, Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität Münster, Albert-Schweitzer-Strasse 33, D-48129 Münster, FRG.
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Abstract |
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Abstract Two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) identifies distinct apoA-I– or apoE-containing subclasses of high-density lipoproteins (HDLs), each of which plays a different role in reverse cholesterol transport. In this study we used 2D-PAGGE to investigate the role of apoA-IV–containing lipoproteins in reverse cholesterol transport in native plasma. Incubation of 2D electrophoretograms with anti–apoA-IV antibodies identified up to three subclasses of particles. The smaller particle subclasses, LpA-IV-1 and LpA-IV-2, were found in every plasma sample. The largest particle subclass, LpA-IV-3, was observed in fewer than 10% of the plasmas analyzed. 2D-PAGGE of apoA-I–deficient plasma and apoA-I–depleted plasma and anti–apoA-I immunosubtracting 2D-PAGGE of normal plasma revealed that LpA-IV-1 and LpA-IV-2 do not contain apoA-I. The importance of LpA-IV-1 and LpA-IV-2 for uptake and esterification of cell-derived cholesterol was investigated using pulse-chase incubations of plasma with [3H]cholesterol-labeled fibroblasts followed by anti–apoA-I immunosubtracting 2D-PAGGE. During 1-minute pulse incubation with cells, [3H]cholesterol was taken up by
-LpE
>LpA-IV-1 >pre-ß1-LpA-I >LpA-IV-2 (">" denotes
"more than"). During subsequent chase incubation without cells,
proportionately less radioactivity disappeared from LpA-IV-1 and
LpA-IV-2 than from pre-ß1-LpA-I and -LpE. During
5-minute pulse incubations, radioactive cholesteryl esters were formed
in pre-ß3-LpA-I >
-LpA-I >LpA-IV-1 >LpA-IV-2. The
fractional esterification rate was highest in
pre-ß3-LpA-I and lowest in -LpA-I. Subsequent chase
led to the disappearance of [3H]cholesteryl esters from
pre-ß3-LpA-I and, to a lesser extent, from LpA-IV-1 and
LpA-IV-2 but to an increase of [3H]cholesteryl esters in
-LpA-I and LDL. Similar pulse-chase experiments with
apoA-I–deficient plasma revealed that LpA-IV-1 and LpA-IV-2 take up
and esterify cell-derived cholesterol even more
effectively than in normal plasma. We conclude that LpA-IV-1 and
LpA-IV-2 are apoA-I–free lipoproteins that are important contributors
to reverse cholesterol transport.
Key Words: lecithin:cholesterol acyltransferase • HDL subfractions • reverse cholesterol transport • cholesterol efflux • nondenaturing two-dimensional electrophoresis
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Introduction |
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The reverse cholesterol transport model is widely used to explain the protective role of HDLs in atherogenesis. In this model, HDLs mediate the efflux of excess cholesterol from peripheral cells into plasma as well as the esterification of cholesterol in plasma by LCAT. From HDL, CEs are subsequently delivered to the liver by several mechanisms. In humans, the most important pathway apparently is the exchange of CEs with triglycerides from LDL and VLDL that is mediated by CETP (reviewed in References 1 through 31 2 3 ). HDLs, however, encompass structurally and functionally heterogeneous particles that can be classified according to a variety of properties, including hydrated density, apolipoprotein composition, and charge (reviewed in Reference 44 ). 2D-PAGGE of plasma in the sequence agarose gel electrophoresis followed by PAGGE and subsequent immunoblotting characterizes HDL-subclasses by charge, size, and antigenicity.4 Immunodetection with anti–apoA-I antibodies differentiates pre-ß1-LpA-I, pre-ß2-LpA-I, pre-ß3-LpA-I, and
-LpA-I.2 5 6 7 Previously, pulse-chase experiments
identified the quantitatively minor HDL subclass
pre-ß1-LpA-I as being the initial acceptor of
cell-derived cholesterol.2 5 6 From
pre-ß1-LpA-I, which contains apoA-I as the only
apolipoprotein, cell-derived cholesterol is rapidly
transferred to LDL via
pre-ß2-LpA-I
pre-ß3-LpA-I-LpA-I.2 5 6
LCAT esterifies cholesterol either directly during its
passage through pre-ß3-LpA-I or in -LpA-I after
recycling from LDL.2 6 8 9
The absence of premature atherosclerosis in some
patients with apoA-I deficiency10 11 stimulated us to
search for HDL subclasses that are free of apoA-I but that fulfill
important functions in reverse cholesterol transport. We
previously identified -LpE, a lipoprotein that contains apoE as the
only protein and that also serves as an initial acceptor of
cell-derived cholesterol.12 Other
apoA-I–free lipoproteins that may play some role in reverse
cholesterol transport are those containing apoA-IV
(reviewed in Reference 1313 ). ApoA-IV is secreted by enterocytes into the
lymph fluid as a component of chylomicrons.14 15 16 17 18 In
fasting plasma, apoA-IV is found in HDL and in the lipoprotein-free
fraction.16 17 18 19 20 21 22 23 Sequential anti–apoA-IV and
anti–apoA-I immunoaffinity chromatography has
identified apoA-IV–containing particles that also contain apoA-I
(LpA-I/A-IV) and particles containing ApoA-IV alone
(LpA-IV).23 The latter particle contains more than 90% of
the apoA-IV in fasting plasma. Both LpA-I/A-IV and LpA-IV are poor in
lipids, which are almost entirely phospholipids and
triglycerides.23 Both particles contain
activities for LCAT and CETP and stimulate cholesterol
efflux from cells in vitro.23 24 To better understand the
in vivo relevance of apoA-IV–containing lipoproteins in reverse
cholesterol transport, we analyzed the contribution
of LpA-IV to the uptake, esterification, and transfer of
cell-derived cholesterol in native plasma. Similar to
previously reported experiments that helped to investigate the role of
apoA-I- and apoE-containing HDL subclasses, we used pulse-chase
incubations of native plasma with
[3H]cholesterol-labeled fibroblasts and
subsequent 2D-PAGGE.2 4 5 6 12 Since LpA-IV contains little
UC,23 nonspecific equilibration of labeled and unlabeled
molecules does not contribute much to the appearance of radioactivity
in these lipoproteins. However, since apoA-IV–containing lipoproteins
have electrophoretic properties similar to those of -LpA-I and
pre-ß1-LpA-I,4 we modified the 2D-PAGGE
system by introducing an electrophoretic immunoadsorption step. This
anti–apoA-I immunosubtracting 2D-PAGGE allowed us to investigate
the role of LpA-IV in reverse cholesterol transport in
native plasma. In addition, to test this procedure against a valid
control and to demonstrate the importance of apoA-I–free lipoproteins
in reverse cholesterol transport, we also analyzed
the plasma of an apoA-I–deficient patient.11
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Methods |
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Plasma Samples
Electrophoretic analyses and pulse-chase experiments were performed on plasma samples from three normolipidemic individuals and a woman with apoA-I deficiency.11 Table 1
summarizes characteristics of the lipid
metabolism of the normolipidemic proband and the
apoA-I–deficient patient whose data have been selected for
presentation in Tables 2 through 5. All four subjects had the
phenotypes apoA-IV-1/1 and apoE3/3.25 Moreover, we
analyzed 20 different plasma samples by nondenaturing 2D
electrophoresis and subsequent anti–apoA-IV immunoblotting to
investigate possible effects of the apoA-IV polymorphism on the
electrophoretic appearance of apoA-IV–containing lipoproteins. Blood
samples were collected after overnight fasting and immediately cooled
on ice. Plasma and sera were obtained by centrifugation
at 4°C (3000g, 15 minutes), divided into aliquots, and
frozen at -70°C. For the experiments in which plasma was incubated
with cells, streptokinase was used as the anticoagulant at a final
concentration of 150 U/mL blood.
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Quantification of Lipids, Apolipoproteins, and HDL
Subfractions
Serum concentrations of triglycerides and
cholesterol were quantified enzymatically with an
autoanalyzer (Hitachi/Boehringer). HDL
cholesterol was measured after precipitation of
apoB-containing lipoproteins with phosphotungstic
acid/MgCl2 (Boehringer Mannheim). LDL
cholesterol was calculated by using the Friedewald
formula.26 Concentrations of apoA-I, apoA-II, and apoB
were determined with a modified commercially available turbidimetric
assay (Boehringer Mannheim).27 ApoA-IV was
quantified by electroimmunodiffusion using a rabbit anti-human
apoA-IV antiserum.28 Plasma activities of LCAT and CETP
were determined as described previously.25
Isolation of Lipoproteins
LpA-IV and LpA-I/A-IV were isolated from plasma by sequential
anti–apoA-IV and anti–apoA-I immunoaffinity
chromatography.23
Nondenaturing 2D Electrophoresis
The distribution of apoA-I–, apoE-, and apoA-IV–containing
lipoproteins in normal plasma was analyzed by 2D-PAGGE, whereby
agarose gel electrophoresis was followed by PAGGE.5 6
Briefly, in the first dimension, 20 µL of each plasma sample was
separated by electrophoresis at 4°C in a 0.75% agarose gel using a
50 mmol/L merbital buffer (pH 8.7, Serva). Bromphenol blue was added to
a standard sample to visualize albumin in the native gel. The
electrophoresis was stopped when the albumin/bromphenol blue
marker had migrated 6 cm. Agarose gel strips containing the
preseparated lipoproteins were then transferred to a 2% to 20%
polyacrylamide gradient gel. Separation in the second dimension
was performed at 40 mA for 4 to 5 hours at 10°C. During this time,
the endogenous plasma albumin, which because
bromphenol blue had been added to the cathodic buffer (300 µL/L
buffer) was visible in the native gel as a faint blue band, had
migrated 10 cm. The proteins separated in the PAGGE were electroblotted
onto a nitrocellulose membrane. ApoA-I– and apoE-containing
lipoproteins were detected by the use of sheep antibodies against human
apoA-I or human apoE (Boehringer Mannheim), respectively, which
had been biotinylated following the manufacturer's recommendations
(Sigma), and streptavidin-biotinylated horseradish peroxidase
complex (Amersham). ApoA-IV–containing lipoproteins were identified by
the use of a rabbit anti-human apoA-IV antiserum,28 a
biotinylated donkey anti-rabbit antiserum (Amersham), and
streptavidin–horseradish peroxidase.
Demonstration of LpA-IV by Anti–ApoA-I Immunosubtracting
2D-PAGGE
To circumvent contamination of LpA-IV with -LpA-I and
pre-ß1-LpA-I, we modified the 2D-PAGGE procedure
described above. Agarose gel electrophoresis was followed by
immunosubtracting electrophoresis in a 2% to 20%
polyacrylamide gradient gel that was covered by a 4-cm broad
0.6% agarose gel that contained the -globulin fraction of a
goat anti–apoA-I antiserum (Boehringer Mannheim) at a
final concentration of 4% (vol/vol). This immunoprecipitating gel
prevented all apoA-I–containing lipoproteins from migrating into the
polyacrylamide gel. Electrophoresis and Western blotting were
performed under the conditions described for 2D-PAGGE. Complete removal
of apoA-I–containing lipoproteins was ascertained by anti–apoA-I
immunoblotting.
Cell Culture
Normal human skin fibroblasts were cultured in Dulbecco's
modified Eagle's medium (DMEM) containing 10% fetal calf serum using
dishes of 3-cm diameter as described previously.6 At the
state of near-confluence, fibroblasts were labeled for 72 hours at
37°C with 0.5 mCi [(1,2-3H)]cholesterol
(New England Nuclear, 51.7 Ci/mmol), which had been complexed with
fetal calf serum. The final specific radioactivity in the labeled cells
amounted to 1.3±0.4x107 counts per minute (cpm)/mg cell
UC (mean±SD).
Cholesterol Efflux Experiments
To determine the cholesterol efflux from cells into
the different lipoprotein subfractions, 1 mL plasma was incubated for
different time intervals with
[3H]cholesterol-labeled fibroblasts that
had been washed six times with PBS (pH 7.4). To measure radioactivity
in LpA-IV, 20 µL plasma was separated by anti–apoA-I
immunosubtractive 2D-PAGGE. To determine radioactivity in -LpE,
pre-ß1-LpA-I, and -LpA-I, 20 µL plasma was separated
by 2D-PAGGE. In parallel, an aliquot of 20 µL unlabeled plasma from
the same donor was run in the same gel. After completion of 2D
electrophoresis, one half of the gel containing the labeled sample was
stored at 4°C. Proteins in the other half of the gel were
electroblotted onto a nitrocellulose membrane to immunolocalize
apoA-I–, apoE-, or apoA-IV–containing lipoproteins. These immunoblots
were then used as templates to localize pre-ß1-LpA-I,
pre-ß3-LpA-I, -LpA-I, -LpE, LpA-IV-1, LpA-IV-2, and
LDL in the native gels. Since pre-ß3-LpA-I was not
detectable in every anti–apoA-I immunoblot, positive immunoblots
of previous experiments were used for localization. The lipoproteins
were cut out and their lipids extracted by incubation with
chloroform/methanol (2:1, vol/vol) for 72 hours.29 In some
experiments UC and CEs were separated by thin-layer
chromatography on silica gel plates (Merck) as the
immobile phase and hexane/ether (6:4, vol/vol) as the mobile phase. The
radioactivity in the lipids was counted by scintillation
spectrometry.
Every experiment was performed three times on each plasma sample from three normolipidemic donors and the apoA-I–deficient patient. For every experiment, separate dishes with labeled fibroblasts were used. Interassay coefficients of variation in the recovery of radioactivity in the various lipoproteins of a specific plasma were below 20%. As reported previously,30 about 90% of the radioactivity in apoB-free plasma was recovered in apoA-I– and apoE-containing HDL subfractions separated by nondenaturing 2D-PAGGE.
Other Procedures
Protein concentrations were measured according to Lowry's
method31 using bovine serum albumin as the
standard. SDS-PAGE was performed according to the protocol published by
Laemmli.32 For statistical comparisons we used the
Student's t test.
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Results |
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Characterization of ApoA-I–, ApoE-, and ApoA-IV–Containing Lipoproteins in Normal Plasma by 2D-PAGGE
Fig 1
presents the distribution of apoA-I–,
apoE-, and apoA-IV–containing lipoproteins, respectively, after
separation of normal plasma by 2D-PAGGE. As described
previously,2 5 6 7 apoA-I was detected in a major HDL
subfraction with -mobility as well as in three minor
subfractions with pre-ß mobility that differ by their size
(pre-ß1-LpA-I, pre-ß2-LpA-I, and
pre-ß3-LpA-I) (Fig 1a). Pre-ß3-LpA-I was
immunodetectable in only a few Western blots. LDL also reacted with
anti–apoA-I antibodies. ApoE was detectable in a particle that is
mainly colocalized with -LpA-I and in a quantitatively minor
subfraction with electrophoretic -mobility that we previously
termed -LpE.10 In some Western blots, LDL immunoreacted
with anti-apoE (Fig 1b). ApoA-IV was visualized in three particles
(Fig 1c and 1d). Two of them, termed LpA-IV-1 and LpA-IV-2, form sharp
bands that exhibit electrophoretic "slow -mobility"; ie,
on agarose gel electrophoresis they migrate faster than pre-ß-LpA-I
but slower than -LpA-I (Fig 1c). We observed these two particles in
all of 23 analyzed normolipidemic samples. The third particle,
termed LpA-IV-3, forms a diffuse spot, has electrophoretic
-mobility, and is larger than LpA-IV-1 and LpA-IV-2. In contrast
to LpA-IV-1 and LpA-IV-2, LpA-IV-3 was observed in only 2 of 23
normolipidemic plasmas analyzed (Fig 1d).
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Electrophoretic Differentiation of LpA-IV and
LpA-I/A-IV
To analyze which of the electrophoretic LpA-IV particles
correspond to LpA-I/A-IV and LpA-IV, both LpA-I/A-IV and LpA-IV were
isolated from plasma by sequential immunoaffinity
chromatography and separated by either 2D-PAGGE or
SDS-PAGE. 2D-PAGGE and subsequent anti–apoA-IV immunoblotting of
LpA-I/A-IV did not lead to the visualization of any particle (Fig 2a). After SDS-PAGE of 100 µg lyophilized LpA-I/A-IV,
apoA-I was identified on protein staining (Fig 3a,
lane 1), whereas apoA-IV became detectable only by anti–apo-IV
immunoblotting (Figure 3b, lane 1). Thus, LpA-I/A-IV appears to contain
only trace amounts of apoA-IV that escaped detection by 2D-PAGGE and
anti–apoA-IV immunoblotting. In contrast, 2D-PAGGE and
anti–apoA-IV immunoblotting of LpA-IV revealed the presence of
LpA-IV-1 and LpA-IV-2 (Fig 2b). On SDS-PAGE of LpA-IV, apoA-IV was
already detectable in Coomassie Blue–stained gels. No apoA-I was
detectable in this particle either on protein staining (Fig 3a, lane 2)
or immunoblotting (Fig 3b, lane 2). In another attempt to differentiate
LpA-IV and LpA-I/A-IV, plasma of an apoA-I–deficient patient was
separated by 2D-PAGGE. As expected, we did not find any
anti–apoA-I–immunoreactive particles (Fig 4a),
whereas apoE-containing particles including -LpE were present
(Fig 4b). We observed apoA-IV–containing particles with
electrophoretic properties similar to those of LpA-IV-1, LpA-IV-2, and
LpA-IV-3 (Fig 4c). Together these data indicate (1) that LpA-IV-1 and
LpA-IV-2 do not contain apoA-I and (2) that apoA-I does not appear to
be an obligate constituent of LpA-IV-3. Moreover, LpA-I/A-IV could not
be localized in our nondenaturing 2D electrophoretograms.
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Cholesterol Efflux From Cells Into LpA-IV-1 and
LpA-IV-2
LpA-IV-1 and LpA-IV-2 exhibit electrophoretic mobilities that are
similar to those of pre-ß1-LpA-I and -LpA-I (cf Fig 1
). To isolate LpA-IV-1 and LpA-IV-2 from 2D-PAGGE-gels without
contamination of apoA-I–containing lipoproteins, we used
anti–apoA-I immunosubtracting 2D-PAGGE. This method led to the
detection of LpA-IV-1 and LpA-IV-2 but not of LpA-IV-3 (Fig 5a). The complete removal of apoA-I by the
immunoadsorbing agarose gel was confirmed by anti–apoA-I
immunoblotting (Fig 5b).
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Table 2 presents the distribution of radioactive
cholesterol in various lipoproteins of a normal plasma
after a 1-minute pulse incubation with
[3H]cholesterol-labeled fibroblasts and a
1-minute chase incubation without cells. -LpE,
pre-ß1-LpA-I, -LpA-I, and LDL were isolated from the
gel after 2D-PAGGE, LpA-IV-1 and LpA-IV-2 after anti–apoA-I
immunosubtracting 2D-PAGGE. LpA-IV-1 took up 30% more radioactivity
than did pre-ß1-LpA-I (P<.05) and 30% less
than -LpE (P<.01). Only small amounts of
[3H]cholesterol appeared in LpA-IV-2. During
the subsequent chase incubation only 25% and 33% of the initial
radioactivity disappeared from LpA-IV-1 and LpA-IV-2 (NS),
respectively, whereas the radioactivity decreased by 65% in -LpE
and pre-ß1-LpA-I (both P<.01). In parallel
radioactivity increased by about 80% in LDL and about 30% in
-LpA-I.
Table 3 presents the distribution of radioactive
cholesterol in various lipoproteins of apoA-I–deficient
plasma. After a 1-minute pulse, similar amounts of radioactivity were
detectable in LpA-IV-1 and -LpE. Significantly higher amounts of
[3H]cholesterol accumulated in LpA-IV-1 of
apoA-I–deficient plasma compared with LpA-IV-1 of normal plasma
(P<.05; cf Table 2). Considerably less radioactivity was
detectable in LpA-IV-2; only background radioactivity was found in
pre-ß1-LpA-I. As described previously,32
considerable amounts of radioactivity were also found in a particle
with the electrophoretic properties of -LpA-I; however, that
particle did not contain apoA-I. Because LpA-IV-1 and LpA-IV-2
comigrate with this fraction, the radioactivity is at least partially
attributable to LpA-IV-1 and LpA-IV-2. During a 1-minute chase, only
25% of the initial radioactivity disappeared from LpA-IV-1 (NS) but
75% disappeared from -LpE (P<.01). Almost all of the
radiolabel appeared in LDL.
Esterification of Cell-Derived Cholesterol in
LpA-IV
Table 4 summarizes the outcomes of our studies on
the relative contribution in normal plasma of
pre-ß3-LpA-I, -LpA-I, LpA-IV-1, LpA-IV-2,
and LDL to the esterification of cell-derived
[3H]cholesterol and transfer of
[3H]CEs. After a 5-minute pulse-incubation with
[3H]cholesterol-labeled fibroblasts,
radioactive CEs were recovered in pre-ß3-LpA-I,
-LpA-I, LpA-IV-1, LpA-IV-2, and LDL. Pre-ß3-LpA-I
contained the highest amount of [3H]CE and had the
highest FER (ie, ratio of [3H]CE to
[3H]UC+[3H]CE). -LpA-I contained twice
as much [3H]CE as did LpA-IV-1 and LpA-IV-2
(P<.01). However, the FER was higher in apoA-IV–containing
particles. During the subsequent 5-minute chase the incubation without
cells but in the presence of the LCAT-inhibitor DTNB, 65%
of [3H]CE initially present in
pre-ß3-LpA-I disappeared (P<.01) compared
with 37% and 21% of the [3H]CE initially present in
LpA-IV-1 and LpA-IV-2, respectively (both P<.01). In
contrast, -LpA-I and LDL accumulated significant amounts of
[3H]CE during this chase incubation.
Table 5 summarizes the results of pulse-chase
experiments that were performed to investigate cholesterol
esterification and CE transfer in apoA–deficient plasma. Both LpA-IV-1
and LpA-IV-2 contributed to the esterification of
cholesterol in apoA-I–deficient plasma. Neither the
amounts of radioactive CE nor the FERs in LpA-IV-1 and LpA-IV-2
differed significantly between normal and apoA-I–deficient plasmas (cf
Table 4). LpA-IV-3 also accumulated cell-derived
[3H]cholesterol, but significantly lower
proportions were esterified compared with LpA-IV-1 and LpA-IV-2
(P<.01). The transfer of [3H]CE from LpA-IV-1
and LpA-IV-2 to other lipoproteins was faster in apoA-I–deficient
plasma than in normal plasma. In contrast, LpA-IV-3 accumulated
[3H]CE during the chase incubation.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Previous studies with apoA-IV–containing lipoproteins either isolated from plasma22 23 or reconstituted in vitro24 33 34 35 36 37 38 39 have provided considerable evidence that apoA-IV contributes to reverse cholesterol transport. In fasting plasma, apoA-IV is associated with lipoproteins of high or very high density.22 23 Incubation of apoA-IV with phospholipids or with cell membranes leads to the formation of small HDL-like particles.33 34 In vitro, apoA-IV activates LCAT.35 36 As an activator of a putative HDL-conversion factor, apoA-IV is involved in remodeling of HDL.37 ApoA-IV in both native and reconstituted lipoproteins has been shown to promote cholesterol efflux from various cells.22 23 24 38 39 Since apoA-IV easily dissociates from lipoproteins both in vivo and in vitro,22 23 24 40 41 42 little is known of the role of apoA-IV–containing lipoproteins in reverse cholesterol transport in vivo. Thus, it is unclear how much apoA-IV is to be found in apoA-I–containing lipoproteins21 22 23 43 and whether apoA-IV interacts with cellular HDL-binding sites.22 24 44 45 46 In this study we investigated for the first time the role of apoA-IV–containing lipoproteins in the uptake, esterification, and transfer of cell-derived cholesterol in native plasma.
2D-PAGGE and subsequent anti–apoA-IV immunoblotting discriminated
three particles: LpA-IV-1, LpA-IV-2, and LpA-IV-3. Like
pre-ß1-LpA-I and -LpE but unlike -LpA-I, both
LpA-IV-1 and LpA-IV-2 form sharp bands, probably because they are
homogeneous lipid-poor particles.23 Since
LpA-IV-1 and LpA-IV-2 were detectable in LpA-IV, in apoA-I–free, and
in apoA-I–deficient plasmas but not in LpA-IV/A-I, we conclude that
these particles do not contain apoA-I. Our data do not allow any
definite conclusion on the electrophoretic appearance of LpA-I/A-IV.
The absence of LpA-IV-3 in apoA-I–depleted plasma might indicate that
LpA-I/A-IV occurs principally as LpA-IV-3. However, we found an
LpA-IV-3–like particle in the plasma of an apoA-I–deficient patient
and were unable to detect LpA-IV-3 in LpA-I/A-IV. ApoA-I is probably
not an obligate constituent of LpA-IV-3. The finding of LpA-IV-1 and
LpA-IV-2 in every plasma sample, including those from apoA-I–deficient
individuals, supports the data of Duverger et al, who concluded that
most of the apoA-IV in fasting plasma resides on particles free of
apoA-I.23 This is in contrast to other authors who
reported that apoA-IV predominantly occurs in apoA-I–containing
particles.21 22 43
In combination with prior incubation of plasma with
[3H]cholesterol-labeled fibroblasts,
2D-PAGGE has previously helped to improve our understanding of how
important reverse cholesterol transport is to the
quantitatively minor HDL-subfractions such as
pre-ß1-LpA-I, preß3-LpA-I, and
-LpE.2 4 5 6 12 30 Since these particles are very poor in
UC, any appearance of radioactivity within them indicates the specific
movement of [3H]cholesterol from cell
membranes to the particles rather than nonspecific exchange of
radiolabeled and unlabeled cholesterol molecules. LpA-IV is
also a lipid-poor particle.23 Therefore, it is
unlikely that uptake of [3H]cholesterol by
LpA-IV-1 and LpA-IV-2 reflects nonproductive equilibration but
rather indicates that these particles serve as initial acceptors of
cell-derived cholesterol into the plasma compartment.
Interestingly, LpA-IV-1 of normal plasma took up more radioactivity
than pre-ß1-LpA-I and less radioactivity than -LpE
(Table 2). In this context it is also noteworthy that we previously
found that the plasmas of patients with Tangier disease or apoA-I
deficiency accumulate considerable amounts of cell-derived
cholesterol in a fraction with the electrophoretic
properties of small -LpA-I (ie, -LpA-I3), although
apoA-I was immunologically undetectable in this region of the
gel.30 Because the electrophoretic properties of LpA-IV-1
and LpA-IV-2 are similar to those of -LpA-I3, it is
likely that these apoA-IV–containing particles contributed to this
phenomenon. This also indicates that apoA-IV–containing lipoproteins
are important contributors to the residual activity of HDL-deficient
plasmas to release cholesterol from
cells.30
During chase-incubations of either normal or apoA-I deficient
plasma, significantly smaller proportions of
[3H]cholesterol were removed from LpA-IV-1
and LpA-IV-2 than from pre-ß1-LpA-I and -LpE. Unlike
pre-ß1-LpA-I and -LpE, which transfer cell-derived
cholesterol to other particles for further esterification
and catabolism,4 5 6 8 9 12 47 both LpA-IV-1 and LpA-IV-2
are involved in the esterification of cholesterol (Table 4). With respect to the FER, LpA-IV-1 and LpA-IV-2 are less effective
than pre-ß3-LpA-I but more effective than -LpA-I, two
other lipoproteins containing LCAT activity.6 8 9 Like
pre-ß3-LpA-I, LpA-IV-1 and LpA-IV-2 serve as net donors
of CE to other lipoproteins.6 8 However, the fractional
transfer rate is significantly lower in LpA-IV-1 and LpA-IV-2 than in
pre-ß3-LpA-I. This raises the question of whether
LpA-IV-1 and LpA-IV-2 catabolize cholesterol by mechanisms
other than CETP-mediated transfer of CE to apoB-containing
lipoproteins, which has been thought to be the most important pathway
for removal of CEs from HDL.48 In this regard it is
noteworthy that turnover studies have shown that apoA-IV is catabolized
much more rapidly than apoA-I.49 Since apoA-IV mediates
binding of lipoproteins to liver cells,44 45 46 it is
possible that the short-lived LpA-IV catabolizes
cholesterol of peripheral cells by hepatic
uptake and degradation, whereas the long-lived LpA-I catabolizes
cholesterol by transfer to LDL and VLDL, which are more
rapidly removed by the liver than HDL.
In conclusion, this study demonstrates for the first time the
importance of apoA-IV–containing lipoproteins in reverse
cholesterol transport in native plasma. Other than -LpE,
LpA-IV-1 and LpA-IV-2 provide further examples of apoA-I–free HDL
subclasses help to release cholesterol from cells. Unlike
pre-ß1-LpA-I, and -LpE, LpA-IV-1 and LpA-IV-2 are
initial acceptors of cell-derived cholesterol and are
also involved in the production and removal of CEs. LpA-IV-1
and LpA-IV-2 may catabolize cell-derived cholesterol
directly instead of by transferring CEs to apoB-containing
lipoproteins. Like -LpE, LpA-IV-1 and LpA-IV-2 may serve as a backup
system for reverse cholesterol transport and may contribute
to the antiatherogenic role of HDL when apoA-I–containing particles
are absent or severely reduced in
concentration.10 11 12 23
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the grants from Wissenschaftsministerium Nordrhein-Westfalen (Benningsen-Foerder-Preis) and Deutsche Forschungsgemeinschaft to Dr von Eckardstein (Ec 116,3-1) and to Dr Steinmetz (Ste 381,5-1). We gratefully acknowledge the excellent technical assistance of Anja Merschjann, Sabine Motzny, and Martina Plüster. We are grateful to Dr G. Noseda for providing us with plasma of the apoa-I–deficient patient and to Dr Paul Cullen for skillful review of the manuscript.
Received June 8, 1995; accepted August 2, 1995.
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References |
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