The Enhanced Association of Apolipoprotein E With Apolipoprotein B–Containing Lipoproteins in Serum-Stimulated Hepatocytes Occurs Intracellularly
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.
- Received June 9, 1994.
- Accepted February 24, 1995.
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 α-migrating to pre–β- and β-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.
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.
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.
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 α-migrating lipoproteins (Fig 1A⇓, lanes 3 and 4), and there was no detectable apoE associated with large, pre–β-migrating lipoproteins (Fig 1A⇓, lanes 1 and 2). However, when cells were incubated with medium supplemented with 10% FBS, apoE-rich pre–β-migrating bands became visible (Fig 1B⇓, lanes 1 and 2). The amount of apoE associated with α-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).
To determine whether the redistribution of apoE from α- to pre–β- and β-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.
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.
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.
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).
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%.
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.
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 α-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 β band. This particle resembles the recently described γ-migrating, apoE-rich HDL identified in both mouse plasma and in the medium of hepatoma cells.18 Although the function of the γ-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.
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.
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