Apoprotein (apo) ()E plays an important role as a ligand for receptor mediated endocytosis of triglyceride-rich lipoprotein particles(1) . ApoE is an integral component of very low density lipoproteins (VLDL), chylomicron remnants and some subclasses of high density lipoprotein. ApoE functions as a ligand for the low density lipoprotein (LDL) receptor on peripheral cells and the LDL and remnant receptor on hepatocytes(1) . In vitro, models of remnant-like emulsion particles are capable of imitating the metabolism of particles in vivo since they rapidly acquire apoE in plasma (2) and then compete with endogenous lipoproteins for receptor-mediated uptake(3, 4) .

In addition to the receptor-mediated uptake pathways, the existence of a non-apoE-specific uptake of triglyceride-rich particles by scavenger cells (e.g. macrophages) has been proposed(5) . Triglyceride-rich particle uptake in the absence of any apoproteins has also been shown in tissue culture experiments in different cells(6) .

ApoE mRNA and apoE is found intracellularly in more cell types than any other apoprotein(1, 7, 8) . In hepatocytes apoE is found intracellularly in Golgi cisternae and vesicles and in peroxisomes as well as in mitochondria(9) . Unlike most apoproteins that are synthesized solely in the liver and intestine, apoE is synthesized also in a number of peripheral tissues. Although the role of apoE in these tissues is still not clear, it has been suggested that apoE may facilitate local redistribution of lipids among cells within a tissue or play a role in intracellular lipid metabolism(9) .

In mice, lack of apoE has been generated by homologous recombinant inactivation of the apoE gene(10, 11) . These apoE ``knock out'' models were normal in weight and reproduced normally. However, significant differences in their lipid and lipoprotein profiles were observed between normal and genetically manipulated animals. Accumulation of cholesterol-rich remnants was demonstrated, and the animals developed extensive lipid loaded atherosclerotic lesions in the absence of plasma and tissue apoE(10, 11) . This suggests that uptake of lipoprotein particles into lesions can occur in the absence of apoE, and this is accompanied by abnormal cell metabolism of the internalized lipoprotein lipids.

We questioned, therefore, whether exogenous apoE would modulate cellular lipid metabolism. In this study our approach was to isolate effects of apoE on particle uptake, from effects of apoE on cellular lipid metabolism. A macrophage cell line which does not synthesize apoE but does show apoB-E receptor specificity for lipoprotein uptake was selected(12) . Effects of exogenous apoE on cellular triglyceride and cholesterol metabolism were determined by incubating in vitro generated remnant-like particles of small VLDL or IDL size with J774 macrophages. At physiologic concentrations of apoE remnant-like particles we found increases of triglyceride hydrolysis and utilization, increase of cholesteryl ester hydrolysis, and modulation of cholesterol homeostasis in the presence of exogenous apoE, and these effects were distinct from apoE-induced effects on particle uptake.

EXPERIMENTAL PROCEDURES

Materials

Emulsions

The dried lipids were resuspended in 10 ml of a preheated (60 °C) buffer (150 mM NaCl, 0.24 mM EDTA, pH 8.4, density 1.006). To remove excess phospholipid liposomes, sucrose (1 g/10 ml of buffer) was added before sonication. The cloudy suspension was sonicated for 1 h, 50 °C, at 40 watts, under a stream of nitrogen using a Bronson sonifier model 450 equipped with a horn tip. After sonication the sonicate was dialyzed against the above buffer for 16 h with three changes of dialysate to remove free sucrose.

The lipid mixture was centrifuged at 40,000 rpm, 4 °C, in a swinging bucket rotor (Beckman 50.1) for 20 min. After centrifugation, there was a separation between larger particles (white top), smaller particles (cloudy middle part), and titanium particles (gray-black bottom). The cloudy middle fraction was collected and respun at 28,000 rpm, 4 °C, for 12 h in a swinging bucket Beckman 50.1 rotor. The resulting creamy top layer containing emulsion particles was collected while the sucrose containing liposomes were trapped at the bottom. Emulsions were used for experiments within 5 days of preparation but were stable for longer periods (>10 days) as judged by column chromatography (see below).

The final emulsion triglyceride concentrations were determined by the triglyceride GPO-PAP test, the cholesteryl oleate was determined by the cholesterol/HP test. The ratio of triglyceride:cholesteryl ester was 1.07 ± 0.19:1 for the emulsions used herein (n = 11). The specific activity of the markers for the core lipids was determined by liquid scintillation counting. Emulsion phospholipid was measured by the Bartlett (14) method.

As a tool for sizing particles we used column chromatography. Aliquots of the emulsion particles were loaded onto a Sepharose CL-2B column (1.6 50 cm). Fractions were collected, and radioactivity was monitored. The particles eluted before LDL similar to IDL or remnant-size particles (Fig. 1). The ratio of the emulsion core lipid triolein and cholesteryl oleate to phospholipid was in the range of IDL or remnant-size particles (2.0-3.0/1). Incubation of emulsion particles with apoE (containing trace amounts of I-apoE) (13) in the cell culture medium used in these experiments (see below) had no significant effect on the emulsion elution profile, and no new population of particles appeared.

Figure 1: Elution of emulsion particles by Sepharose CL-2B gel column chromatography. 1.0 mg of radiolabeled lipid ([H]cholesteryl ether, 4194 cpm/µg of core lipid, core lipid/phospholipid ratio, 2.2/1) emulsion was placed on a 1.6 50-cm column. Emulsion particles eluted slightly before LDL, similar to small VLDL or IDL particles.

Cells

Incubations

Prior to the incubation the cells were washed with PBS at 37 °C. Then the PBS was exchanged with experimental medium. The incubation was performed at 37 °C on a rocker (Lab-Line Instruments, Inc., Melrose Park, IL), usually for 4-6 h. At the end of the incubation, medium was removed. The cells were chilled on ice and washed twice with ice-cold PBS containing 0.2% BSA (1- and 5-min washes) and twice with PBS alone. Then cells were incubated with heparin (1400 units/ml of PBS) at 4 °C on a rocker for 1 h. The heparin solution was removed and the cells were washed twice with PBS.

Assays

The fate of particle cholesteryl oleate was studied using particles labeled with [H]cholesteryl oleate. In these experiments, the cell extract was assayed for H-free cholesterol and [H]cholesteryl ester by TLC. Cellular cholesteryl ester resynthesis was determined by incubations with particles containing [C]triolein. After extraction with hexane/isopropanol the newly formed cholesteryl-[C]oleate was separated from the other lipids containing [C]oleate by TLC in the solvent system described above. Radioactivity was determined by liquid scintillation counting in a scintillation counter (Tri-Carb liquid scintillation spectrometer 3255, Packard Instrs., Meriden, CT) with Ultima Gold scintillation fluid (Packard). Either actual counts normalized by protein or normalized counts, recalculated in micrograms by the appropriate specific activity of the marker under consideration of the efficiency of the scintillation counter, are presented. Cells were dissolved in 2 ml of 0.1 N NaOH. A 100-µl aliquot was used for protein concentration determination by the Lowry et al. (18) method using bovine serum albumin as a standard. Cell total cholesterol and free cholesterol were assayed by gas liquid chromatography as previously detailed(3) .

Fluorescence Microscopy and Fluorescence Quenching Experiments

Images were recorded with a Photometrics cooled CCD camera or a JVC 665V video camera. All images used for quantitation were taken with the digital CCD camera. Image processing was done using the ISee program (Innovation Corp., Research Triangle Park, NC) running on a SPARC workstation (Sun Microsystems Inc., Mountain View, CA). Customized median filtering and spot-defining routines described in Dunn et al.(24) were added to the ISee package. Image processing to determine fluorescence spot intensities was carried out as described previously(24) . Briefly, diffuse background fluorescence was subtracted out, and spots were defined using a threshold value. The brightness of objects that contained between 4 and 60 pixels (0.14 0.14 µm per pixel) after this procedure were then measured.

Statistical Analysis

RESULTS

ApoE Particle Binding

Particle Uptake and Internalization

Figure 2: Effect of increasing apoE concentration on particle uptake. J774 macrophages were incubated for 4 h at 37 °C in DMEM, 1% BSA containing 300 µg of particle core lipid/ml of incubation medium (particle core lipid/phospholipid, 3.0) with increasing amounts of apoE. Particle uptake (expressed as emulsion triglyceride plus cholesterol uptake) was calculated after lipid extraction of [H]cholesteryl ether (specific activity, 2687 cpm/µg of core lipid) with hexane/isopropanol, 3/2. The inset shows that with appropriate particle concentrations in the absence (solid bars) and presence of 0.01 µg of apoE/µg of core lipid (hatched bars) similar uptake of particles can be achieved. (Compare uptake of particle at a particle concentration of 1200 µg/ml in the absence of apoE, with uptake at 300 µg/ml in the presence of apoE.) Results are means ± S.D. of three parallel experiments.

Fig. 3demonstrates that the time course of uptake of emulsion particles without and with apoE (at respective core lipid concentration ratios of 4/1) is very similar over 6 h. Therefore, we were able to achieve similar particle uptake of model TGRP both in the absence and presence of apoE by varying the concentration of each in the incubation media.

Figure 3: Time course of particle uptake without and with apoE. Under experimental conditions designed to provide equivalent uptake emulsion particles (core lipid/phospholipid, 2.2; specific activity of [H]cholesteryl oleate, 1918 cpm/µg of core lipid) were incubated with J774 macrophages in the absence (1100 µg of core lipid/ml (-)) and presence of apoE (2.75 µg of apoE and 275 µg of core lipid/ml (- - - - -)) over incubation periods of 15-360 min. Particle uptake was calculated from cell-associated [H]cholesteryl oleate + H-free cholesterol. Results are means ± S.D. of three parallel experiments.

Using fluorescence microscopy techniques, we demonstrated that particles were internalized into the cells in the absence and presence of apoE (Fig. 4). Cells were incubated for 5 min with DiI-labeled emulsions, the emulsions were removed from the medium, and cells were fixed and treated after 2 min with TNBS, a DiI-quenching agent. Only particles that are internalized maintain their fluorescence. By incubating higher particle concentrations without apoE, equivalent intracellular fluorescence intensity was achieved as compared to particles with apoE. Both by direct visualization (Fig. 4, A and B) and by fluorescence quantitation (Fig. 4C), particle internalization was very similar in the absence and presence of apoE. Fig. 4shows data at 2 min after the initial 5-min incubation; very similar data for fluorescence quantitation (and particle internalization) were obtained immediately after the initial 5-min incubation, as well as after 10- and 15-min incubations.

Figure 4: Equivalent uptake demonstrated by fluorescence microscopy. J774 macrophages on coverslip bottom dishes were incubated with DiI emulsions (core lipid/phospholipid, 2.2/1) without (A) or with (B) apoE for 5 min at 37 °C, washed as indicated under ``Experimental Procedures,'' and chased in emulsion-free medium for an additional 2 min. Cells were fixed, surface DiI fluorescence-quenched using 5 mM TNBS, and post-quenching images were taken using a cooled CCD camera and a 63 objective. A, labeled cells in the absence (1200 µg of core lipid/ml) of apoE; B, labeled cells in the presence of apoE (300 µg of core lipid, 3 µg of apoE/ml). Bar = 15 µm. C, a representative experiment showing total brightness of DiI-labeled spots in background-corrected images of fields labeled as described above and image-processed as described under ``Experimental Procedures.'' Brightness was normalized to the number of nuclei present in corresponding phase contrast images. Each bar shows an average for multiple dishes containing a total of 50-100 cells ± S.D. (solid bar, absence of apoE, n = 6 dishes; hatched bar, presence of apoE, n = 4 dishes).

Cell Triglyceride Metabolism

Figure 5: Net triglyceride mass accumulation compared to particle triglyceride uptake in the absence and presence of apoE at equivalent particle uptake. J774 macrophages were incubated with emulsion particles in the absence (1200 µg of core lipid/ml (solid bars)) and in the presence of apoE (3.0 µg of apoE and 300 µg of core lipid/ml (hatched bars)). Emulsion particles contained 4050 cpm of [H]cholesteryl oleate/µg of triglyceride and had a core lipid/phospholipid ratio of 2.2. Values of uptake and mass were obtained after 4 h incubation at 37 °C from three parallel experiments.

Figure 6: Cellular triglyceride mass over a range of different particle uptake in the absence and presence of apoE. Incubations with emulsion particles (core lipid/phospholipid was 2.7, specific activity 3454 cpm of [H]cholesteryl oleate/µg of particle triglyceride) were performed in the absence of apoE () at various core lipid concentrations and in the presence of 300 µg of core lipid and varying apoE concentrations () between 0.01 and 0.04 µg of apoE/µg of core lipid. Points labeled 1, 2, and 3 represent incubations in the absence of apoE at particle concentrations of 300, 600, and 1200 µg of core lipid/ml, respectively. Points labeled 4, 5, 6, and 7 represent incubations at apoE/particle ratios of 0.01, 0.02, 0.03, and 0.04 µg of apoE/µg of core lipid, respectively. The correlation coefficient between triglyceride uptake and mass was 0.88 in the absence and 0.92 in the presence of apoE, and the slopes were 0.81 and 1.10, respectively.

In experiments using emulsions radiolabeled with [C]triolein (rather than [H]cholesteryl ether), the percent of [C]triolein recovered as cellular C-free fatty acid over varying particle uptake obtained by incubating cells at increasing particle concentrations (300, 600, and 1200 µg/ml) without apoE was 12.7, 11.4, and 11.4%, respectively. This was similar to percent cell C-free fatty acid after incubation the presence of apoE, at a particle concentration of 300 µg/ml with varying apoE particle ratios, i.e. 12.3, 13.7, 12.2, and 13.6%, for apoE ratios of 0, 0.01, 0.02, and 0.04 µg of apoE/µg of core lipid, respectively. Thus, there are no apparent differences in mass of free fatty acid accumulating in cells in the absence versus the presence of apoE.

The triglyceride mass remaining in the cell is a result of triglyceride uptake plus new triglyceride synthesis minus triglyceride utilization. We questioned if the smaller deposition of triglyceride in the presence of apoE could be due to lower synthetic rates of new triglyceride, higher utilization, or both (Table 1). Assessing the contribution of new triglyceride synthesis to cell triglyceride mass over a range of particle uptakes, we found that in the absence of apoE net new triglyceride synthesis could account for 5.6 ± 4.8% of net triglyceride mass (n = 21) and in the presence of apoE, 7.4 ± 4.9% (n = 21). Net triglyceride synthesis was very similar at equivalent particle uptake in the absence and presence of apoE (Table 1). This indicates that the smaller triglyceride accumulation in the presence of apoE was due to increased utilization rather than differences in new triglyceride synthesis.

Utilization of triglyceride was estimated from triglyceride uptake after subtracting triglyceride mass and adding triglyceride synthesis in the absence and presence of apoE (Table 1). The formation of diglycerides/monoglycerides, and phospholipid measured by [H]glycerol incorporation was not significantly different under conditions of equivalent uptake (data not shown). Triglyceride utilization was over 2-fold higher in the presence of apoE as compared to its absence at similar ranges of particle uptake.

Cholesteryl Ester Hydrolysis

Figure 7: Cellular hydrolysis of particle cholesteryl ester to free cholesterol. J774 macrophages were incubated for 4 h at 37 °C in DMEM, 1% BSA with emulsion particles (core lipid/phospholipid 2.7, specific activity of [H]cholesteryl oleate, 3559 cpm/µg of cholesteryl oleate). After incubation the cells were extracted in hexane/isopropyl alcohol (3/2, v/v), and then cell-free cholesterol (- - - - -) was separated from cholesteryl ester (-) by thin layer chromatography, as detailed under ``Experimental Procedures.'' A, cell free cholesterol and cholesteryl ester radioactivity at increasing particle concentrations in the absence of apoE. B, cell free cholesterol and cholesteryl ester radioactivity after incubation with 300 µg of core lipid/ml and increasing apoE concentrations. Results are means ± S.D. of three parallel experiments.

To assess the effects of apoE on hydrolysis and redistribution of internalized cholesteryl ester between free (FC) and esterified cholesterol (CE) we first compared [H]FC/[H]CE at equal particle uptake (Fig. 8A). In the presence of apoE release of free cholesterol results in a 2-fold greater increase in this ratio compared to the absence of apoE. We then compared the effects of apoE on cell distribution of cholesteryl ester relative to free cholesterol over a range of apoE/particle ratios and particle concentrations (Fig. 8B). Of interest, at different degrees of lipid loading obtained with increasing apoE/particle ratios (from 0.01 to 0.10 µg of apoE/µg of core lipid) the percent of [H]cholesteryl oleate recovered as cell H-free cholesterol after incubation was very similar (range 83-85%). Clearly in the presence of apoE much less internalized cholesteryl ester remains as cholesteryl ester in the cell. ApoE leads to a major shift of cellular cholesteryl ester toward free cholesterol.

Figure 8: Cell free cholesterol relative to cholesteryl ester accumulation in the absence and presence of apoE. A, J774 macrophages were incubated with emulsion particles in the absence (1200 µg of core lipid/ml (solid bars)) and in the presence of apoE (3.0 µg of apoE and 300 µg of core lipid/ml (hatched bars)). Emulsion particles contained 3642 cpm of [H]cholesteryl oleate/µg of cholesteryl oleate and had a core lipid/phospholipid ratio of 2.2. After incubation the cells were extracted in hexane/isopropanol, 3/2, and then cell-free cholesterol was separated from cholesteryl ester by thin layer chromatography, as detailed under ``Experimental Procedures.'' Cellular H-free cholesterol and [H]cholesteryl ester values were summed to determine uptake of emulsion particles. Further, from the H-free cholesterol (FC) and [H]cholesteryl oleate (CE) values, the ratio of radiolabeled free cholesterol and cholesteryl ester indicates cholesteryl ester hydrolysis. Values of uptake and FC/CE are means ± S.D. after 4 h of incubation at 37 °C from five parallel determinations. B, cellular cholesteryl ester compared to free cholesterol in the absence and presence of apoE. Incubations with emulsion particles (core lipid/phospholipid, 2.7, specific activity 3559 cpm of [H]cholesteryl oleate/µg of particle cholesteryl oleate were performed in the absence of apoE () at various core lipid concentrations (300, 600, 1200 µg of core lipid/ml) and in the presence of 300 µg of core lipid and varying apoE concentrations () between 0.01 and 0.04 µg of apoE/µg of core lipid. The correlation coefficient between free cholesterol and cholesteryl ester was 0.94 in the absence and 0.90 in the presence of apoE, and the slopes were 0.31 and 0.18, respectively.

We also questioned if differences in free cholesterol efflux in the absence and presence of apoE might account for some of the above results. This is unlikely to be a significant contributor to our data since in measurements of actual cell cholesterol masses using gas liquid chromatography mass assays, no differences in FC/CE mass ratios were observed between control cells incubated with only 1% BSA, or cells incubated with particles with apoE (300 µg of core lipid/ml, 3 µg of apoE/ml) and particles without apoE (1200 µg of core lipid/ml). (FC/CE mass ratios were 1.87 ± 0.34, 1.77 ± 0.18, and 1.82 ± 0.22, respectively; n = 6.) As well, when comparing results of particle uptake measured by [H]cholesteryl ether assays with results of uptake calculated from the sum of [H]FC + [H]CE (from cellular [H]cholesteryl oleate hydrolysis), no real differences were noted. Thus, differences in cholesterol efflux cannot account for our results.

Contribution of Acyl-CoA/Cholesterol Acyltransferase (ACAT)

We next examined whether this apoE-related increase in ACAT activity contributed substantially to our results on redistribution of internalized particle cholesteryl ester (albeit in the opposite direction). To address this we performed experiments incubating emulsions under conditions of equivalent particle uptake without and with apoE in the absence and presence of 58.035, a well described inhibitor of ACAT(15) . Fig. 9A (in the absence of apoE) and Fig. 9B (in the presence of apoE) compare production of H-free cholesterol relative to cholesteryl ester over time in the presence and absence of ACAT inhibition. Repeated experiments did show effects of ACAT, but with only slightly higher values of free cholesterol in the presence of ACAT inhibition as in its absence, independent of the absence or presence of apoE. Nevertheless, these differences were consistently small. This suggested that, while some re-esterification of free cholesterol did occur, these effects were relatively minor, and, thus, most of the effects of apoE on redistribution of internalized cholesteryl ester were due to effects on cholesteryl ester hydrolysis and not on re-esterification of free cholesterol.

Figure 9: Cholesteryl ester hydrolysis over time: effect of apoE and ACAT inhibition. Under experimental conditions designed to provide equivalent uptake of emulsion particles (core lipid/phospholipid, 2.1, specific activity of [H]cholesteryl oleate 1288 cpm/µg of core lipid) were incubated with J774 macrophages in the absence (A) (1100 µg of core lipid/ml) and presence (B) (2.75 µg of apoE and 275 µg of core lipid/ml) of apoE over incubation periods of 10-360 min. After incubation the cells were extracted in hexane/isopropanol, 3/2, and then cell-free cholesterol was separated from cholesteryl ester by thin layer chromatography, as detailed under ``Experimental Procedures.'' A, in the absence of apoE, particle cholesteryl ester hydrolysis was calculated by the ratio of H-free cholesterol/[H]cholesteryl oleate with (- - - - -) and without (-) 58.035 (1 µg/ml of incubation medium), an ACAT inhibitor, and in B, in the presence of apoE the ratio of H-free cholesterol/[H]cholesteryl oleate with (- - - - -) and without (-) 58.035. Results are means ± S.D. of three parallel experiments.

Lysosomal Catabolism

We next questioned whether particles without apoE may have less targeting to the lysosomal compartment than particles with apoE. Again, under conditions of equivalent particle uptake, DiI-labeled emulsions in medium containing Fm (which rapidly targets to the lysosomal compartment) (23) were examined by fluorescence microscopy after 10-min incubations. Fig. 10shows that the great majority of particles without and with apoE colocalize with Fm indicating that even in the absence of apoE, particles rapidly reach the lysosomal compartment. These results indicate that effects of apoE on intracellular triglyceride and cholesteryl ester metabolism are not due mainly to effects of apoE on delivering particles to the lysosomal compartment but suggest that apoE affects transport and metabolism of free fatty acids and free cholesterol released from the lysosomes.

Figure 10: Intracellular localization of DiI-labeled emulsion particles in the absence and presence of apoE. A, J774 macrophages on coverslip bottom dishes were incubated with DiI-labeled emulsions (core lipid/phospholipid, 1.7/1) without or with apoE continuously for 10 min (A and B) and with Fm (40 µg/ml) (C and D). Cells were then washed and fixed in 2% formaldehyde in PBS. Surface DiI fluorescence was quenched using 5 mM TNBS, and pairs of images were obtained using DiI and fluorescein filter sets. Bar is 15 µm. A and C show J774 cells labeled with apoE emulsions at 300 µg/ml and Fm. B and D show J774 cells labeled with emulsions at 1200 µg/ml without apoE and Fm. J774 cells are imaged using DiI optics in A and B and fluorescein optics in C and D. Arrows indicate examples of co-localization between corresponding DiI emulsion and Fm images in A and C, and in B and D, respectively.

DISCUSSION

The role of apoE in mediating triglyceride-rich particle uptake is well documented. Our findings, herein, suggest additional specific effects of apoE on intracellular lipid metabolism and that these effects are distinct from apoE effect on enhancing cell particle uptake. ApoE increases triglyceride hydrolysis and utilization, and it influences intracellular processing of internalized cholesteryl ester. Cholesteryl ester hydrolysis is more efficient in the presence of apoE. Effects on cholesteryl ester resynthesis catalyzed by ACAT were small.

Our results are of particular interest in view of recent studies of apoE in a number of metabolic pathways. The synthesis and presence of apoE, in a majority of tissues, suggests an important role of this apoprotein in local redistribution of lipids between tissues, within a tissue, and possibly within a single cell. ApoE synthesis and mass is enhanced during tissue repair and healing of neurological lesions(13) . Changes in apoE synthesis have been associated with bile acid synthesis, steroidogenesis, and cell division, differentiation, and proliferation(1, 7, 8, 28, 29) . It has been proposed that apoE may modulate mechanisms regulating intracellular cholesterol utilization by directly altering cholesterol trafficking, or by interacting with specific signal transduction pathways(9, 30) . Our results suggest that exogenous apoE also has an intracellular role, not only in cholesterol, but also in triglyceride and fatty acid metabolism.

Metabolism of triglyceride-rich lipoproteins is dependent on particle size and apoprotein and lipid compositions(16, 17, 20, 26) . Certainly, apoE is not the only modulator of triglyceride-rich particle metabolism. Still, apoE does increase triglyceride-rich particle uptake by receptor mediated endocytosis. Nevertheless, as we have previously shown, uptake of triglyceride-rich particles, especially when present in cell culture media at near physiological concentrations can be substantial in the absence of apoE(6) . Physiologically, triglyceride-rich lipoproteins likely never exist without apoE. In the present study, we have utilized approaches to assure equal uptake of model triglyceride-rich lipoproteins, both in the absence and the presence of apoE. Thus, differences measured were due to effects of exogenous apoE once internalized, and not due to differences in cellular particle uptake. Effects of endogenous apoE were excluded by choosing J774-A2 macrophages, a cell line that does not synthesize apoE (12) .

Our previous (6) and current data show that particles are internalized in the absence of apoE. As further evidence for internalization of particles in the absence of apoE, we also show that not only are cholesteryl esters and triglycerides hydrolyzed in the absence of apoE (albeit less than in its presence), but also that chloroquine, a lysosomal hydrolase inhibitor, completely abolishes these hydrolytic effects whether apoE is present or not.

Our experiments in which chloroquine essentially abolishes cholesteryl ester hydrolysis, both in the presence and absence of apoE, confirm that the lysosome is a key step in the metabolic pathway of the triglyceride-rich particles. Thus, the question remains whether the effects of apoE on intracellular triglyceride and cholesteryl ester metabolisms are mediated through pre- as compared to post-lysosomal pathways. Tabas et al.(20) have previously shown that -VLDL of large size distributes initially to a peripheral compartment while smaller size -VLDL, like LDL proceeds more rapidly to a lysosomal compartment. It is possible that apoE may effect subtle differences in delivery of particles to lysosomes or to specific groups of lysosomes. Also, lysosome composition may be different in the presence versus absence of apoE. At this point, without data available, these possibilities cannot be ruled out. In our experiments using fluorescent-labeled emulsions with and without apoE, even in the absence of apoE, after 10-15 min, a substantial amount of particles is rapidly located in a lysosomal compartment. Of interest, in a separate study assessing mechanisms of uptake of apoE containing triglyceride-rich particles by receptor-dependent versus non-receptor pathways, we found no differences in particle cholesteryl ester hydrolysis in incubations from 1 to 8 h(31) . Therefore, while apoE may have some effects on the rate of triglyceride-rich particles delivery to lysosomes, it is unlikely that, in our experiments which were generally carried out over periods of 4 h or more, a very short delay could be responsible for a substantial part of the observed effects of apoE intracellularly.

Although we do not provide direct data for this, it is likely that apoE has effects on post lysosomal trafficking of lipid molecules to other cellular compartments, e.g. peroxisomes, as has been suggested by others(9) . For the latter some apoE, at least, would have to be spared from lysosomal degradation. Of interest, our own recent data ()and that of others (32, 33) suggest that, in comparison to apoB, substantially less apoE undergoes intracellular degradation, after internalization.

Animals made apoE-deficient by homologous recombination techniques have plasma elevation of VLDL and remnant-like particles as well as large lipid accumulation in arteries (10, 11) and in liver(10) . This suggests that, in these mice models, VLDL and remnant particles enter cells by apoE-independent pathways, and once internalized, lipoprotein triglycerides and cholesteryl esters may not be efficiently hydrolyzed, resulting in lipid storage. Because of the relative ubiquitous distribution of apoE in cells of greatly varying functions, it is also possible that in different cell lines apoE may have different metabolic roles. The basic mechanisms whereby apoE may influence these putative differences in intracellular lipid metabolism are still not known. One possibility is that apoE may act as an intracellular carrier or ``chaperone'' for lipid or other molecules. Different apoE isoforms are also likely to behave differently intercellularly as indicated by the association of the apoE-4 isoform with amyloid-like S4 protein and a markedly increased risk for Alzheimer's disease(34) . In addition to the different abilities of different apoE isoforms to modulate binding and internalization of lipoprotein particles, we question whether different apoE isoforms carried into the cells on triglyceride-rich lipoproteins will also demonstrate isoform-specific effects on intracellular triglyceride and cholesterol metabolism.

FOOTNOTES

*

This work was supported by National Institutes of Health grants HL-40404, HL-21006, and DK-27083 and by Belgian Fonds de la Recherche Scientifique Medicale Grant 3-4632-92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Pediatrics, Columbia University, BHN 702, 630 W. 168th St., New York, NY 10032. Tel.: 212-305-7082; Fax: 212-305-8995.

()

The abbreviations used are: apo, apoprotein; TGRP, triglyceride-rich particle; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; TG, triglyceride; FC, free cholesterol; CE, cholesteryl ester; ACAT, acyl-CoA/cholesterol acyltransferase; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; DiI, 1,1`-dioctadecyl-3,3,3`,3`-tetramethyl-indocarbocyanine perchlorate; Fm, fluorescein-labeled -macroglobulin; TNBS, trinitryl benzoyl sulfonic acid.

()

M. Al-Haideri, N. M. Galeano, B. Schwiegelshohn, and R. J. Deckelbaum, unpublished observations.

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