From the Lipoprotein and Atherosclerosis Research
Group, Departments of Pathology & Laboratory Medicine and Biochemistry,
Microbiology, and Immunology, University of Ottawa Heart Institute,
Ottawa, Ontario K1Y 4W7, Canada and the
Centre d'Immunologie
INSERM-CNRS de Marseille Luminy, 13288 Marseille, France
Received for publication, January 7, 2003, and in revised form, January 17, 2003
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ABSTRACT |
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The pathways of hepatic intra- and
peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by
infecting primary mouse hepatocytes from either apoA-I-deficient or
ABCA1-deficient mice with a recombinant adenovirus expressing the human
apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes
with exogenously added hapoA-I (exo apoA-I) and examining the
hapoA-I-containing lipoproteins formed. The cells, maintained in
serum-free medium, were labeled with
[3H]choline, and the cell medium was
separated by fast protein liquid chromatography or
immunoprecipitated to quantify labeled choline phospholipids
specifically associated with hapoA-I. With the apoA-I-deficient hepatocytes, the high density lipoprotein fraction formed with endo
apoA-I contained proportionally more phospholipids than that formed
with exo apoA-I. However, the lipoprotein size and electrophoretic mobility and phospholipid profiles were similar for exo apoA-I and endo
apoA-I. Taken together, these data demonstrate that a significant
proportion of hapoA-I is secreted from hepatocytes in a
phospholipidated state but that hapoA-I is also phospholipidated peri-cellularly. With primary hepatocytes from ABCA1-deficient mice,
the expression and net secretion of adenoviral-generated endogenous
apoA-I was unchanged compared with control mice, but 3H-phospholipids associated with endo apoA-I and exo apoA-I
decreased by 63 and 25%, respectively. The lipoprotein size and
electrophoretic migration and their phospholipid profiles remained
unchanged. In conclusion, we demonstrated that intracellular and
peri-cellular lipidation of apoA-I represent distinct and additive
pathways that may be regulated independently. Hepatocyte
expression of ABCA1 is central to the lipidation of newly synthesized
apoA-I but also contributes to the lipidation of exogenous apoA-I.
However, a significant basal level of phospholipidation occurs in the
absence of ABCA1.
The hepatic and intestinal origins of the major high density
lipoprotein (HDL)1
apolipoproteins, apolipoprotein (apo)A-I and apoA-II, are well defined
(1, 2). In contrast, HDL lipid constituents have complex and
multiple origins that include secretion as nascent lipoproteins
containing apoA-I (3, 4), acquisition of lipids from remnant
lipoproteins arising from lipolysis of triglyceride-rich lipoproteins
(5-7), and from cellular lipid efflux (8). The relative contributions
of the different pathways are not well understood, particularly the
secretion of nascent lipoproteins and the contribution of the efflux
pathway. The ATP-binding cassette transporter, ABCA1, was recently
shown to control the efflux of cellular phospholipids and cholesterol
(9-12) and through this pathway to maintain HDL in the circulation.
Impairment of ABCA1, as in Tangier disease, leads to extremely low
levels of HDL (13-16). The major tissues affected in this disease are
rich in macrophages, which express high levels of ABCA1 (17-19). This
was first interpreted as evidence that excess lipids accumulated in
scavenger receptor-expressing cells were a major source of HDL lipids
(20, 21), but recent evidence shows that macrophage contribution to
HDL-cholesterol concentrations is minor (22). ABCA1 is expressed in
many tissues and at high levels in liver, brain, and small intestine,
but also testis, lung, spleen, and kidney (17-19). This suggests that
in both liver and intestine, where apoA-I synthesis is also high, ABCA1
may contribute to the lipidation of newly secreted or nascent lipoproteins. Previously, work with hepatocytes from chicken (23, 24)
or rat (25) had suggested that apoA-I was lipidated intracellularly. However, Hamilton et al. (26), using electron
microscopy, failed to identify any lipidated apoA-I particles in
hepatocytes, putting the intracellular lipidation hypothesis in
dispute. Recently, Chisholm et al. (27) investigated the
secretion and lipidation of apoA-I from HepG2 cells. They concluded
that some apoA-I acquired lipid intracellularly and was then secreted
along with lipid-poor apoA-I. Subsequently, the secreted apoA-I could
acquire lipids extracellularly to form buoyant HDL particles. In those
studies, which support the model of intracellular lipidation of apoA-I, HDL particles were obtained by carbonate extraction of cell
homogenates. Despite careful quantitation and inclusion of controls,
mixing of cell contents and artificial lipidation of apoA-I may occur.
Here we have characterized the hepatic lipidation of apoA-I by using
adenoviral expression of human apoA-I (hapoA-I) (28, 29) in primary
hepatocytes of apoA-I-deficient and ABCA1-deficient mice. We also
characterized the nascent lipoproteins formed by primary hepatocytes
cultured in lipoprotein-free medium compared with those formed by
interaction of exogenous apoA-I with the same cells. In both conditions
hepatocytes generate a lipidated pool of apoA-I-containing lipoproteins
via a pathway dependent on ABCA1. However, the lipidation of apoA-I is
reduced but not abolished in experiments with ABCA1-deficient
hepatocytes, suggesting the existence of alternate lipidation pathways.
Animals and Primary Hepatocyte Cultures--
ApoA-I-deficient
(Apoa1tm1Unc) C57BL/6J mice were obtained from
Jackson Laboratories (Bar Harbor, ME). ABCA1-deficient mice were generated according to Hamon et al. (30). The mice were
maintained on a 12 h light/12 h dark schedule on a normal chow
diet. Primary hepatocytes were prepared from these mice according to
established protocols (31, 32). Briefly, the cells were seeded in
fibronectin-coated (25 µg/well) 6-well plates at an initial density
of 1-2 × 106 cells per well in William's medium
containing penicillin (100 units/ml), streptomycin sulfate (100 units/ml), Fungizone® (250 ng/ml; Invitrogen) and 10% fetal
bovine serum (Sigma).
Cell Labeling--
Six h following the initial plating, the
cells were washed in William's medium without fetal bovine serum
(2 × 2 ml) and incubated with Hepatozyme® medium (Invitrogen)
containing 10 µCi/well of [3H]choline (PerkinElmer Life
Sciences). The following day (24 h) the labeled medium was removed and
the cells were infected for 1 h with either the recombinant
hapoA-I encoding Ad5 adenovirus (AdAI) or luciferase adenovirus (AdLuc)
at a multiplicity of infection of 75:1 plaque-forming units per cell in
William's medium without fetal bovine serum (28, 29). After the 1 h
infection, the hepatocytes were incubated for an additional 24 h
with fresh labeling medium as described above. The third day (18-24 h
after adenovirus infection), following 2 × 2 ml washes in
non-radioactive medium, the cells were incubated with unlabeled
Hepatozyme® medium (1 ml per well) in the absence or presence of 5 µg of hapoA-I. The cells were returned to the 37 °C incubator (5%
CO2) for 3.5 h, and the medium was subsequently
collected and spun down to pellet any cell debris. The medium with
newly secreted apoA-I (AdAI-infected cells) or with exogenously added
hapoA-I (AdLuc-infected cells) was analyzed as described below. In some
experiments, 10 µM 9-cis-retinoic acid, a
retinoid X receptor (RXR) ligand, was added to the hepatocytes 12 h prior to and during the 3.5 h incubation. Glyburide (100 µM), a known inhibitor of ABCA1-mediated lipid efflux,
was added only during the 3.5 h incubation.
Distribution of Secreted ApoA-I in the Various Lipoprotein
Pools--
The medium from four 6-well plates (24 wells) were pooled
and concentrated down to 2 ml (12-fold concentrated with Amicon 10K
filter units). The samples were immediately loaded on two calibrated
Superdex 200 columns connected in a series similar to that described
previously for isolation of lipoproteins from plasma samples (28). Very
low density lipoprotein (VLDL)- and low density lipoprotein (LDL)-sized
species elute in the void volume on these columns. HDL2/3
particles and smaller very high density lipoprotein (VHDL) fractions
containing albumin ( The Heterogeneity and Charge of ApoA-I Secreted from Primary
Hepatocytes--
The charge and size of apoA-I secreted from the
primary hepatocytes was determined by agarose gel (Beckman Lipogel,
Beckman Coulter, Fullerton, CA) and 4-20% non-denaturing
polyacrylamide gradient gel electrophoresis (PAGGE) (Novex,
Invitrogen), respectively, as described previously (28, 29). Briefly,
following transfer of proteins from the gels to nitrocellulose, the
membranes were probed with biotinylated monoclonal antibodies directed
against human apoA-I (a combination of 4H1 (against the extreme N
terminus) and 5F6 (against the central region)) (33). The antibodies
were biotinylated with Sulfo-NHS-Biotin (Pierce) and visualized by chemiluminescence following treatment with Streptavidin-conjugated horseradish peroxidase (Amersham Biosciences). The size of the apoA-I
species were compared with biotinylated molecular weight markers of
known hydrodynamic diameter, and the charge of secreted apoA-I was
compared with lipid-free apoA-I and HDL both isolated from human plasma.
Immunoprecipitation of ApoA-I and Associated Choline-containing
Phospholipids--
ApoA-I secreted from hepatocytes was
immunoprecipitated under native conditions either directly from the
medium or from lipoprotein fractions isolated by FPLC as follows. The
immunoprecipitations were carried out with a polyclonal anti-human
apoA-I antiserum from sheep (Roche Molecular Biochemicals) and protein
G-Sepharose (Amersham Biosciences). An equal volume of an anti-human
apoB antiserum from sheep, which does not cross-react with murine apoB, was used as a control where indicated. The immunoprecipitates were
collected following centrifugation (10 min at 3000 × g) and washed three times with 10 ml of phosphate-buffered
saline (no detergents) and resuspended in a final volume of 1 ml of
phosphate-buffered saline. These immunoprecipitates were either
subjected directly to scintillation counting or were further analyzed
by Bligh and Dyer lipid extraction (34) and thin layer chromatography
(TLC). TLC separation was performed on silica gel plates and a solvent system (chloroform/methanol/acetic acid/formic acid/water,
70:30:12:4:2) for separation of phosphatidylcholine and
sphingomyelin. The TLC bands corresponding to phosphatidylcholine and
sphingomyelin were excised and counted for radioactivity.
Alternatively, cells were labeled with 32P-phosphate
(Amersham Biosciences) to label all cellular phospholipids. Cells were
treated as with labeling with [3H]choline, except that
32P-phosphate in Hepatozyme medium was only added after
adenoviral infection on the second day (not also on the first day as
for [3H]choline). On the third day, the hepatocytes were
washed as before and incubated in fresh Hepatozyme in the absence or
presence of apoA-I for 3.5 h. The hapoA-I-containing lipoproteins
were immunoprecipitated as described above, and then phospholipids were
extracted by the method of Bligh and Dyer (34). The phospholipids were
separated by TLC in the solvent system of chloroform, methanol, acetic
acid, formic acid, water (at a volume ratio of 70:30:12:4:2). The TLC plate was exposed to a phosphorimaging plate and the relative amounts
of phospholipids were determined by densitometry scanning (BioRad
software, Quantity One, version 4.11). Results are expressed as the
average of at least three replicates to control for variable loading
and extraction efficiency.
Lipidation of a newly synthesized apoA-I can occur both
intracellularly during transport from the endoplasmic reticulum
to the cell surface and peri-cellularly through lipid efflux. To document the relative contribution to the lipidation observed, we
compared endogenously synthesized apoA-I and exogenously added apoA-I.
Primary hepatocytes were isolated from 4-6-month-old mice by liver
collagenase perfusion, and isolated cells were cultured on
fibronectin-coated plates in the presence of serum-free media (Hepatozyme). The following day, cells were infected with either an
adenoviral construct encoding human apoA-I (AdAI) or luciferase (AdLuc) for 1 h, washed, and then returned to Hepatozyme media. The next day, the cells were washed and then incubated with fresh Hepatozyme media in the absence or presence of exogenous hapoA-I for
3.5 h. This time period was chosen to allow sufficient secretion of hapoA-I for analysis, and yet minimize peri-cellular interactions. We chose a concentration of exo apoA-I that approximated the amount of
hapoA-I secreted during the same time period. The hapoA-I-containing lipoproteins in the media were then analyzed by a number of methods. The adenoviral vector was selected specifically to ensure apoA-I synthesis and secretion independent of experimental factors.
Electrophoretic Migration of hapoA-I-containing
Lipoproteins--
The electrophoretic migration on agarose gels of
hapoA-I newly secreted from primary hepatocytes (hereafter referred to
as endogenously synthesized apoA-I or "endo apoA-I") or exogenously added hapoA-I (referred to as "exo apoA-I") was assessed (Fig. 1). Endo apoA-I and exo apoA-I from
apoA-I-deficient mice were both found to have exclusively pre- ApoA-I Is Found in Different Lipoprotein Pools--
To evaluate
the lipoprotein size distribution of apoA-I secreted by the hepatocytes
expressing hapoA-I, the medium was concentrated and immediately
fractionated by FPLC and analyzed. The distribution of immunoreactive
human apoA-I and murine apoB (both apoB100 and apoB48) in the FPLC
fractions were analyzed by slot blot. Immunoreactive hapoA-I segregated
into 3 well separated peaks (Fig.
2A). The largest
apoA-I-containing lipoproteins eluted at a position previously calibrated for VLDL (fractions 10-14). This fraction also overlapped with the largest peak of murine apoB-containing lipoproteins (fractions 9-13; data not shown). The second peak of immunoreactive
apoA-I-containing lipoproteins eluted at the position of
HDL2/3 (fractions 19-23) and the third corresponded to
lipid-poor VHDL and apoA-I (fractions 25-29). The results presented
are representative of three separate experiments.
The distribution of immunoreactive apoA-I after FPLC separation of
medium lipoproteins shows that endo apoA-I and exo apoA-I form
lipoproteins of similar sizes ranging from VLDL/LDL to VHDL (see Fig.
2, A and B, and the distribution obtained with
control hepatocytes in Fig. 3,
A and B). Furthermore, when exo apoA-I was added
to the medium of hepatocytes infected with AdAI, the amount of label
associated with apoA-I was additive (data not shown). This result
clearly indicates that lipidation occurs both peri-cellularly and
intracellularly.
ABCA1+/+ control and ABCA1 ApoA-I in the HDL2/3 Pool Is Heterogeneous in
Size--
The different apoA-I-containing lipoprotein populations
generated by the primary hepatocytes and separated by FPLC (VLDL, HDL,
and VHDL) were further analyzed by non-denaturing PAGGE and Western
blot analysis (Fig. 4). Similar amounts
of immunoreactive hapoA-I were loaded from each lipoprotein pool. The
same lipoprotein pool for different hepatocyte samples was similarly
concentrated and loaded. The endo apoA-I present in VLDL (lane
1), HDL2/3 (lane 2), and VHDL fractions
(lane 3) are well separated from one another. The
HDL2/3 and lipid-poor apoA-I yield distinct bands, which
are compatible with the known formation of lipoproteins with varying numbers of apoA-I and with varying degrees of lipidation. A large amount of hapoA-I is secreted as HDL2/3-sized species (Fig.
2A), with a significant size heterogeneity, which in this
pool can reach 10.4 nm (Fig. 4, lane 2). The three
lipoprotein fractions from the media of AdLuc-infected hepatocytes
incubated with exo apoA-I (Fig. 2B) were also analyzed by
non-denaturing PAGGE: VLDL (lane 4), HDL (lane
5), and VHDL (lane 6). Interestingly, in comparison to
endo apoA-I, lipidation of exo apoA-I produced profiles of similarly as
well as differently sized hapoA-I-containing lipoproteins (comparing
lanes 2 and 5 and 3 and 6).
This suggests differences in how endo apoA-I and exo apoA-I
lipoproteins are speciated and lipidated; it also indicates that our
experiments distinguish between lipidation associated with secretion
and efflux. Furthermore, when hepatocytes infected with AdLuc and
incubated with exo apoA-I were incubated with 9-cis-retinoic
acid, a retinoid X receptor ligand, the resulting hapoA-I-containing
lipoproteins, VLDL (lane 7), HDL (lane 8), and
VHDL (lane 9), were similar to control exo apoA-I fractions.
An increased amount of larger-sized HDL particles is evident,
suggesting that 9-cis-retinoic acid can enhance lipidation of exogenous hapoA-I. The 9-cis-retinoic acid effect was not
observed with endo apoA-I (data not shown).
Non-denaturing PAGGE and Western blot analysis were performed on the
hapoA-I-containing lipoprotein fractions generated by ABCA1+/+ control
and ABCA1 Distribution of apoA-I and Phospholipids in the Lipoprotein
Fractions Separated by FPLC--
From the apoA-I-deficient
hepatocytes, the calculated relative distribution of endo apoA-I in the
different lipoprotein fractions is shown in Fig.
6A. Interestingly, ~20% of
the total endo apoA-I secreted was found in HDL2/3-sized
fractions. As well, a smaller but significant percentage of secreted
apoA-I was also found associated with the VLDL pool. This result is in
good general agreement with previous results in monkey hepatocytes (4)
and in HepG2 cells, although the latter do not secrete VLDL and
therefore have no apoA-I-containing lipoproteins in this lipoprotein
size (27, 35).
The association of [3H]choline phospholipids with hapoA-I
in the three-lipoprotein pools was estimated by immunoprecipitation of
hapoA-I under native conditions. Equal volumes of the pooled FPLC
lipoprotein fractions (identified as VLDL, HDL2/3, and VHDL in Fig. 2) were immunoprecipitated with an anti-hapoA-I antibody raised
in sheep. An anti-hapoB antibody also raised in sheep, which does not
cross-react with murine apoB, was used and subtracted as nonspecific
binding. The results show that although the majority of secreted
hapoA-I is in the lipid-poor fraction (Fig. 2), a significant amount of
the phospholipid associated with apoA-I (16.8%) is in the
HDL2/3 lipoprotein pool (Fig. 6B). Therefore, this demonstrates that apoA-I can be secreted with significant quantities of phospholipid, which is consistent with the size and
heterogeneity of hapoA-I in the HDL2/3 pool as determined by 4-20% non-denaturing PAGGE (Fig. 4).
Lipoproteins formed by lipidation of exo apoA-I were analyzed in the
same manner. Fractions corresponding to VLDL, HDL, and VHDL were pooled
and immunoprecipitated with antibodies against hapoA-I (Fig.
6C) and the radioactivity associated with apoA-I measured as
described above (Fig. 6D). A smaller proportion of apoA-I
(6.7%) was found in the HDL fraction for exo apoA-I compared with endo
apoA-I. A significant amount (11.9%) of the
[3H]choline-labeled phospholipids were associated with
the HDL2/3 pool. Therefore, this demonstrates that apoA-I
can acquire significant quantities of phospholipid peri-cellularly,
which is consistent with the size and heterogeneity of hapoA-I in the
HDL2/3 pool as determined by 4-20% non-denaturing PAGGE
(Fig. 4).
Levels of hapoA-I and [3H]choline phospholipids found in
the different lipoprotein fractions of ABCA1+/+ control and ABCA1
[3H]choline-labeled phospholipids were extracted,
separated by TLC, and quantified. For both endo apoA-I and exo apoA-I,
over 90% of the [3H]choline label was found in
phosphatidylcholine species with the remainder in sphingomyelin species
(data not shown). For a more complete evaluation of all phospholipid
species, hepatocytes were incubated with 32P-phosphate to
label all phospholipids, as described under "Experimental Procedures." 32P-labeled phospholipids sphingomyelin
(SPM), phosphatidylcholine (PC), phosphatidylinositol (PI),
phosphatidylserine (PS), and phosphatidylethanolamine (PE) were
separated by TLC and quantified by densitometry scanning. PC
constituted between 85-90% of the total phospholipid, whereas SPM,
PE, PI, and PS contributed the additional 10-15%. There were no
significant differences between the ratio of the minor phospholipid
species associated with endo apoA-I and exo apoA-I for apoA-I ( ABCA1 Contributes to the Lipidation of Nascent apoA-I-containing
Lipoproteins--
Hepatocytes infected with AdAI or AdLuc were labeled
with [3H]choline and release of
3H-phospholipids into the medium after a 3.5 h
incubation were measured. Hepatocytes were incubated with
9-cis-retinoic acid and glyburide to alter ABCA1 activity
and evaluate the role of ABCA1 in the lipidation of endo apoA-I or exo
apoA-I. The result shown is the mean (± S.D.) of four experiments,
each performed in triplicate. In AdAI-infected hepatocytes,
9-cis-retinoic acid did not significantly alter the amount
of 3H-phospholipid (Fig. 8)
associated with hapoA-I, but glyburide treatment resulted in a modest
but consistent decrease in the lipidation of newly secreted apoA-I. In
general, neither 9-cis-retinoic acid nor glyburide showed a
remarkable effect on lipidation of endo apoA-I. AdLuc-infected cells
were incubated for the 3.5-h period with 50, 15, 5, or 2.5 µg/well of
exogenously added hapoA-I and released 3H-phospholipids
were measured. Varying amounts of exo apoA-I were added to investigate
the effect of increasing amounts of hapoA-I protein on lipidation.
Addition of 9-cis-retinoic acid resulted in a significant
increase in lipidation of hapoA-I, whereas glyburide treatment resulted
in a more modest but significant decrease in hapoA-I-associated
lipidation (Fig. 8). Increasing amounts of exo apoA-I increased the
total labeled phospholipid associated with hapoA-I, but did not change
the ratio of VLDL/HDL/VHDL-associated lipids, nor the effects of
9-cis-retinoic acid and glyburide (data not shown). Results
shown are the mean (± S.D.) of four independent experiments at 5 µg
of exo apoA-I/well and are typical of results with varying
concentrations of exo apoA-I.
The goals of the present study were to characterize the
contribution of secretion and efflux pathways to the lipidation of apoA-I by hepatocytes and determine the role of hepatocyte ABCA1 in
these pathways. ABCA1 deficiency did not affect the net secretion of
apoA-I expressed by the adenoviral construct. Therefore the concentrations of apoA-I, whether endogenously expressed or exogenously added, allow the direct comparison of the relative distribution of
apoA-I and phospholipids in the different lipoprotein fractions. Overall, ABCA1 deficiency significantly reduced the proportion of
apoA-I found in the HDL fraction, reduced the proportion of lipid
associated with the HDL fraction, and significantly reduced the total
3H-phospholipids released from hepatocytes, but not the
amount of apoA-I secreted, compared with control hepatocytes. We found that HDL formed by endogenously synthesized apoA-I was more profoundly reduced by ABCA1 deficiency than that formed by exogenously added apoA-I. Therefore, ABCA1 contributes to lipidation of apoA-I not only
by the well documented extracellular efflux mechanism (9-12), but
also, and more significantly, to the lipidation of newly secreted apoA-I. ABCA1 has been shown to be present in intracellular
compartments, specifically early endosome and late endosome
compartments (36). Our results suggest that intracellular ABCA1 may
contribute to the lipidation of newly synthesized apoA-I, although
current evidence only correlates lipid efflux with ABCA1 activity at
the cell surface (36). In the liver, ABCA1 contributes significantly to
lipidation of hepatic apoA-I, although we do not know exactly the
physiological contribution of liver ABCA1 to circulating plasma HDL
levels. Macrophages have been shown to contribute only a minor
proportion of HDL (22) and it is likely that liver ABCA1 may provide a larger proportion. There are also tissue-specific transcripts of ABCA1
that may provide alternative regulation of ABCA1 in different tissues
(18, 19, 22, 37). Although there has been much speculation and indirect
evidence for a major contribution of hepatic ABCA1 to circulating
levels of HDL, the present study provides direct evidence for the role
of ABCA1 in the liver.
Our study also highlighted significant differences between lipidation
of apoA-I by secretion and efflux mechanisms. Comparison of HDL and
VHDL fractions formed by exo apoA-I and endo apoA-I to each other in
apoA-I-deficient (Fig. 4) and ABCAI-deficient hepatocytes (Fig. 5)
showed distinct size profiles, as secretion and efflux pathways
generated similar but non-identical HDL and VHDL species. Agarose gel
electrophoresis and phospholipid analysis demonstrated that the charge
and composition of HDL species formed by endo apoA-I and exo apoA-I
were unchanged, despite the difference in the distribution profile.
Thus, HDL formed by endogenous apoA-I synthesis and secretion and by
exogenous apoA-I and efflux mechanisms are subtly different, and may
involve different pathways and proteins necessary to lipidate apoA-I.
This assertion is reinforced by the observation that exo apoA-I was
sensitive to stimulation by 9-cis-retinoic acid (Figs. 4 and
8), whereas endo apoA-I was relatively insensitive.
9-cis-retinoic acid is a ligand for the RXR, and RXR can
heterodimerize with peroxisome proliferator-activated receptor
(PPAR)- Glyburide has been documented to be an inhibitor of ATP-binding
cassette transporters, including ABCA1, and can inhibit lipid efflux
effectively in fibroblasts, endothelial cells and macrophages (11, 12,
45, 46). Glyburide was surprisingly ineffective in hepatocytes.
Plasma HDL and apoA-I levels are reduced to practically undetectable
levels in Tangier disease due to the hypercatabolism of poorly
lipidated apoA-I. It has been documented that apoA-I secretion is not
affected in Tangier disease subjects and in an animal model of Tangier
disease (Wisconsin Hypo-Alpha Mutant (WHAM) chicken) (47-52).
In our model system, we also observed no decrease of
adenoviral-mediated apoA-I expression in the ABCA1-deficient hepatocytes compared with control hepatocytes. Instead, ABCA1 deficiency significantly reduced the amount of HDL formed by impairing the lipidation of apoA-I. However, there is still a significant basal
level of lipidation of apoA-I in the absence of ABCA1, for both endo
apoA-I and exo apoA-I. If our model system is an accurate depiction of
hepatic apoA-I lipidation, it is unclear if this degree of impairment
of lipidation by the isolated hepatocyte would be sufficient to
generate an HDL/apoA-I profile of Tangier disease. Possibly, defective
HDL lipidation in other cells or within the circulation could
contribute to the phenotype. The source of the residual lipidation
remains unknown. It is possible that other ABC transporters could be
playing a minor role in hepatocytes. A retroendocytotic mechanism may
also be involved (53). We have discovered a novel apoA-I-binding site
on the extracellular matrix of macrophages (54) and
hepatocytes2 involved in
ABCA1-mediated efflux, but, as yet, we have not attributed any specific
physiological role to this binding site. Hepatocytes also secrete apoE,
but the mechanism of lipidation of newly secreted apoE remains unclear
with evidence for and against an ABCA1-independent mechanism (55-58).
In any case, our study demonstrates the existence of an
ABCA1-independent lipidation pathway.
The absence of Hepatocytes (primary and transformed) and enterocytes are the
physiological models used to study apoA-I secretion. Thrift et
al. (61) studied lipoprotein secretion from transformed HepG2 (hepatocyte) cells into serum-free medium. Similar to the results found
in this study, analysis of concentrated HepG2 cell medium by PAGGE
revealed a broad immunoreactive-apoA-I band between 7.1-12.2 nm with
the majority of apoA-I in the lipid-free or lipid-poor form (< 8 nm).
However, HepG2 cells do not secrete normal VLDL-sized particles, and
not surprisingly no apoA-I was detected in this lipoprotein pool (61).
In our study, between 3-8% of total apoA-I and 0.8-2.4% of total
3H-phospholipids associated with the VLDL fraction. ABCA1
deficiency did reduce, but not significantly, endo apoA-I and
phospholipid associated with VLDL, but not exo apoA-I. We intend to
evaluate further the nature of apoA-I association with VLDL secreted
from hepatocytes from control and ABCA1-deficient mice. We will also analyze the role of ABCA1 in the secretion of VLDL and its associated apolipoproteins, in view of the increase in VLDL noted in patients with
Tangier disease. Cynomolgus monkey hepatocytes in culture also secreted
nascent apoA-I particles (4). Unlike the results presented here (Fig.
4), very little heterogeneity in secreted apoA-I was observed, even
after 3 d in culture. The results from that study are, however,
difficult to interpret due to the extended period of time that secreted
apoA-I was in the medium and perhaps due to the lack of sensitivity in
the assay required for detection of minor subpopulations of apoA-I.
Similar to the role of microsomal triglyceride transfer protein
in apoB secretion, hepatocytes and enterocytes might also express
proteins that facilitate the lipidation of newly secreted apoA-I, such
as ABCA1. Therefore, it is difficult to interpret studies from 3T3
cells (62), polarized Madin-Darby canine kidney cells (63), and
mouse C127 cells (64), which have each been transfected with apoA-I
cDNAs and found to secrete lipid-poor apoA-I-containing particles.
Furthermore, most of these studies isolated apoA-I by
ultracentrifugation rather than non-denaturing techniques that do not
dissociate apolipoproteins. A recent study by Chisholm et
al. (27) examined the lipidation state of newly secreted apoA-I
from HepG2 cells. The authors concluded that ~20% of newly secreted
apoA-I is lipidated intracellularly and another 30% is minimally
lipidated shortly thereafter extracellularly. In our study, the most
physiologically relevant cell model, primary hepatocyte, was used to
address nascent HDL secretion, and care was taken to separate the
secreted lipoproteins by non-denaturing techniques, such as FPLC on
calibrated Superdex 200 columns and immunoprecipitation. With this
approach, a significant amount (~20% of total) of hapoA-I is found
secreted as mature-sized HDL particles with pre- The discovery of a major role for ABCA1 in phospholipid and cholesterol
efflux to nascent HDL or lipid-poor apoA-I has established a renewed
interest in the processes by which apoA-I is lipidated. Most reports
have studied ABCA1 in peripheral cells (i.e. fibroblasts and
macrophages) and shown that this transporter is responsible for efflux
of cholesterol and phospholipid to apoA-I. Neufeld et al.
(65) demonstrated that an expressed ABCA1-GFP fusion protein was
localized to the basolateral surface of WIF-B cells, a polarized
hepatocyte cell line, and stimulated apoA-I-mediated cholesterol
efflux. Overexpression of ABCA1 in transgenic mice led to increased
circulating HDL levels, although expression was not targeted
specifically to the liver (66). A major role of hepatocyte ABCA1 in
apoA-I lipidation agrees with the reports of ABCA1 being most abundant
in the liver (17-19). Recently, Basso et al. (67)
demonstrated that injection of a liver-targeted adenoviral ABCA1
construct into C57Bl/6 mice resulted in increased apoA-I-mediated
cholesterol efflux and HDL-cholesterol concentration. Our study is the
first to evaluate the role of hepatocyte ABCA1 in the process of apoA-I lipidation.
In conclusion, we demonstrated that intracellular and peri-cellular
lipidation of apoA-I represent distinct and additive pathways that may
be regulated independently. Hepatocyte expression of ABCA1 is central
to the lipidation of newly synthesized apoA-I, but an ABCA1-independent
pathway may also contribute to the lipidation of apoA-I. Our future
work will focus on the lipidation of apoA-I by cholesterol and the
potential regulation of lipidation of apoA-I by hepatic cholesterol levels.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7.1 nm diameter) were carefully separated.
Aliquots (200 µl) from each fraction were analyzed for apoA-I by
Western blot analysis following transfer to nitrocellulose with a slot
blot apparatus (BioRad Bio-Dot SF unit) as described previously (28).
No background signal could be detected as indicated by analysis of
medium collected from hepatocytes infected with the AdLuc. The relative
distribution of apoA-I in the VLDL, HDL2/3, and VHDL pools
was determined by densitometric scanning (BioRad software, Quantity
One, version 4.11). For comparison, the relative distribution of murine
apoB (apoB48 and apoB100) was also measured using a polyclonal
anti-mouse apoB antibody (BIODESIGN International, Kennebunk, ME) and
visualized by chemiluminescence (Pierce West Pico SuperSignal
substrate, Pierce) after incubation with horseradish
peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
migration, and no
-migrating immunoreactive apoA-I band appeared
even with prolonged exposure. This is in contrast with a previous study
in monkey hepatocytes (4), where apoA-I-containing lipoproteins were
found to segregate into two pre-
- and one
-migrating fractions.
Similarly, lipoproteins formed by hepatocytes from ABCA1
/
mice endo
apoA-I or exo apoA-I (Fig. 1) possessed only pre-
migration. This
result demonstrates that all HDL formed, whatever the source, have
similar pre-
electrophoretic mobility, and thereby lack a
significant hydrophobic core.
View larger version (51K):
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Fig. 1.
ApoA-I-associated lipoproteins have
pre- migration. Medium samples from endo
apoA-I and exo apoA-I from apoA-I (
/
) and ABCA1 (
/
) hepatocytes
were applied to an agarose gel and electrophoresed. Proteins were
transferred to nitrocellulose and probed with anti human apoA-I. Human
plasma HDL and lipid-free apoA-I act as standards for
-migrating and
pre-
-migrating lipoproteins.
View larger version (25K):
[in a new window]
Fig. 2.
FPLC separates apoA-I-associated lipoproteins
into three lipoprotein classes. After the 3.5-h incubation,
medium samples were immediately loaded on two calibrated Superdex
200 columns connected in series. VLDL/LDL eluted in the void volume
(fractions 9-15), HDL2/3 eluted between fractions 17-23,
and lipid-poor apoA-I/VHDL eluted between fractions 24-33. Results are
presented as the percent of human apoA-I present in each fraction.
A, endo apoA-I. B, exo apoA-I. Distribution
profiles presented are typical of at least three separate
experiments.
View larger version (30K):
[in a new window]
Fig. 3.
Buoyant lipoprotein classes are reduced in
ABCA1-deficient hepatocytes. Medium samples were separated by FPLC
as in the legend to Fig. 2. VLDL/LDL eluted in the void volume
(fractions 8-13), HDL2/3 eluted between fractions 14-19,
and lipid-poor apoA-I/VHDL eluted between fractions 19-27.
A, endo apoA-I. B, exo apoA-I. ABCA1+/+ control
(dotted line) and ABCA1 /
(solid line) are
superimposed on the same graph and demonstrate the redistribution of
apoA-I for both endo apoA-I and exo apoA-I. Distribution profiles
presented are typical of at least three separate experiments.
/
mouse hepatocytes were also infected
with AdAI or AdLuc and then analyzed by FPLC for size separation of the
endo apoA-I- and exo apoA-I-containing lipoproteins. Comparing the FPLC
profiles of hapoA-I-containing lipoproteins from control and
ABCA1-deficient hepatocytes, a reduction can be seen in the proportion
of hapoA-I found in the buoyant VLDL and HDL fractions for both endo
apoA-I (Fig. 3A) and exo apoA-I (Fig. 3B). These results demonstrate the important contribution of ABCA1 to both intracellular and peri-cellular lipidation of apoA-I.
View larger version (28K):
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Fig. 4.
Endo apoA-I and exo apoA-I in
apoA-I-deficient hepatocytes produce similar but non-identical HDL and
VHDL species. Lipoproteins from endo apoA-I or exo apoA-I from
apoA-I-deficient hepatocytes were separated by FPLC and analyzed by
non-denaturing 4-20% polyacrylamide gradient gel electrophoresis,
transferred to nitrocellulose, and probed for human apoA-I.
Lipoproteins from endo apoA-I, VLDL (lane 1), HDL
(lane 2), and VHDL (lane 3), were compared with
lipoproteins from exo apoA-I, VLDL (lane 4), HDL (lane
5), and VHDL (lane 6). Lipoproteins obtained from
hepatocytes treated with 9-cis-retinoic acid (abbreviated
9-cis RA in the figure), VLDL (lane 7), HDL
(lane 8), and VHDL (lane 9), were also compared.
9-cis-retinoic acid increased the amount of hapoA-I found in
larger, more buoyant HDL species. Standards are indicated on the
figure.
/
hepatocytes (Fig. 5). VLDL
(lane 1), HDL (lane 2), and VHDL (lane
3) from control hepatocytes incubated with exo apoA-I are
identical to VLDL (lane 7), HDL (lane 8), and
VHDL (lane 9) from exo apoA-I in ABCA1
/
hepatocytes.
Similarly, for endo apoA-I, the VLDL (lane 4), HDL
(lane 5), and VHDL (lane 6) for the control
hepatocytes were very similar to the VLDL (lane 10), HDL
(lane 11) and VHDL (lane 12) from ABCA1
/
hepatocytes. We know that the quantity of hapoA-I found in VLDL and HDL
fractions is significantly reduced in ABCA1
/
hepatocytes (Fig. 3,
A and B), but, importantly, the nature of the
lipoprotein particles formed is unchanged.
View larger version (32K):
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Fig. 5.
Lipoproteins from ABCA1+/+ and ABCA1 /
hepatocytes are qualitatively similar. Lipoproteins from endo
apoA-I or exo apoA-I from ABCA1 control or ABCA1-deficient hepatocytes
were separated by non-denaturing 4-20% polyacrylamide gradient gel
electrophoresis, transferred to nitrocellulose and probed for human
apoA-I. Lipoproteins from exo apoA-I for ABCA1+/+ control: VLDL
(lane 1), HDL (lane 2), and VHDL (lane
3); or ABCA1-deficient hepatocytes: VLDL (lane 7), HDL
(lane 8), and VHDL (lane 9), showed identical
bands. Similarly, lipoproteins from endo apoA-I for ABCA1+/+ control:
VLDL (lane 4), HDL (lane 5), and VHDL (lane
6); and ABCA1-deficient hepatocytes: VLDL (lane 10),
HDL (lane 11), and VHDL (lane 12), were also very
similar. Standards are indicated on the figure. Human HDL was included
for size comparison.
View larger version (30K):
[in a new window]
Fig. 6.
Endo apoA-I forms a substantial amount of
HDL. Medium samples were separated by FPLC. ApoA-I and
3H-phospholipid were determined from each lipoprotein
fraction, and endo apoA-I and exo apoA-I are compared for apoA-I /
hepatocytes. A, endo apoA-I in the VLDL, HDL, and VHDL
lipoprotein fractions was quantified, and results are presented as the
mean percent of total human apoA-I for three separate experiments (± S.D.). B, 3H-phospholipid associated with endo
apoA-I for each lipoprotein fraction was determined, and results are
presented as mean percent of total 3H-phospholipid secreted
(± S.D.). C, exo apoA-I in the VLDL, HDL, and VHDL
lipoprotein fractions was quantified, and results are presented as the
mean percent of total human apoA-I for three separate experiments (± S.D.). D, 3H-phospholipid associated with exo
apoA-I for each lipoprotein fraction was determined, and results are
presented as mean percent of total 3H-phospholipid secreted
(± S.D.).
/
hepatocytes were also quantified (Fig.
7). There was a significant decrease
(82%; p < 0.05) in endo apoA-I associated with the
HDL in ABCA1
/
hepatocytes compared with control hepatocytes (Fig. 7A). A decrease in HDL-associated
3H-phospholipids was also demonstrated, although to a
lesser extent (35%, p < 0.10; Fig. 7B).
There was also a significant decrease (63%, p < 0.05;
data not shown) in total hapoA-I-associated
3H-phospholipids released from the ABCA1
/
hepatocytes
compared with control hepatocytes. There was a slight reduction of
apoA-I and 3H-phospholipid found in the VLDL fraction of
ABCA1
/
hepatocytes compared with control hepatocytes, which did not
reach statistical significance. For exo apoA-I, there was a decrease
(65%, p < 0.10) in association of exo apoA-I with the
HDL fraction for ABCA1
/
hepatocytes compared with control
hepatocytes (Fig. 7C), with a smaller decrease (42%,
p < 0.07; Fig. 7D) in HDL-associated 3H-phospholipids. Total hapoA-I-associated
3H-phospholipids released were also decreased (25%,
p < 0.10; data not shown). Importantly, the total
amount of hapoA-I secreted by AdAI-infected control and ABCA1
/
hepatocytes was the same, demonstrating that ABCA1 deficiency did not
impair secretion of apoA-I (ABCA1 (+/+) control = 1.33 ± 0.22 µg/h/well; ABCA1 (
/
) = 1.33 ± 0.11 µg/h/well).
View larger version (37K):
[in a new window]
Fig. 7.
HDL protein and lipid are reduced in ABCA1
deficiency. ApoA-I and 3H-phospholipid were determined
from each lipoprotein fraction, and ABCA1+/+ control hepatocytes were
compared with ABCA1 /
hepatocytes. A, endo apoA-I in the
VLDL, HDL, and VHDL lipoprotein fractions was quantified, and results
are presented as the mean percent of total human apoA-I for three
separate experiments (± S.D.). B,
3H-phospholipid associated with endo apoA-I for each
lipoprotein fraction was determined, and results are presented as mean
percent of total 3H-phospholipid secreted (± S.D.).
C, exo apoA-I in the VLDL, HDL, and VHDL lipoprotein
fractions was quantified, and results are presented as the mean percent
of total human apoA-I for three separate experiments (± S.D.).
D, 3H-phospholipid associated with exo apoA-I
for each lipoprotein fraction was determined, and results are presented
as mean percent of total 3H-phospholipid secreted (± S.D.).
/
),
ABCA1 (+/+) control, and ABCA1 (
/
) hepatocytes (data not shown).
This result is a critical demonstration that despite the differences in
the amount of hapoA-I or [3H]choline phospholipids
present in the lipidated buoyant fractions, the nature and composition
of the lipoproteins are unchanged in the absence of ABCA1.
View larger version (43K):
[in a new window]
Fig. 8.
Treatment of apoA-I /
hepatocytes with
9-cis-retinoic acid increases association of
3H-phospholipid with exo apoA-I but not endo apoA-I.
Hepatocytes were treated with 9-cis-retinoic acid (12 h
prior to and including the 3.5-h incubation) or glyburide (during the
3.5-h incubation). Then hapoA-I was immunoprecipitated from the medium,
and associated 3H-phospholipid was determined. Each
condition was performed in triplicate and normalized to 100% for the
no additions condition. The results shown here are the mean
of four separate experiments (± S.D.).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, -
, and -
, liver X receptor-
and -
, and farnesoid X receptor (and others) to regulated many genes involved in
lipid metabolism. Liver X receptor (38-40), PPAR
(41),
PPAR
(41, 42), and PPAR
(43) in complex with RXR have been
documented to increase ABCA1 expression in macrophages but not
hepatocytes. There are many targets of RXR regulation that may affect
apoA-I lipidation (44). We do not wish to comment on the mode of RXR activation, but rather use 9-cis-retinoic acid as a tool to
differentiate between lipidation of endo apoA-I and exo apoA-I. Our
results show that ABCA1 deficiency most strongly affects lipidation of endo apoA-I (Fig. 7), whereas 9-cis-retinoic acid strongly
affects only lipidation of exo apoA-I. One possible explanation may be that lipidation of endo apoA-I is maximal in the wild-type hepatocytes, i.e. limited by lipid availability (Fig. 7A), but
lipidation of exo apoA-I is not (Fig. 7C), which allows the
stimulatory effect of 9-cis-retinoic acid to be observed.
There exist other possibilities, and we are currently investigating the
mechanism of 9-cis-retinoic acid stimulation.
-migrating HDL assessed by agarose gel
electrophoresis, reflects the absence of a significant hydrophobic core. Our results3 show that free
cholesterol associates with apoA-I-containing lipoproteins and
lecithin:cholesterol acetyltransferase is known to be secreted
by the hepatocytes (59, 60). The question is raised as to why
-migrating HDL is not formed. Even extended incubations of apoA-I
with hepatocytes (24 h, data not shown) were not sufficient to generate
a hydrophobic core. Another factor necessary for HDL maturation may not
be present on hepatocytes but possibly on other cell types
(i.e. peripheral cells).
migration, in
accordance with the work of Chisholm et al.
(27).
![]() |
ACKNOWLEDGEMENT |
---|
We thank Ruth McPherson for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a group grant from the Canadian Institutes of Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ Supported by a postdoctoral scholarship from the Heart and Stroke Scientific Research Corporation of Canada.
** Supported by a postgraduate scholarship from the Heart and Stroke Foundation of Canada.
To whom correspondence should be addressed: Lipoprotein and
Atherosclerosis Research Group, University of Ottawa Heart Inst., Rm.
H460, 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Canada. Tel.: 613-761-5255; Fax: 613-761-5281; E-mail: ymarcel@ottawaheart.ca.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M300137200
2 J. W. Burgess, M. D. Wang, R. S. Kiss, and Y. L. Marcel, unpublished observations.
3 H. Zheng, M. D. Wang, R. S. Kiss, V. Franklin, E. M. Rubin, and Y. L. Marcel, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HDL, high density lipoprotein; ABCA1, ATP binding cassette transporter A1; Ad5, adenovirus serotype 5; AdAI, Ad5 adenoviral construct expressing human apolipoprotein A-I; AdLuc, Ad5 adenoviral construct expressing firefly luciferase; apo, apolipoprotein; endo, endogenously synthesized; exo, exogenously added; FPLC, fast protein liquid chromatography; hapoA-I, human apolipoprotein A-I; LDL, low density lipoprotein; PAGGE, polyacrylamide gradient gel electrophoresis; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; SPM, sphingomyelin; TLC, thin layer chromatography; VHDL, very high density lipoprotein; VLDL, very low density lipoprotein..
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