The Recycling of Apolipoprotein E in Primary Cultures of Mouse Hepatocytes

EVIDENCE FOR A PHYSIOLOGIC CONNECTION TO HIGH DENSITY LIPOPROTEIN METABOLISM*

Monica H. FarkasDagger §, Larry L. SwiftDagger , Alyssa H. Hasty||, MacRae F. Linton||**DaggerDagger, and Sergio FazioDagger ||DaggerDagger

From the Departments of Dagger  Pathology, || Medicine, and ** Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received for publication, August 6, 2002, and in revised form, December 18, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Internalization of apoE-containing very low density protein (VLDL) by hepatocytes in vivo and in vitro leads to apoE recycling and resecretion. Because of the role of apoE in VLDL metabolism, apoE recycling may influence lipoprotein assembly or remnant uptake. However, apoE is also a HDL protein, and apoE recycling may be related to reverse cholesterol transport. To investigate apoE recycling, apoE-/- mouse hepatocytes were incubated (pulsed) with wild-type mouse lipoproteins, and cells and media were collected at chase periods up to 24 h. When cells were pulsed with VLDL, apoE was resecreted within 30 min. Although the mass of apoE in the media decreased with time, it could be detected up to 24 h after the pulse. Intact intracellular apoE was also detectable 24 h after the pulse. ApoE was also resecreted when cells were pulsed with HDL. When apoA-I was included in the chase media after a pulse with VLDL, apoE resecretion increased 4-fold. Furthermore, human apoE was resecreted from wild-type mouse hepatocytes after a pulse with human VLDL. Finally, apoE was resecreted from mouse peritoneal macrophages after pulsing with VLDL. We conclude that 1) HDL apoE recycles in a quantitatively comparable fashion to VLDL apoE; 2) apoE recycling can be modulated by extracellular apoA-I but is not affected by endogenous apoE; and 3) recycling occurs in macrophages as well as in hepatocytes, suggesting that the process is not cell-specific.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Apolipoprotein E (apoE)1 is a 299 amino acid protein that is a constituent of all plasma lipoproteins with the exception of the smallest low density lipoproteins (LDL) (1). After secretion, apoE readily distributes among lipoproteins within the plasma compartment and plays a role in receptor-mediated clearance of these particles (2-5). In addition to its extracellular role as a ligand, apoE has several intracellular functions. It modulates intracellular lipid metabolism (6, 7), promotes cholesterol efflux from macrophages (8), plays a role in the routing of internalized lipoprotein remnants (9, 10), and is involved in the assembly (11, 12) and secretion (13, 14) of very low density lipoproteins (VLDL). Finally, apoE is a major HDL protein and may have a role in HDL formation, maturation, and hepatic uptake (1, 15-17). Based on the multiple and critical roles for apoE in the metabolism of lipids and lipoproteins, it has been hypothesized that apoE may follow unique pathways of secretion and internalization, allowing it to have maximum impact on cellular functions.

Recent studies from our laboratory have given support to this hypothesis and have shown that a portion of apoE internalized on triglyceride-rich lipoproteins is spared degradation and is resecreted (18, 19). Radiolabeled apoE was found associated with nascent hepatic Golgi lipoproteins after injecting [125I]VLDL into C57BL/6 mice (18). We also found apoE with nascent Golgi lipoproteins recovered from the livers of apoE-/- mice reconstituted with bone marrow from C57BL/6 mice (19). Furthermore, primary cultures of hepatocytes from these transplanted apoE-/- mice secreted apoE, a clear indication that internalized apoE can survive degradation and find its way out of the cell. Studies from other laboratories have also shown that apoE internalized on triglyceride-rich lipoproteins by rat liver, hepatoma cell lines, or fibroblasts is not completely degraded and is resecreted (20-22). The results from these studies establish the presence of a unique intracellular processing pathway for apoE-containing triglyceride-rich lipoproteins leading to recycling.

Although the evidence supporting apoE recycling is convincing, little is known regarding the cell biology and physiologic relevance of the process. It is not clear whether apoE recycles from specific lipoprotein particles or whether recycling is dependent upon the entry pathway into the cell. Furthermore, the possibility that the recycling process can be modulated has not been explored. To address these questions, we have developed a model utilizing primary hepatocytes from apoE-/- mice and a pulse-chase protocol using unlabeled lipoproteins from wild-type mice. Using this model, we demonstrate apoE recycling when either VLDL or HDL is the source of the apoprotein. In addition, we report that resecretion of apoE is increased by the presence of apoA-I in the chase media. Using hepatocytes from wild-type mice, we demonstrate that apoE recycles from human VLDL and that apoE is recycled in the presence of endogenous apoE production. Finally, we show that apoE recycles from mouse peritoneal macrophages, demonstrating that recycling is not specific for the liver cell.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mice-- ApoE-deficient mice (apoE-/-) on the C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, ME). ICR mice were purchased from Harlan (Indianapolis, IN). All of the mice were kept on a 12-h light/dark cycle and were fed a normal mouse chow diet (RP5015, PMI Feeds Inc., St. Louis, MO). Food and water were available ad libitum. All of the animal procedures were carried out in accordance with institutional guidelines with approval from the Animal Care Committee of Vanderbilt University.

Isolation and Culture of Primary Mouse Hepatocytes-- Hepatocytes were isolated from apoE-/- and ICR mice as described previously (19). Cells were plated onto 6-well (35 mm) BioCoat mouse collagen IV-coated dishes (BD Biosciences) at a density of 4.5 × 105 viable cells/ml in low glucose Dulbecco's modified Eagle's medium (Invitrogen) containing 1% bovine serum albumin, 0.8 mM oleate, 0.167 µg/ml insulin (4 milliunits/ml), 0.02 µg/ml dexamethasone, 100 units of penicillin, and 100 µg of streptomycin/ml. This medium was used for isolation and plating of hepatocytes in all of the experiments and did not contain serum to avoid introduction of exogenous apoE into the system.

Isolation of Plasma Lipoproteins-- Mice were anesthetized using a ketamine/xylazine mixture, and blood was collected from the inferior vena cava. EDTA was added to each sample to a final concentration of 12.5 mM. Plasma lipoprotein fractions (d < 1.019 g/ml and d 1.100-1.210 g/ml) were isolated and washed using TLA 100.4 and 120.2 rotors in an Optima TLX Tabletop Ultracentrifuge (Beckman). Protein concentrations were determined by the BCA method (Pierce) using bovine serum albumin as standard and modified to eliminate interference by lipid (23). Apoprotein composition of the fractions was determined by SDS-PAGE as described above. Gels were stained with 0.01% Coomassie Brilliant Blue (5% acetic acid, 50% methanol), destained (5% acetic acid, 7.5% methanol), and dried.

Human VLDL was isolated at d < 1.019 g/ml from healthy donors and washed as described above. The protocol was approved by the Vanderbilt Institutional Review Board, and informed consent was obtained prior to obtaining blood. Media d < 1.006 g/ml lipoproteins for the degradation experiment were isolated using the same rotors as described above.

Hepatocyte Recycling Experiments-- Primary hepatocytes were isolated and cultured as described above. After overnight culture (12-16 h), fresh media supplemented with apoE-containing lipoproteins (d < 1.019 g/ml or d 1.10-1.21 g/ml) were added and cells were pulsed for 2 h. Medium was aspirated, and hepatocytes were washed three times with ice-cold phosphate-buffered saline (PBS), incubated with heparin (10 mg/ml in PBS) for 3 min, and washed two times with ice-cold PBS. Fresh medium without lipoproteins was added, and hepatocytes were chased for 0, 30, 60, 120, and 240 min and 24 h. In some experiments, purified human apoA-I was added to the chase media (25 µg/ml, Calbiochem). Medium was collected at each time period, and cells were washed with PBS. The PBS wash was added to the medium for analysis of resecreted apoE. Cells were collected by the addition of TriZOL reagent (Invitrogen). Protease inhibitors were added to the medium, and samples were stored at -20 °C until analysis. TriZOL-treated cells were stored at -80 °C until analysis.

Macrophage Recycling Studies-- Peritoneal macrophages were collected from apoE-/- mice 3 days after an intraperitoneal injection of thioglycollate. Cells were plated onto 6-well dishes at 3 × 106 cells/well in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics. The following day cells were washed and pulsed with media containing 7.7 µg of VLDL collected from C57BL/6 mice or control media with no VLDL. After 2 h, the cells were washed four times with PBS and 1.2 ml of fresh media containing 5% lipoprotein-deficient serum was placed on the cells. At 0, 10, 30, 60, 120, and 240 min and 24 h after placement in pulse media, cells were washed twice with 400 µl of PBS, and the pulse media and washes were pooled. Cells were collected by addition of TriZOL reagent, and protein was prepared as described below.

Isolation of Lipoproteins from Cell Cultures-- Lipoproteins were adsorbed from the media using Liposorb (PHM-L Liposorb, Calbiochem) as described previously (19). The recovery efficiency of medium apoE by Liposorb was determined to be ~70% with equal adsorption of lipoprotein classes (data not shown). Adsorbed proteins were separated by SDS-PAGE as described below.

Analysis of Cellular Proteins-- Cellular proteins were isolated using TriZOL reagent per the manufacturer's instructions. Protein pellets were dried under nitrogen gas, 1% SDS was added, and samples were incubated at 60 °C for 50 min with vigorous vortexing every 10 min. Unsolubilized material was pelleted, and the supernatant was used for SDS-PAGE.

SDS-PAGE and Western Blotting-- Proteins were separated using the NuPage system (Invitrogen) with 4-12% bis-tris gels and MOPS-SDS buffer. Samples were solubilized in lithium dodecyl sulfate sample buffer. Proteins were transferred to nitrocellulose using the NuPage system. Membranes were blocked with 5% nonfat dry milk, incubated with primary antibodies, washed extensively, and incubated with horseradish peroxidase-conjugated secondary antibodies (Promega). The primary antibody to mouse apoE was produced by the Protein Immunology Core of the Clinical Nutrition Research Unit at Vanderbilt University. The anti-human apoE antibody was purchased from Medical and Biological Laboratories International Corporation (Watertown, MA). Protein bands were visualized using enhanced chemiluminescence (Amersham Biosciences). Films were scanned using a Bio-Rad GS700 imaging densitometer, and relative amounts of protein were estimated using Molecular Analyst software.

Degradation Experiment-- Hepatocytes from wild-type ICR and apoE-/- mice were isolated and cultured in Dulbecco's modified Eagle's medium containing bovine serum albumin and oleic acid as described above. After overnight culture, medium was collected from ICR hepatocytes, and the d < 1.006 g/ml fraction was isolated. The lipoprotein fraction was incubated with conditioned media (24 h) from apoE-/- hepatocytes in the presence or absence of protease inhibitors in a 35-mm dish at 37 °C. Aliquots were collected at 0, 8, 16, and 24 h, and lipoproteins were adsorbed using Liposorb. Proteins were analyzed by SDS-PAGE and Western blotting.

Analysis of ApoE on HDL and VLDL Particles-- Primary hepatocytes from apoE-/- mice were isolated and cultured as described above. After overnight culture, medium was removed and the cells were incubated in fresh medium for 1 or 2 h. HDL (150 µg/ml) isolated from ICR mice was added to the 1-h and 2-h conditioned media, and the samples were incubated at 37 °C for 1 and 2 h, respectively. Lipoprotein fractions (d < 1.019 g/ml, d > 1.019 g/ml) were isolated by centrifugation for 3 h at 120,000 rpm in the 120.2 rotor. Aliquots of the d < 1.019 g/ml and d > 1.019 g/ml were diluted in 1 ml of PBS. Lipoproteins were adsorbed using Liposorb, proteins separated by SDS-PAGE, blotted to nitrocellulose, and probed for apoE.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A Coomassie Brilliant Blue stained gel of typical VLDL and HDL preparations is shown in Fig. 1. ApoE was clearly present in the VLDL fraction (lane 2), and there was no albumin contamination. The HDL fraction (lane 4) also contained apoE and only trace amounts of albumin.


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Fig. 1.   Apoprotein composition of mouse plasma lipoproteins. Mouse plasma was isolated as described under "Experimental Procedures." Apoproteins (10 µg) from each lipoprotein fraction (d < 1.019 g/ml, d 1.10-1.21 g/ml) were separated by SDS-PAGE, stained with Coomassie Brilliant Blue, destained, and dried. Lanes 1, 3, and 5, molecular weight markers; lane 2, d < 1.019 g/ml; lane 4, d 1.10-1.21 g/ml.

To demonstrate apoE recycling in the in vitro model, hepatocytes from apoE-/- mice were incubated with VLDL for 2 h. Cells and media were collected after various chase periods up to 24 h and analyzed for the presence of apoE by Western blotting (Fig. 2). ApoE was present in the media at all time points (Fig. 2A). Densitometric analyses of the films showed that approximately equal amounts of apoE are present in the media in the 30-, 60-, and 120-min samples. ApoE was seen in the cells for up to 240 min in this experiment (Fig. 2B). An analysis of cellular apoE showed that one-half of the total uptake (0 min) of apoE is present in the cells at the 30-min time point. Cellular apoE decreased over the course of the experiment, although the change was less dramatic than from 0 to 30 min. In some experiments, apoE was found inside the cells at 24 h. To exclude the possibility that the decrease in apoE in the media resulted from extracellular degradation, conditioned medium (24 h) from apoE-/- hepatocytes was incubated with apoE-containing VLDL for 0, 8, 16, and 24 h. As shown in Fig. 2C, there was no significant decrease in apoE with time, indicating that extracellular degradation does not play a role in the reduced appearance of apoE at later time points in the recycling experiments.


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Fig. 2.   Resecretion of apoE from primary hepatocytes from apoE-/- mice using triglyceride-rich lipoproteins. Hepatocytes were isolated from apoE-/- mice and cultured as described under "Experimental Procedures." Cells were pulsed for 2 h with apoE-containing d < 1.019 g/ml lipoproteins (40 µg/ml) isolated from ICR mice. Hepatocytes were chased in media without lipoproteins for different times. Media and cells were collected at each time point. Media lipoproteins were adsorbed using Liposorb, and cell protein was isolated using TriZOL. Proteins were separated by SDS-PAGE, blotted to nitrocellulose, and probed for apoE. Films were analyzed by densitometry, and apoE was expressed as relative amount present at each time point. A, media. B, cells. Films show a representative experiment. Densitometric analyses are shown as mean ± S.E., n = 3 experiments. C, conditioned media (24 h) from apoE-/- hepatocytes were incubated at 37 °C with d < 1.006 g/ml lipoproteins from wild-type cells in a 35-mm culture dish in the presence or absence of protease inhibitors. Aliquots were removed at 0, 8, 16, and 24 h. Lipoproteins were adsorbed with Liposorb, and proteins were separated by SDS-PAGE, blotted to nitrocellulose, and probed for apoE. M, stock media; E-/-, no VLDL added. ser, serum; pi, protease inhibitors.

To determine whether apoE recycling was unique for triglyceride-rich lipoproteins, pulse-chase experiments were carried out using apoE-containing HDL. The results of these experiments are shown in Fig. 3. ApoE appeared in the media up to 240 min after the pulse with relatively similar amounts present at each chase period (Fig. 3A). Cellular apoE decreased during the 30-min incubation by almost 50% and then remained constant through 240 min. There was little apoE remaining within the cell at the 24 h period (Fig. 3B). Because apoE-/- hepatocytes secrete large apoE-deficient VLDL and because apoE can easily exchange from HDL to VLDL, consideration was given to the possibility that recycling apoE in the HDL experiments was actually internalized as part of VLDL particles. To test this possibility, HDL was incubated in conditioned media from apoE-/- hepatocytes. The d < 1.019 g/ml and d > 1.019 g/ml fractions were isolated by density gradient ultracentrifugation, and equal aliquots from each lipoprotein fraction were separated by SDS-PAGE. The results from the Western blot show that the apoE is contained almost exclusively in the d > 1.019 g/ml fraction (Fig. 3C).


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Fig. 3.   Resecretion of HDL-derived apoE from primary mouse hepatocytes. Hepatocytes were isolated from apoE-/- mice and cultured as described under "Experimental Procedures." Cells were pulsed for 2 h with apoE-containing d 1.10-1.21 g/ml lipoproteins (150 µg/ml) isolated from ICR mice and chased in media without lipoproteins for 0, 30, 60, 120, 240 min, and 24 h. Media and cells were collected at each time point. Cells were harvested using TriZOL. Media (A) and cells (B) were treated as described in Fig. 2. Densitometric analyses are the mean ± S.E. of two experiments. C, conditioned media was collected from apoE-/- hepatocytes after 2 h. HDL (150 µg/ml) was added to each sample and incubated at 37 °C for 0, 1, and 2 h, respectively. Lipoprotein fractions were isolated by ultracentrifugation, treated with Liposorb, and solubilized for SDS-PAGE and immunoblotting as described in "Experimental Procedures." Lanes 1, 3, and 5, d < 1.019 g/ml; lanes 2, 4, and 6, d >1.019 g/ml; lane 7, serum. E-/-, no VLDL added; ser, serum.

To determine whether apoE resecretion is enhanced by the presence of extracellular acceptors of cholesterol, apoA-I was added to the chase media after pulsing the cells with VLDL (Fig. 4). The presence of apoA-I increased the resecretion of apoE by ~4-fold at 30, 60, 120, and 240 min (Fig. 4A). The amount of apoE recovered in cells chased for 30 min in the absence of apoA-I was greater than that found in hepatocytes chased in the presence of apoA-I (Fig. 4B). With longer chase periods, the mass of apoE in the cells was similar regardless of whether the cells were chased in the presence or absence of apoA-I.


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Fig. 4.   Effect of apoA-I on the resecretion of apoE. Hepatocytes were isolated from apoE-/- mice and cultured as described under "Experimental Procedures." Cells were pulsed for 2 h with apoE-containing d < 1.019 g/ml lipoproteins (35 µg/ml) isolated from ICR mice and chased in media without lipoproteins in the presence or absence of purified human apoA-I (25 µg/ml) for different times. Media (A) and cells (B) were treated as described in Fig. 2. Films show a representative experiment. Densitometric analyses are shown as mean ± S.E., n = 3 experiments. E-/-, no VLDL added; ser, serum.

Because the previous experiments utilized hepatocytes from apoE-/- mice, we also investigated apoE recycling in wild-type liver cells to determine whether this phenomenon is driven or modulated by endogenous apoE. To investigate apoE recycling in the presence of endogenous apoE production, hepatocytes from ICR mice were incubated in the presence or absence of human d < 1.019 g/ml lipoproteins for 2 h and chased in media without lipoproteins for 0 and 30 min. Resecretion of human apoE was monitored using an antibody specific to human apoE. Human apoE appeared in the media during the 30-min chase (Fig. 5). ApoE was also present in the cells at both the 0 and 30 min time points (Fig. 5).


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Fig. 5.   Resecretion of human apoE from primary mouse hepatocytes. Hepatocytes were isolated from ICR mice and cultured as described under "Experimental Procedures." Cells were pulsed for 2 h in the presence or absence of apoE-containing human d < 1.019 g/ml lipoproteins (40 µg/ml). Hepatocytes were chased in media without lipoproteins for 0 and 30 min. Media and cells were collected at each time point and treated as described for Fig. 2. Results are representative of three experiments. ser, serum.

To determine whether the process of apoE recycling is specific for the hepatocyte, peritoneal macrophages from apoE-/- mice were pulsed with apoE-containing VLDL from wild-type mice. Cells and media were collected after various chase periods up to 24 h and analyzed for the presence of apoE by Western blotting (Fig. 6). ApoE was present in the media at all time points and was present in the cells up to 24 h after the pulse.


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Fig. 6.   Resecretion of apoE from mouse macrophages. Peritoneal macrophages from apoE-/- mice were collected, pulsed for 2 h with apoE-containing d < 1.019 g/ml lipoproteins, and chased for various times with fresh Dulbecco's modified Eagle's medium. Media lipoproteins were adsorbed using Liposorb, and cell protein was isolated using TriZOL. Proteins were separated by SDS-PAGE, blotted to nitrocellulose, and probed for apoE. C, control samples from cells with no VLDL added.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ApoE recycling has been explored using a new model in which hepatocytes from apoE-/-mice are pulsed with apoE-containing lipoproteins and chased in the absence of lipoproteins. Using this model, we have shown that apoE recycles when either VLDL or HDL is the source of the apoprotein. Furthermore, we show that resecretion of apoE is enhanced when apoA-I is included in the chase medium, providing the first evidence that the recycling process can be modulated and that it is related to HDL metabolism. Finally, we demonstrate recycling of human apoE in the presence of endogenous apoE production. In addition, we provide the first evidence that the recycling of exogenous apoE also occurs in peritoneal macrophages from apoE null mice.

These studies demonstrate clearly that apoE internalized with VLDL is spared degradation within hepatocytes and is then resecreted (Fig. 2). The apoprotein appears in the media within 30 min of instituting the chase, suggesting that recycling occurs rapidly. With time, there is a decrease in the amount of apoE found in the media. This decrease does not result from extracellular degradation (Fig. 2C) but presumably reflects intracellular degradation. We hypothesize that recycling is a continual process in which apoE is internalized and then either targeted for degradation or routed back out of the cell. With each cycle, a portion of apoE is degraded, thus explaining the reduction of apoE in the media over time. Clearly, the amount of apoE within the cell decreases with time, but a fraction of the protein can remain intact for up to 24 h. It is unclear whether this intracellular apoE represents a pool of apoE retained within a specific storage compartment or whether it represents a fraction of apoE that is continually recycled.

Previous studies from our laboratories (18, 19) as well as from other investigators (20-22) have provided evidence that apoE recycles when presented to cells on triglyceride-rich particles. Our current study is the first to demonstrate that apoE also recycles when HDL is the source of the apoprotein (Fig. 3). Pulse-chase studies with apoE-/- hepatocytes and mouse HDL led to the appearance of apoE in the media. Because apoE is an exchangeable apoprotein and apoE-/- hepatocytes secrete large triglyceride-rich VLDL, we were concerned that apoE may exchange from HDL to newly synthesized VLDL, which in turn was internalized, leading to apoE recycling. However, the incubation of apoE-containing HDL with conditioned media from apoE-/- hepatocytes demonstrated that apoE remains associated almost exclusively with the HDL fraction (Fig. 3C). Thus, apoE was not recycling from VLDL but was indeed recycling from HDL. The pathway for HDL-apoE recycling may or may not be the same as that for VLDL-apoE recycling. In fact, there may be multiple recycling pathways based on entry point into the cell. Importantly, the discovery that apoE recycles even when associated with HDL adds significantly to our knowledge of this process, as it indicates that this phenomenon is not specific for triglyceride-rich lipoproteins and may be physiologically correlated to reverse cholesterol transport.

ApoE mediates endocytosis of lipoprotein particles by binding to the LDL receptor (2, 24) and the LDL receptor-related protein (LRP) (3, 25). ApoE may also be internalized by binding to heparan sulfate proteoglycans with (4, 26) or without (5, 27) binding to the LRP. ApoE-containing HDL has been shown to be taken up by the LDL receptor (1, 17) LRP (15) and heparan sulfate proteoglycans (16). The cell entry of apoE through these different pathways may result in alternate trafficking routes with different subcellular destinations and different recycling times. Studies by Rensen et al. (22) suggest that apoE internalized on VLDL by the LDL receptor is spared degradation and is resecreted, whereas Heeren et al. (20) provide evidence that LRP may be the predominant recycling pathway of apoE on triglyceride-rich lipoproteins. We have shown previously that apoE recycles in the absence of the LDL receptor (18). Although these studies do not exclude recycling of apoE through the LDL receptor, they suggest that there may be alternate recycling pathways. Our working hypothesis is that apoE recycles because it binds more tightly to its receptor than it does to the lipoprotein at the point of pH drop in the endosome. In this scenario, apoE could engage the LDL receptor, LRP, or heparan sulfate proteoglycans from the surface of either VLDL or HDL. Conversely, given the significant functional difference between VLDL and HDL in delivering lipid cargo to the liver cell, one could postulate that when HDL docks to the membrane via SR-BI (28), apoE diffuses away from the particle and becomes membrane-bound and internalized as such or as part of an incoming remnant lipoprotein. Clearly additional studies are required to determine whether the point of entry into the cell is important for the intracellular routing and recycling of apoE.

The addition of apoA-I to the chase medium resulted in a 4-fold increase in the appearance of apoE in the media (Fig. 4), suggesting an effect on either the rate or mass of apoE recycling. The mechanism through which apoA-I enhances the recycling of apoE is unknown. It is interesting to speculate that it may be related to cholesterol efflux from the cell as both apoE and apoA-I have been shown to play crucial roles in this process. ApoE has been shown to promote cholesterol efflux from the cell (8), whereas apoA-I serves as an acceptor for cholesterol efflux (29, 30) in a process probably mediated through the ATP-binding cassette A1 (ABCA1) pathway (31-33). In addition, there is evidence that binding of HDL/apoA-I to the cell surface mobilizes cholesterol from intracellular pools to the cell surface (34, 35) where the lipid could be transported out of the cell via ABCA1. We speculate that the addition of apoA-I to the media mobilizes intracellular cholesterol and activates the ABCA1 pathway that in turn stimulates apoE resecretion. Huang et al. (36) have presented evidence that cholesterol efflux from macrophages mediated by endogenous apoE production is not dependent on ABCA1; however, this does not preclude a role for recycling apoE in cholesterol efflux via the ABCA1 pathway. On the other hand, Remaley et al. (37) demonstrated that HeLa cells transfected to express ABCA1 had increased the efflux of cholesterol and phospholipid to apoE, suggesting a role for ABCA1 in apoE-mediated cholesterol efflux. Rees et al. (38) demonstrated that apoA-I stimulates the secretion of apoE by macrophages but concluded that apoA-I primarily stimulated the secretion of newly synthesized apoE with little effect on a cell surface pool. However, it should be noted that stimulation of apoE secretion was seen only after 8 h of incubation with apoA-I. In contrast, we found a 4-fold increase in apoE resecretion within 30 min, suggesting that recycling apoE may respond more rapidly to extracellular signals than does newly synthesized apoE.

Our studies also demonstrate the recycling of human apoE internalized on triglyceride-rich particles by wild-type mouse hepatocytes (Fig. 5). These results are significant for two reasons. They provide preliminary evidence suggesting that apoE recycling may be an important process in humans and show that recycling is not an aberrant process induced by the absence of apoE. However, it is possible that the recycling pathways for apoE in the presence and absence of endogenous apoE production are not the same.

Finally, our studies demonstrate that apoE recycling is not limited to hepatocytes but occurs in macrophages as well. This finding is important in light of the role of apoE in cellular cholesterol homeostasis and the reverse cholesterol transport mentioned above (8, 35), as macrophages trapped in the arterial intima would benefit from the potential to recycle apoE to maintain adequate cholesterol efflux. Analyses of the influence of HDL and apoA-I on macrophage apoE recycling will provide further insight into this important process.

In summary, our studies demonstrate that apoE recycling is not limited to uptake via triglyceride-rich lipoproteins but occurs after internalization on HDL particles as well. This finding suggests that there may be multiple entry pathways into the cell that lead to recycling and proposes a possible role for recycling apoE in HDL metabolism. The finding that apoA-I enhances apoE recycling suggests that the process can be modulated and may provide a key to understanding its physiologic relevance. Because apoA-I increases cholesterol efflux from the cell through an ABCA1-mediated mechanism and because ABCA1 plays an obligatory step in HDL metabolism, it is possible that apoE recycling is linked to reverse cholesterol transport both by serving as a signaling mechanism for HDL cholesterol entry into the cell and by increasing cholesterol efflux out of the cell via an ABCA1-mediated mechanism. Whereas the former mechanism might be at play in the hepatocyte, it is tempting to speculate that the latter is used by the macrophage to control intracellular cholesterol homeostasis.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL68114, HL57986, and HL65709. The Protein Immunology Core that developed the antibody to apoE is part of the Clinical Nutrition Research Unit (DK26657).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.

§ Supported by National Institutes of Health Vascular Biology Training Grant HL07751 and a predoctoral fellowship awarded by the American Heart Association Southeast Affiliate.

To whom correspondence may be addressed: Dept. of Medicine, 315 Preston Research Bldg., Vanderbilt University School of Medicine, Nashville, TN 37232-6300. Tel.: 615-936-1450; Fax: 615-936-1872; E-mail: Sergio.Fazio@Vanderbilt.edu, MacRae.Linton{at}Vanderbilt.edu, or Larry.Swift{at}Vanderbilt.edu.

Dagger Dagger Established Investigator of the American Heart Association.

Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M208026200

    ABBREVIATIONS

The abbreviations used are: apo, apolipoprotein; VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; LRP, LDL receptor-related protein; ABCA1, ATP-binding cassette A1; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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