© 1995 American Heart Association, Inc.
Articles |
The Enhanced Association of Apolipoprotein E With Apolipoprotein B–Containing Lipoproteins in Serum-Stimulated Hepatocytes Occurs Intracellularly
From the Gladstone Institute of Cardiovascular Disease (S.F.), Cardiovascular Research Institute, University of California, San Francisco, and the Lipid and Lipoprotein Research Group and Department of Biochemistry (Z.Y.), University of Alberta, Edmonton, Canada.
Correspondence to Sergio Fazio, MD, PhD, Division of Endocrinology, Vanderbilt University, School of Medicine, Medical Center North, AA 4206, Nashville, TN 37232-2250. E-mail fazios@ctrvax.vanderbilt.edu.
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Abstract |
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Abstract Synthesis and secretion of VLDL in HepG2 cells are stimulated by several lipogenic factors, including serum. We previously found that the amount of apolipoprotein (apo) E associated with large lipoproteins such as VLDL increased under conditions of stimulated lipogenesis. The present study was designed to determine if the increased apoE association with VLDL occurs intracellularly or after secretion. In addition to HepG2, we studied rat hepatoma McA-RH7777 cells for production of endogenous rat apoE and transfected human apoE3. In both hepatoma cell lines stimulation of lipogenesis and production of large apoB-containing lipoproteins by serum resulted in increased apoE association with these particles and in decreased apoE association with HDL without affecting the total apoE output. Although evidence of apoE redistribution was seen among lipoproteins in the media, the apoE newly secreted under conditions of stimulated lipogenesis mainly associated with apoB-containing lipoproteins at the expense of its association with HDL. However, this effect was not attributable to reduced HDL lipid and apoA-I mass. Finally, redistribution of apoE from HDL to apoB-containing lipoproteins was also observed intracellularly in both HepG2 and transfected McA-RH7777 cells expressing human apoE3. These data suggest that the redistribution of apoE from HDL to apoB-containing lipoproteins upon activated lipogenesis in hepatoma cells occurs intracellularly and is not attributable to a decrease in HDL production.
Key Words: apoE • HepG2 • rat hepatoma • apoB • lipoprotein secretion
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Introduction |
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Human apolipoprotein (apo) E is a 299-residue protein that is found in several classes of plasma lipoproteins, including hepatic apoB-100–containing VLDL.1 2 3 In the human hepatoma cell line HepG2, enhanced association of apoE with large, apoB-containing lipoproteins occurs when cellular lipogenesis and VLDL production are stimulated.4 The increased apoE association with the enhanced production of triglyceride-rich VLDL particles also occurs when rat primary hepatocytes are used.5 6 However, although the secreted apoB-containing lipoproteins are enriched in apoE upon stimulation of lipogenesis, the mechanism regulating the synthesis and secretion of apoE appears to be independent of that regulating synthesis and secretion of apoB.4 Conditions that stimulate total apoB secretion almost fivefold do not change the secretion of total apoE.4 The enhanced association of apoE with large lipoproteins reflects a redistribution of the secreted apoE from HDL to the large, triglyceride-rich VLDL.4 An important question is whether the redistribution of apoE occurs intracellularly during lipoprotein assembly or extracellularly after apoE secretion.
Unlike apoB, which is extremely hydrophobic and does not exchange among different lipoprotein particles, apoE can exchange.7 Because of this ability to exchange, it has been difficult to ascertain whether apoE is secreted with the larger lipoproteins or associates with these particles after secretion. Moreover, such studies are complicated by the introduction of exogenous lipoproteins when cells are cultured in the presence of serum. We have shown that the changes in apoE distribution among lipoproteins in HepG2 media do not result from postsecretion redistribution to serum lipoproteins.4 However, the possibility has not been excluded that apoE might associate with the increased amount of VLDL secreted by HepG2 cells after secretion. In addition, it is unknown whether the redistribution of apoE from HDL to VLDL reflects a physical preference of apoE for larger lipoprotein particles during intracellular assembly or is attributable to a reduction in HDL production.
In this study we sought to determine if the intracellular association
of apoE with newly formed VLDL is enhanced upon stimulated lipogenesis.
To this end, we monitored both the association of apoE with newly
formed VLDL and the distribution of apoE among secreted lipoproteins in
the media of two hepatoma cell lines, human HepG2 and rat McA-RH7777
cells, before and after stimulation with serum. Our data demonstrate
that apoE distribution changes from 
-migrating to pre–ß- and
ß-migrating lipoproteins upon the activation of VLDL secretion and
that the enhanced apoE association with apoB-containing lipoproteins
occurs intracellularly rather than after secretion. Additionally, the
stimulation of VLDL production is not accompanied by reduced HDL
cholesterol or HDL apoA-I levels, suggesting that the decreased
association of apoE with HDL is not a consequence of a decreased
production of HDL. Moreover, we observed the same phenomenon of apoE
association with large lipoproteins upon the stimulation of lipogenesis
in stably transfected McA-RH7777 cells that overexpress human
apoE3.
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Methods |
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Materials
Culture media were obtained from Life Technologies, Inc. Fetal bovine serum (FBS) and horse serum were obtained from HyClone. [35S]Methionine (Tran 35S-label) was obtained from ICN Biomedicals, Inc. Amplify, [14C]acetate, and ECL western blotting kits were from Amersham/Searle. The Superose 6 column (Pharmacia Fine Chemicals) was used on a Gilson fast-performance liquid chromatography system. Rabbit polyclonal antibodies against human apoE, rat apoA-I, or rat apoE were gifts from Dr K. Weisgraber of the Gladstone Institute of Cardiovascular Disease, San Francisco, Calif.
Cell Culture
HepG2 cells4 and McA-RH7777 cells8 9
were obtained from American Type Culture Collection (ATCC) and cultured
under standard conditions. Briefly, HepG2 cells (ATCC HB 8065) were
grown in Eagle's minimum essential medium with nonessential amino
acids, sodium pyruvate, and 10% FBS. McA-RH7777 cells (ATCC CRL 1601)
were cultivated in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% FBS and 10% horse serum. Both cell lines were
maintained by replacing the medium every other day, and the cells were
split 1:10 at confluence. Generation and characterization of stable
transformants of McA-RH7777 cells that overexpress human apoE3 have
been described.10 Cells were grown in T75 flasks to
approximately 70% confluence prior to experiments.
Chromatography of Cell Media
After a 15-hour incubation, conditioned media (10 mL) were
collected in the presence of 1 mmol/L phenylmethylsulfonylfluoride
(PMSF), concentrated by using Centricon filters (Amicon) to a final
volume of 200 µL, and injected onto a Superose 6 column to separate
lipoproteins.4 For western blot and immunoprecipitation
assays, fractionated media samples were analyzed either individually or
after pooling the major lipoprotein classes that had been separated by
column chromatography. In this system lipoproteins eluted in fractions
16 through 33. Fractions 16 through 20 were pooled as VLDL, 21 through
24 as IDL, 25 through 29 as LDL and large HDL (HDL1), and
30 through 33 as HDL. The pooled samples were again concentrated by
using Centricon filters and analyzed using sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) or agarose gel
electrophoresis assays. To determine the recovery of apoE from media
after chromatography, all lipoprotein-containing fractions (16 through
33) were pooled, concentrated, and separated by SDS-PAGE. Comparisons
were made with the apoE levels in equivalent amounts of media before
chromatography.
Western Blot Analysis
The concentrated Superose 6 fraction samples were
electrophoresed on either a 12% polyacrylamide gel to separate
apolipoproteins or an agarose gel to resolve
lipoproteins.4 10 After electrophoresis proteins were
transferred to a nitrocellulose membrane and probed with rabbit
antibodies specific to human apoE, rat apoE, or human apoA-I; the
secondary antibody was anti-rabbit antiserum raised in goat. The
immunoreactive bands were visualized by using a chemiluminescent
reaction (ECL) after exposing the x-ray film for 1 to 15 seconds.
Metabolic Labeling and Immunoprecipitation
Cells were incubated with [35S]methionine (100
µCi/dish) for 2 hours in methionine-free medium. The conditioned
media (10 mL) were concentrated to 200 µL by using Centricon filters
and fractionated on a Superose 6 column.4 The 0.5-mL
fractions were mixed with polyclonal antibodies to precipitate human or
rat apoE.4 10 In the reuptake experiments cells were
incubated with [35S]methionine in the presence or absence
of serum for 4 hours. These conditioned media containing labeled
proteins were extensively dialyzed against 0.15 mol/L NaCl and 0.01
mmol/L EDTA, mixed 1:1 with fresh medium, added to new McA-E3 cells
(stable transformants of McA-RH7777 cells that overexpress human apoE3)
in 60-mm dishes, and incubated for 1, 2, and 16 hours. The media
collected at different times were adjusted to a density of 1.063 g/mL
by adding solid KBr and subjected to ultracentrifugation. Lipoproteins
in the d<1.063 g/mL fraction were concentrated with fumed
silica (Sigma Chemical Co) and subjected to SDS-PAGE followed by
fluorography.4
Preparation of Microsomes From Cultured Hepatocytes
For analysis of intracellular lipoproteins, cells were
incubated for 2 hours in the absence or presence of 10% FBS and 10%
horse serum. Cells from one T75 flask were washed with
phosphate-buffered saline containing 0.2% albumin, scraped off the
flask, collected with 3 mL phosphate-buffered saline, and resuspended
in 1 mL homogenization buffer (10 mmol/L Tris, pH 7.4, 250 mmol/L
sucrose, 100 mmol/L leupeptin, 0.5 mmol/L PMSF, and 1 mmol/L
dithiothreitol). The cell suspension was sonicated 10 times for 5
seconds each by using a Branson Sonifier 450 at setting 5. The
homogenized sample was spun in a clinical centrifuge for 10 minutes to
remove unbroken cells and cell debris and then subjected to
ultracentrifugation at 90 000 rpm for 15 minutes at 4°C in a Beckman
TL100.2 rotor to collect the microsomal membranes. The membrane
fraction was resuspended in 0.5 mL sodium carbonate, pH 11.3, mixed for
1 hour at 4°C, and dialyzed overnight at 4°C against 1
mmol/L ethylenediamine. After the density was adjusted to 1.1 g/mL
with solid KBr, the sample was ultracentrifuged at 100 000 rpm for 2.5
hours at 4°C in a Beckman TL100 tabletop ultracentrifuge. The top
100-µL fraction was collected, and lipoproteins were precipitated
with fumed silica.8 Electrophoresis and subsequent western
blot analysis of the precipitated apolipoproteins were
performed.4 10
Analysis of Secreted Lipoprotein Lipids
Cells were incubated for 2 hours with [14C]acetate
(25 µCi/mL) in the presence or absence of serum. After Centricon
concentration and Superose 6 fractionation of the conditioned media,
fractions containing the major lipoprotein classes were pooled. Lipids
were extracted with chloroform/methanol (2:1) and separated by
thin-layer chromatography.4 The bands corresponding to
triglycerides, cholesteryl esters, and cholesterol were scraped off the
thin-layer chromatography plate, mixed with liquid scintillation
cocktail, and counted.
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Results |
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Redistribution of ApoE From HDL to VLDL in HepG2 Media Upon Stimulated Lipogenesis
Lipoproteins secreted into the media were separated into four major classes (VLDL, IDL, LDL/HDL1, and typical HDL) by Superose 6 column chromatography. Lipoproteins in each of the four classes were further resolved on agarose gels, and the distribution of apoE-containing lipoproteins was determined by western blot analysis (Fig 1
). When HepG2 cells were incubated
with serum-free medium for 2 hours the secreted apoE associated mainly
with the -migrating lipoproteins (Fig 1A, lanes 3 and 4), and there
was no detectable apoE associated with large, pre–ß-migrating
lipoproteins (Fig 1A, lanes 1 and 2). However, when cells were
incubated with medium supplemented with 10% FBS, apoE-rich
pre–ß-migrating bands became visible (Fig 1B, lanes 1 and 2). The
amount of apoE associated with -migrating lipoproteins in the
serum-containing medium decreased to 70% (as determined by
densitometry) compared with the serum-free medium (Fig 1B, lanes 2
through 4). It is relevant to mention that in this experimental setting
the antiserum against human apoE did not cross-react with the bovine
apoE that might be present in FBS (not shown).
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To determine whether the redistribution of apoE from - to pre–ß-
and ß-migrating lipoproteins occurred intracellularly or after
secretion, we performed experiments in which these two possibilities
could be distinguished. First, to study the distribution of apoE in the
nonstimulated state, we metabolically labeled the cells by using
[35S]methionine in the absence of serum for 2 hours. Half
of this medium was used to analyze apoE distribution under serum-free
conditions; the other half was dialyzed to remove the
[35S]methionine, mixed with fresh medium (1:5) containing
10% FBS, and reincubated with cells for an additional 2 hours. The
labeled apoE was used as a marker to detect the redistribution of
preexisting apoE to serum or HepG2 lipoproteins. The unlabeled, newly
secreted apoE was analyzed by western blotting, and its distribution
was considered representative of both intracellular association
and postsecretion redistribution. The data obtained from these
experiments are summarized in Fig 2. Panel A shows the
distribution of the secreted 35S-labeled apoE obtained from
the conditioned serum-free media. As expected, the majority of apoE was
in the HDL fractions (lanes 5 through 7). The conditioned media
containing prelabeled apoE was dialyzed to remove free
[35S]methionine, diluted with medium containing 10% FBS,
and added to HepG2 cells for another 2 hours. Because the medium
containing the prelabeled apoE was diluted, the labeled apoE was not
detectable within the short exposure time (1 to 10 seconds) necessary
for the ECL western blot. The western blot in panel C clearly
demonstrates that the newly made, nonradioactive apoE secreted from
HepG2 cells in the presence of 10% serum mainly associated with the
VLDL/IDL/LDL fractions (lanes 2 through 4). A significant decrease of
apoE association with HDL fractions was also observed (compare lanes 5
through 7 of panels C and A). Redistribution of the prelabeled apoE
among lipoproteins in the medium containing serum is shown in panel B;
the association of preexisting apoE with the VLDL/IDL/LDL secreted from
HepG2 cells increased upon the addition of serum into the medium.
However, the distribution pattern of the preexisting apoE is distinct
from the pattern of newly secreted apoE (compare panels C and B). For
example, there was no apoE peak in the VLDL/LDL fractions and no
dramatic decrease in apoE association with HDL.
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Consideration was given to the possibility that the decreased apoE
association with HDL fractions in serum-containing media was due to
reduced production of HDL by HepG2 cells. To test this possibility, we
analyzed the accumulation of 14C-labeled lipids in the
major lipoprotein classes and apoA-I in the media before and after the
addition of serum (Table and Fig 3). In
the nonstimulated condition, the major carriers for the
14C-labeled lipids were LDL-like particles (pool 2), and
very few 14C-labeled lipids were detected in the VLDL
fractions (pool 1). However, after a 2-hour incubation with serum, the
VLDL fractions showed the highest increase in 14C-labeled
lipids (
3.5-fold). Levels of HDL lipids (pool 3) also increased
slightly upon the addition of serum. Western blot analysis of
apoA-I on both agarose gels (Fig 3) and polyacrylamide gels (data not
shown) revealed no change, or in some experiments as much as a 20%
increase, in the amount of apoA-I produced by the stimulation of
lipogenesis. These combined data suggest that the observed decrease in
apoE association with HDL is not a consequence of decreased production
of HDL particles.
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Shift in Distribution of ApoE From HDL to VLDL in McA-RH7777 Cells
Upon Stimulated Lipogenesis
We then analyzed apoE secretion from rat hepatoma McA-RH7777 cells
to assess whether the pattern observed in HepG2 cells is a general
phenomenon of hepatocytes. McA-RH7777 cells synthesize and secrete
lipoproteins similar to those of normal rat plasma.11
Pulse-chase studies of apoE in the McA-RH7777 cells demonstrated that,
as in HepG2 cells, the total secretion of rat apoE was not affected by
serum (10% FBS plus 10% horse serum) (Fig 4). During a
chase time of between 5 minutes and 1 hour, the accumulation of the
pulse-labeled apoE in the McA-RH7777 medium was comparable to that
observed in HepG2 cells.4 This result indicates that the
rate of apoE secretion is unchanged in McA-RH7777 cells under
conditions known to stimulate lipogenesis and VLDL secretion.
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We next examined whether in McA-RH7777 cells supplementing the media
with serum would also result in a shift of the apoE distribution from
HDL to VLDL. The chromatographic separation of media lipoproteins
showed that in the absence of serum apoE coeluted with the HDL
fractions, whereas in the presence of serum the majority of apoE
coeluted with the VLDL fractions (Fig 5). Western blot
analysis of the pooled fractions containing the major lipoprotein
classes showed a clear increase in apoE association with VLDL upon the
addition of serum into the media (Fig 5). The antibody against rat apoE
did not cross-react with the apoE of bovine serum (not shown).
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Using stable transformants of McA-RH7777 cells that overexpress
human apoE3 (McA-E3),10 we performed experiments similar
to those described in the legends for Figs 4
and 5 and observed that
the recombinant human apoE in McA-RH7777 cells underwent the same
pattern of secretion and lipoprotein association as rat apoE in the
McA-RH7777 cells or human apoE in HepG2 cells (ie, the secretion of
transfected apoE was not stimulated by serum, but apoE redistributed
from HDL to VLDL; Fig 6). Thus, the redistribution of
apoE from HDL to large, apoB-containing lipoproteins upon stimulated
lipogenesis occurs with similar characteristics in very different
hepatoma lines; these data suggest the hypothesis that the apoE
association shift might also occur in normal liver cells as a mechanism
to enrich the VLDL with an efficient ligand for lipoprotein removal
from plasma. To demonstrate that the change in the pattern of apoE
association with lipoproteins in the presence or absence of serum was
not due to selective lipoprotein reuptake and/or selective differences
in recovery after chromatography, we performed the experiments
summarized in Figs 7 and 8. Labeled
lipoproteins from McA-E3–conditioned media, with or without FBS, were
not taken up to a significant extent by McA-E3 cells during the first 2
hours of incubation. In the absence of serum there was no appreciable
reuptake of lipoproteins even after 16 hours of incubation (Fig 7, lane
8 versus lanes 5 through 7). In the presence of serum, media apoB-100
and apoE were reduced to 53% and 46% of baseline values,
respectively, after 16 hours of incubation (lane 4 versus lanes 1
through 3). The data in Fig 7 also confirm that in the presence of
serum apoB-100 accumulates while apoE secretion is reduced relative to
serum-free incubations. Similar results showing that no significant
uptake of either apoB or apoE occurs during the first 2 hours of chase
were obtained using HepG2 cells (data not shown), indicating that our
rationale for using short-term incubations was indeed correct. The
recovery of apoE from media lipoproteins after column chromatography in
the presence or absence of serum was the same (Fig 8). Under both
conditions, the recovery of apoE from the lipoprotein-containing
fractions was greater than 90%.
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Association of ApoE With Intracellular Lipoproteins in Hepatoma
Cell Lines
The stably transfected McA-RH7777 cells expressing human
apoE3 were used as a model system with which to study the association
of apoE with microsomal lipoproteins, since high-level expression of
apoE allowed an easier detection of the protein within the cells. In
addition, we observed that overexpression of the recombinant apoE3 did
not affect the efficiency of apoB secretion, nor did it affect the
production of other endogenous rat apolipoproteins, such as apoA-I and
apoE (J. Westerlund and Z. Yao, unpublished data, 1994). Fig 9 shows the increase in apoE content in lipoproteins
isolated from the microsomes of transfected McA-RH7777 cells prepared
by using a sonication/ultracentrifugation procedure. The amount of apoE
associated with the d<1.1 g/mL microsomal lipoproteins in
serum-stimulated cells increased almost threefold compared with that in
cells cultured in serum-free conditions. In independent experiments in
which the microsomes were prepared by lysing the cells with detergents
and isolating the intramicrosomal lipoproteins (d<1.063
g/mL) by ultracentrifugation, a similar increase in apoE association
with lipoproteins following addition of serum was observed (data not
shown). Moreover, elevated apoE association with intramicrosomal
d<1.1 g/mL lipoproteins upon serum stimulation was also
observed in HepG2 cells (data not shown). These data together suggest
that the serum-stimulated increase in apoE content of large
apoB-containing lipoproteins is attributable to intracellular
association of apoE with the apoB-containing lipoproteins during the
formation of lipoproteins rather than to postsecretory association of
apoE with VLDL.
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Discussion |
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The production of hepatic VLDL is a complex process requiring the assembly of lipid and protein components synthesized in different intracellular compartments.2 It is generally believed that apoB, a 550-kD polypeptide, plays an obligatory role in the assembly of VLDL2 even though apoB synthesis alone is not sufficient for VLDL secretion from the liver. In human subjects with abetalipoproteinemia, apoB is present in the liver, but virtually no apoB-containing lipoproteins are found in the plasma.12 Additional factors other than apoB, such as the microsomal triglyceride transfer protein, appear to be involved in the assembly and secretion of apoB-containing lipoproteins.13 14 We are interested in understanding the role apoE plays in the process of hepatic VLDL formation because apoE is an important protein constituent and modulates the metabolism of these lipoproteins. We have observed that in HepG2 cells the synthesis and secretion of apoE and apoB are not regulated by the same mechanism.4 ApoB secretion increases fivefold upon stimulation of lipogenesis, but apoE secretion does not increase, nor do the apoE mRNA levels change.4 During the synthesis of hepatic VLDL, apoE associates with these lipoprotein particles, whereas when VLDL synthesis is low, apoE is secreted with small, HDL-like particles. The present work was designed to determine whether the enhanced apoE association with apoB-containing lipoproteins occurs intracellularly. We also examined rat hepatoma McA-RH7777 cells and the stably transfected McA-RH7777 cells that produce recombinant human apoE3. All the data indicate that despite some postsecretional redistribution of apoE among lipoproteins in the media, the increased apoE association with apoB-containing lipoproteins occurs intracellularly.
The finding of an intracellular shift of apoE from HDL to VLDL upon increased lipogenesis agrees with observations showing increased apoE levels in VLDL from oleate-treated (20-hour incubation) HepG2 cells15 and decreased apoE levels in VLDL from starved rat primary hepatocytes.5 6 Lipoproteins from HepG2 cells are in many ways representative of those of normal human plasma15 16 17 and have been used extensively as a system in which to study the synthesis and secretion of lipoprotein components in liver cells. Similarly, lipoproteins secreted from McA-RH7777 cells are similar to those of normal rat plasma and have been used extensively to study the synthesis and secretion of both endogenous11 and transfected8 apolipoproteins.
Both HepG2 and McA-RH7777 cells secrete small amounts of VLDL of
d<1.006 g/mL and more VLDL of a smaller kind, similar to
LDL in density (d<1.063 g/mL) but richer in
triglycerides.15 16 For these reasons, to increase the
yield of apoB-containing lipoproteins in the intracellular studies we
used density cutoff points higher than 1.006 g/mL. However, we showed
by agarose western blots that the increase in apoE in these experiments
was accounted for by the apoB-containing lipoproteins and not by the
HDL1, which may contaminate the d<1.1
and d<1.063 g/mL preparations (not shown). As is evident
from both Figs 1 and 6, the reduced association of apoE with HDL upon
incubation of the cells with serum is only partially accounted for by
the typical -migrating lipoprotein. A significant reduction in apoE
content is also seen in another HDL particle with a mobility on agarose
between the origin and the ß band. This particle resembles the
recently described 
-migrating, apoE-rich HDL identified in both
mouse plasma and in the medium of hepatoma cells.18
Although the function of the -migrating, apoE-rich HDL is not yet
established, the particle has been shown to strongly promote the efflux
of intracellular cholesterol from fibroblasts.18
We used serum to stimulate lipoprotein production in the hepatoma cells
because it is the most potent lipogenic factor that exerts a
stimulatory effect within 2 hours. An acute stimulatory effect is
important in view of the proposed role of apoE in recapture of
hepatic lipoproteins as a way of increasing the efficiency of
chylomicron remnant removal from plasma.19 20 We elected
to conduct 2-hour chase studies because the reuptake of newly
secreted apoE by cells was expected to be negligible within this time
frame. There is an absence of significant reuptake of
35S-labeled apoB from conditioned media after a 2-hour
incubation with the same McA-RH7777 cells (Z.Y., unpublished data,
1992). In this study, we show that reuptake of both apoB and apoE does
not occur to any detectable extent in McA-E3 cells during the first 2
hours of incubation (Fig 7). Similarly, 2-hour reuptake was not
detectable in HepG2 cells incubated with 35S-labeled
lipoproteins (d<1.063 g/mL) from HepG2 medium (not shown).
We interpret these data to mean that our observations of lipoprotein
and apoprotein changes at 2 hours are not due to reuptake but rather to
differences in secretion rates and patterns. In one study apoE
secretion increased twofold in HepG2 cells incubated with high
concentrations of human LDL.21 We did not observe an
increased secretion of apoE in HepG2 or McA-RH7777 cells cultured with
serum-containing media, but we cannot exclude the
possibility that a minor increase in apoE secretion may occur at
a level below the sensitivity of our western blotting and pulse-chase
assays. Nevertheless, if such an increase in apoE secretion did occur
in our experiment, it was not nearly as large as that observed for VLDL
lipids and apoB.
Despite the fact that apoE is a major protein component of Golgi VLDL
and plasma VLDL,22 it seems that apoE is not a necessary
component for VLDL assembly, nor does it need VLDL as a carrier for
secretion. In fact, individuals with apoE deficiency have apparently
normal plasma VLDL,23 and subjects with
abetalipoproteinemia12 and homozygous
hypobetalipoproteinemia,24 two diseases characterized by
extremely low VLDL production, have normal levels of plasma apoE. Thus,
it would be reasonable to speculate that the increased association of
apoE with VLDL during stimulated VLDL production reflects the physical
preference of apoE for larger particles. Such a result may possibly be
enhanced by sodium carbonate treatment of microsomal particles. It is
difficult to rule out, from a theoretical standpoint, that the
disruption of microsomes might itself be responsible for an artifactual
association of apoE with large lipoproteins. However, sodium carbonate
treatment is a widely accepted technique for studies of lipoprotein
assembly and secretion25 26 27 and has yielded important data
that support the current view of apoB assembly into lipoproteins.
Moreover, Hamilton et al28 have shown that a large portion
of the microsomal vesicles in rat hepatocytes contains nascent VLDL and
reacts positively for the presence of apoE as determined by immunogold
staining. Also, the association of apoE with VLDL does not
represent a mere phenomenon of attraction, since in human
plasma, where apoE is relatively abundant, 25% to 28% of VLDL is not
associated with apoE.29 The choice of conducting these
experiments on transfected cells is justified by the demonstration that
both pattern (Fig 6) and kinetics20 of apoE3 secretion by
McA-E3 cells are identical to those of endogenous rat apoE in
McA-RH7777 cells.
The fact that this shift of apoE from HDL to VLDL occurs intracellularly suggests that the different lipoproteins are processed in the same intracellular compartments. ApoE and probably other apolipoproteins are free to associate with and displace each other from particles of the preferred size. The ability to associate with triglyceride-rich lipoproteins is crucial for the function of apoE. ApoE is the most efficient receptor-binding ligand available to the remnants of VLDL or chylomicrons, and an enrichment in apoE is essential for the removal of these triglyceride-rich remnant particles from the circulation.7 Lipoprotein enrichment in apoE could occur at different sites: in the endoplasmic reticulum and the Golgi compartments during lipoprotein assembly; in the plasma and the extracellular space of extrahepatic tissues, where lipoproteins exchange lipids and apolipoproteins; and in the extracellular space in the liver before lipoprotein capture. The preferential distribution of one of the apoE isoforms (apoE4) and of some apoE variants to VLDL occurs in the plasma,10 30 31 and evidence indicates that apoE in the space of Disse associates with remnant lipoproteins and is involved in their capture by binding to the heparan sulfate proteoglycans on the cell surface.20 32 33 Here we have presented evidence that the VLDL also may become enriched in apoE before its secretion from the liver.
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Acknowledgments |
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Dr Yao was the recipient of a fellowship from the American Heart Association, California Affiliate. The authors wish to thank N. Torres, Y.-L. Lee, and J. McGuire for excellent technical assistance; the technical help of H. Li was crucial for the preparation of the revised manuscript. We are also indebted to Dr K. Weisgraber for all the antibodies used in this project and for helpful comments on the manuscript; Drs J. Westerlund, R. Farese, Jr, J. López, S. Rall, Jr, and R. Mahley for a critical reading of the manuscript; L. Jach and C. Deevy for illustrations; Sylvia Richmond for manuscript preparation; and D. Levy and L. DeSimone for editorial work.
Received June 9, 1994; accepted February 24, 1995.
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References |
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