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J Biol Chem, Vol. 275, Issue 12, 8564-8571, March 24, 2000
From the Apolipoprotein E (apoE) is an
important determinant for the uptake of triglyceride-rich lipoproteins
and emulsions by the liver, but the intracellular pathway of apoE
following particle internalization is poorly defined. In the present
study, we investigated whether retroendocytosis is a unique feature of
apoE as compared with apoB by studying the intracellular fate of very
low density lipoprotein-sized apoE-containing triglyceride-rich
emulsion particles and LDL after LDLr-mediated uptake. Incubation of
HepG2 cells with [3H]cholesteryl oleate-labeled particles
at 37 °C led to a rapid release of [3H]cholesterol
within 30 min for both LDL and emulsion particles. In contrast,
emulsion-derived 125I-apoE was more resistant to
degradation ( Apolipoprotein E (apoE)1
plays a key role in the hepatic metabolism of triglyceride (TG)-rich
lipoproteins such as chylomicrons and very low density lipoproteins
(VLDL) (1-3) and TG-rich emulsions (4-5). In the blood, these
particles are converted into TG-rich remnants through the hydrolysis of
core TG by lipoprotein lipase (LPL) and the concomitant enrichment with
apoE. Various apoE-recognizing systems have been proposed to
participate in remnant removal, including the low density receptor
(LDLr) (6-8), a distinct specific apoE receptor (1, 9), the
LDLr-related protein (8, 10, 11), and heparan sulfate proteoglycans
(HSPG) alone (12, 13) or in concert with LDLr-related protein (5, 14).
However, the LDLr pathway plays a substantially greater role in the
overall clearance of TG-rich lipoprotein remnants in mice than the
non-LDLr pathway (15, 16). It has been shown that the affinity of
TG-rich lipoproteins and emulsions for the LDLr pathway and non-LDLr
pathway is governed by their particle size. Whereas large particles are mainly processed via the liver-specific non-LDLr recognition site, small particles (<50 nm) are almost exclusively recognized and taken
up by the LDLr (17, 18).
The intracellular metabolism of TG-rich remnants may be far more
complex than for LDL, of which both the protein and lipid components
are completely degraded within perinuclear lysosomes while the receptor
recycles back to the cell surface (19, 20). Whereas LDL shows a single
interaction of apoB with the LDLr, four molecules of apoE on
TG-containing lipoproteins can bind a single LDLr (21). In addition,
apoE-containing lipoproteins can interact with multiple LDLrs (22). In
either case, when lipoproteins or emulsions are provided with at least
four molecules of apoE, their affinity for the LDLr is 20-25-fold
higher than for LDL (21-23). The multivalent binding of
apoE-containing particles leading to the cross-linking of LDLrs may
explain the delayed perinuclear lysosomal targeting of Besides its involvement in the cellular recognition and uptake of
TG-rich lipoprotein remnants and emulsions, apoE is also hypothesized
to play a role in the intracellular trafficking of lipids (31-36).
Initial data suggested that apoE affects the transport and metabolism
of free fatty acids and free cholesterol released from the lysosomes
(33). In addition, apoE appeared to have a physiological role in the
assembly and secretion of VLDL (34-36). Although de novo
synthesized apoE in hepatocytes (37) may be used for VLDL assembly,
lipoprotein-derived apoE that is taken up by the cells may also serve
this purpose since apoE from radiolabeled VLDL remnants could be
recovered in hepatic Golgi fractions (exocytotic compartments) after
intravenous injection into mice (38).
The aim of the present study was to investigate whether apoE that is
internalized by hepatocytes can escape from lysosomal degradation and
can be resecreted through retroendocytosis. For this purpose, we
utilized small apoE-containing TG-rich emulsions that mimic the
metabolic fate of TG-rich lipoproteins in the blood (4, 39) and are
exclusively taken up via the LDLr in mice (18). The data show that apoE
is indeed relatively resistant to intracellular degradation as compared
with the cholesteryl oleate (CO) moiety of the emulsion both in
hepatoma cells in vitro (HepG2) and in hepatocytes in
vivo (C57Bl/6KH mice) and that apoE is resecreted at least
in vitro. Since these emulsions mimic TG-rich lipoproteins,
we anticipate that apoE, which has been delivered to the hepatocyte as
a constituent of chylomicron remnants, can possibly be re-used for
intracellular lipid trafficking, such as the assembly and secretion of
VLDL, or may participate in lipoprotein remnant internalization via the
"secretion-recapture" pathway (40, 41).
Animals--
10-12-Week-old male C57Bl/6KH mice weighing 23-24
g (from Broekman Instituut BV, Someren, The Netherlands) fed ad
libitum with regular chow were used for the in vivo experiments.
Chemicals--
Recombinant human apoE (isoform E3) was a
generous gift from Dr. Tikva Vogel, Bio-Technology General Ltd.,
Rehovot, Israel, and was supplied as a lyophilized powder containing
76% apoE, 11.7% L-cysteine, and 12.0% NaHCO3
(42). ApoE was dissolved in phosphate-buffered saline (PBS), pH 7.4 (2.0 mg/ml), and stored under argon at Isolation of Lipoproteins--
Human LDL (1.019 < d < 1.063 g/ml) and HDL (1.063 < d < 1.21 g/ml) were isolated from the blood of healthy
volunteers by differential ultracentrifugation as described (43) and
dialyzed at 4 °C against PBS containing 1 mM EDTA, pH
7.4, with repeated changes of buffer.
Protein Assay--
Protein concentrations were determined
according to Lowry et al. (44) using BSA as a standard.
Radiolabeling of (Lipo)proteins--
LDL was labeled with
[3H]CO by incubation with donor
[3H]CO-containing liposomes, in the presence of human
lipoprotein-deficient serum. In short, liposomes were created by
sonication of 25 mg of EYPC and 200 µCi of [3H]CO using
a Soniprep 150 (MSE Scientific Instruments, Crawley, UK) for 15 min at
18 µm output, and concentrated by density gradient ultracentrifugation. Subsequently, LDL was incubated with the liposomes
(protein:liposomal phospholipid = 1:8, w/w) for 8 h at
37 °C under argon in the presence of 20 mM ethyl
mercurithiosalicylate (45). LDL was purified by density gradient
ultracentrifugation and Superose 6® (Amersham Pharmacia Biotech) gel
filtration. The specific 3H activity was ~15 dpm/ng
protein. [3H]CO-labeled LDL, apoE, and bLf were
radioiodinated at pH 10.0 with carrier-free 125I according
to the ICl method (46). Free 125I was removed by Sephadex
G-50 medium gel filtration. More than 97, 98.5, and 99.5% of the
radiolabels in LDL, apoE, and bLf, respectively, were 10%
trichloroacetic acid-precipitable. The specific
125I-activities of LDL, apoE, and bLf were 114-301,
329-499, and 675 dpm/ng of protein, respectively.
Preparation of (ApoE-Containing) Emulsions--
Emulsions
were prepared according to the sonication and ultracentrifugation
procedure of Redgrave and Maranhao (47) from 100 mg of total lipid at a
weight ratio triolein:EYPC:lysophosphatidylcholine:CO:cholesterol of
70:22.7:2.3:3.0:2.0, using a Soniprep 150 (MSE Scientific Instruments, UK) (4). For synthesis of radiolabeled emulsions, 100-400 µCi of
[3H]CO (67.3 mCi/mg) was added. An emulsion fraction
containing VLDL-sized particles was obtained by consecutive density
gradient ultracentrifugation steps exactly as described (18). The
emulsions were homogeneous with respect to size (low polydispersity of
0.23-0.31) and the mean particle diameter was 44.3 ± 2.6 nm
(mean ± S.D.; n = 6) as determined by photon
correlation spectroscopy using a Malvern 4700 C system (Malvern
Instruments, Malvern, UK) (18). The TG content of the emulsion
fractions was determined with the Roche Molecular Biochemicals
enzymatic kit for triacylglycerols. Association of apoE with the
particles occurred by incubation of emulsions with
(125I-)apoE at TG:apoE = 50:0.3 (low) and 50:3.0
(high) weight ratios for 30 min at 37 °C. Emulsion-bound apoE was
separated from free apoE using density gradient ultracentrifugation as
described (39). The apoE contents of the reisolated emulsions were
4.6 ± 1.1 µg/mg TG (50:0.3) and 34.2 ± 2.6 µg/mg TG
(50:3.0), which corresponds to 4 ± 1 and 27 ± 2 molecules
of apoE per particle. Emulsions were stored at 4 °C under argon and
used for characterization and metabolic studies within 7 days following preparation.
Characterization of (Radiolabeled) ApoE--
The (radiochemical)
purity of apoE as well as the presence of disulfide-linked apoE
homodimers (48) were checked using 10% SDS-PAGE under non-reducing
conditions, using Kaleidoscope prestained molecular weight standards as
reference proteins. Resulting gels were stained for protein with
Coomassie Blue R-250 or assayed for 125I activity by
exposure of dried gels to Kodak X-Omat films. In addition, the
homodimer content and the aggregation state of (emulsion-bound) 125I-apoE was examined by fast protein liquid
chromatography (SMART system; Amersham Pharmacia Biotech), using a
Superdex® 200 column at a flow rate of 50 µl/min and with 50 mM NaPi, 0.15 M NaCl, pH 7.4, in
the absence or presence of 0.5% SDS as eluent.
Culture of HepG2 Cells--
HepG2 cells were cultured at
37 °C in a humidified 5% CO2, 95% air atmosphere in
25-cm2 flasks containing DMEM supplemented with 10% (v/v)
heat-inactivated FCS, 20 mM HEPES, 10 mM
NaHCO3, 100 units/ml penicillin, and 100 µg/ml
streptomycin. At 6-7 days prior to each experiment, cells were seeded
into 2-cm2 12-well dishes. At 24 h before the assays,
cells were washed with DMEM containing 1% BSA and were further
incubated with DMEM containing 10% (v/v) lipoprotein-deficient serum
instead of FCS.
Cell Binding, Association, and Degradation Studies--
Binding,
association, and degradation studies were performed essentially as
described previously (28). For some experiments, cells were pretreated
with heparinase I for 2 h at 37 °C (14). Cells were washed
three times (DMEM + 1% BSA) and were incubated at 4 or 37 °C with
0.5 ml of the same medium with the indicated amounts of radiolabeled
LDL or emulsions (3H or 125I), in the absence
or presence of an excess of unlabeled particles. After incubation, the
cells were cooled to 0 °C, and the incubation media were removed.
The cells were washed three times with PBS + 0.1% BSA, once with PBS,
and were dissolved in 1 ml of 0.2 M NaOH. Aliquots of media
and cell lysates were counted for 3H or 125I
radioactivity, and aliquots of the cell lysates were used for protein
determination. Degradation of 125I-protein in the medium
was determined by separating 125I-protein from degradation
products by 10% trichloroacetic acid precipitation as described (19).
To determine the intracellular hydrolysis of [3H]CO into
[3H]cholesterol, total lipid was extracted according to
Bligh and Dyer (50) and separated using thin layer chromatography
(heptane:diethyl ether:acetic acid = 60:40:1, v/v). CO
(Rf 0.85) and cholesterol (Rf
0.23) were visualized with iodine vapor, scraped off, and counted in 15 ml of Hionic Fluor (Packard Instrument Co.). Using this method, 99.6%
of the emulsion-associated 3H radioactivity appeared as
[3H]CO.
Intracellular Processing and Retroendocytosis--
Cells were
preincubated in the presence of 125I-apoE-containing
emulsions (60 µg of TG/ml) for 3 h at 18 °C, which does not
impair binding and endocytosis, but blocks the fusion of endosomes with lysosomes. As a result, cell-associated emulsions accumulate in the
early endosomal compartment, without being degraded (51, 52). Cells
were washed with DMEM + 1% BSA to remove unbound ligand, and cell
surface-bound apoE was released by a subsequent wash with heparin (770 units/ml in DMEM + 1% BSA) (53, 54). Cells were washed once with PBS + 0.1% BSA and further incubated at 37 °C with 0.5 ml of DMEM + 1%
BSA in the absence or presence of HDL (0.35 mg of protein/ml) or
apoE-deficient emulsion (0.50 mg of TG/ml). After incubation, the media
and cells were treated as described above. In addition, 500-µl
aliquots of media, combined from triplicate samples, were subjected to
density gradient ultracentrifugation at 40,000 rpm for 18 h at
4 °C as described (43). Tubes were fractionated (24 × 0.5 ml)
from top to bottom using a Multiprobe 104DT Robotic System from Packard
Instrument Co., and fractions were counted for 125I
activity. Subsequently, 400-µl fraction aliquots were subjected to
10% trichloroacetic acid precipitation to separate 125I
and 125I-tyrosine from intact 125I-apoE. To
identify the radioactivity within the emulsion and HDL-containing
fractions as intact apoE or degradation products, the fractions were
desalted by dialysis against 100-fold diluted PBS, freeze-dried, and
subjected to 4-20% gradient SDS-PAGE under non-reducing conditions.
The radioactivity on the gel was visualized by imaging using a Packard
Instant Imager (Hewlett-Packard Co., Palo Alto, CA).
Liver Uptake and Serum Decay of Emulsions in Mice--
Mice were
anesthetized by subcutaneous injection of a mixture of ketamine (120 mg/kg body weight), Thalamonal (0.03 mg/kg fentanyl and 1.7 mg/kg
droperidol), and Hypnorm (1.2 mg/kg fluanisone and 0.04 mg/kg fentanyl
citrate), and the abdomens were opened. [3H]CO or
125I-apoE-labeled apoE-containing emulsions (reisolated
after incubation at a ratio TG:apoE = 50:3.0, w/w) were injected
(150 µg of TG, corresponding with 5 µg of apoE) via the inferior
vena cava. At the indicated times, blood samples (<50 µl) and liver
lobules were taken and processed as described in detail (18).
Radioactivity in duplicate serum samples of 10 µl was counted either
directly (125I) or in 2.5 ml of Emulsifier Safe (Packard
Instrument Co.) (3H). The total serum volumes of C57Bl/6KH
mice were 1.068 ± 0.066 ml, as previously determined (18).
Radioactivity in liver samples was also counted directly
(125I) or in 15 ml of Hionic Fluor (Packard Instrument Co.)
after solubilization in 0.5 ml of Soluene®-350 (Packard) for 5 h
at 65 °C (3H). Radioactivity values are corrected for
the serum radioactivity (84.7 µl/g wet weight) present at the time of
sampling (18). Since liver lobules were partly used for determination
of particle degradation, total liver weights were estimated from the
following equation: liver mass (g) = Intrahepatic Processing of Emulsions in Mice--
To determine
the intrahepatic degradation of 125I-apoE, aliquots of
liver lobules were immediately frozen in liquid N2,
homogenized in ice-cold PBS, pH 7.4, and subjected to 10%
trichloroacetic acid precipitation. The intrahepatic conversion of
[3H]CO into [3H]cholesterol was assayed
after similar freezing and homogenization of liver aliquots. Lipids
were extracted and separated as described above.
Purity of Lipid-free and Emulsion-associated ApoE--
The
(radiochemical) purity of apoE was assessed by 10% SDS-PAGE under
non-reducing conditions (Fig.
1A). As expected, apoE appeared to be present mainly as a 34-kDa protein as determined by
staining with Coomassie Blue R-250. In addition, a minor protein band
with an apparent mass of ~100 kDa was observed, which has previously
been shown to represent the disulfide-linked dimer of apoE (48).
Accordingly, this band was not detected after reduction with
Binding of ApoE-containing Emulsions to HepG2 Cells--
To
establish the contribution of HSPG and the LDLr to the recognition of
apoE emulsions by HepG2 cells, binding experiments were conducted at
4 °C (Fig. 2). Removal of HSPG from
the cell surface by treatment with heparinase (2.5 units/ml) resulted
in a 45% reduction of the binding of bLf. In contrast, only a minor effect of heparinase treatment (12-17% reduction) was observed on the
binding of both the [3H]CO-labeled and
125I-labeled apoE-enriched emulsions (incubated at a
TG:apoE = 50:3 weight ratio). It is thus evident that HSPG play
only a minor role in the binding of the apoE-enriched emulsion (Fig.
2A). The binding of both the [3H]CO-labeled
and 125I-labeled apoE-enriched emulsions was
dose-dependently inhibited by an excess of unlabeled
particles (Fig. 2, B and C). LDL also efficiently
inhibited the binding of the radiolabeled apoE emulsions for at least
93-94% (Fig. 2, B and C). Taking these data
together, it is evident that the applied apoE-emulsion particles are
also almost exclusively recognized by the LDLr on HepG2 cells in
vitro, whereas HSPG play only a minor role in particle
recognition.
Association and Degradation of ApoE-containing Emulsions by HepG2
Cells--
To evaluate whether apoE is relatively resistant to
intracellular degradation, the metabolic fate of the
[3H]CO and 125I-apoE moiety of apoE emulsions
(incubated at a TG:apoE = 50:3 weight ratio) in HepG2 cells during
incubation at 37 °C was determined (Fig.
3). The CO and apoE components showed a
similar time-dependent cellular uptake. However, whereas
the hydrolysis of CO started within 30 min after incubation and was
very effective (47% of the total uptake at 4 h), the degradation
of the apoE moiety started slowly (
Previous studies have shown that internalization of LDL by the LDLr
results in complete lysosomal degradation of both its lipid and protein
components (19, 20). Incubation of HepG2 cells with
[3H]CO or 125I-apoB-labeled LDL (20 µg of
protein/ml), which resulted in a similar rate of particle uptake as
compared with apoE emulsions (~1012 particles per mg of
cell protein at 4 h), led to the rapid onset of both
[3H]CO hydrolysis and 125I-apoB degradation
(both within 30 min) (not shown). In Fig.
4 the relative apolipoprotein degradation
rates, as calculated from the fraction of degraded apolipoprotein
(degraded/total uptake 125I-protein) divided by the
fraction of hydrolyzed CO (hydrolyzed/total uptake
[3H]CO), are shown for emulsion-associated apoE and
LDL-associated apoB. It appears that apoE is far more resistant to
intracellular degradation as compared with apoB. These data thus
indicate that apoE emulsions, after LDLr-mediated uptake, may have a
different intracellular fate as compared with LDL, as a result of which apoE may (partially) escape from lysosomal degradation.
Intracellular Processing and Retroendocytosis of ApoE by HepG2
Cells--
Since internalized apoE was shown to be resistant to
intracellular degradation, it was reasoned that intact apoE may be
recovered in the medium after cellular uptake through retroendocytosis. To evaluate this hypothesis, apoE emulsions were incubated with cells
for 3 h at 18 °C (51, 52). Unbound particles were removed by
extensive washing with DMEM/BSA. Residual cell surface-associated 125I-activity was released by 770 units/ml heparin (53,
54), which led to a reduction in total cell association of ~30%. The cells were further incubated at 37 °C in the absence or presence of
protein-free emulsion or HDL in the medium as potential acceptors of
secreted apoE (55-57) (Fig. 5). A
time-dependent decrease in the cellular association of apoE
was observed in the absence of acceptor, with 52 ± 4% of the
radioactivity still associated with the cells after 60 min of
incubation. At this time point, the cell association was reduced to
45 ± 1% (Student's t test; p < 0.05) and 38 ± 3% (p < 0.01) in the presence of
the protein-free emulsion or HDL, respectively. The presence of these
acceptors had no effect on the degradation rate of
125I-apoE but did result in a significantly increased
secretion rate of trichloroacetic acid-precipitable radioactivity
(representing intact protein) as compared with the absence of acceptor
(repeated measures analysis of variance, p < 0.01 and
p < 0.001, respectively). More specifically, whereas
18.6 ± 0.8% of the radioactivity secreted into the medium was
recovered as precipitable protein after 4 h of incubation in the
absence of acceptor, 23.0 ± 0.7 (Student's t test;
p < 0.01) and 29.8 ± 1.9% (p < 0.001) of the radioactivity could be precipitated from the medium in
the presence of emulsion and HDL, respectively (Fig. 5).
Loading the cells with [3H]CO-labeled apoE emulsions at
18 °C led to the hydrolysis of the vast majority of radiolabel into [3H]cholesterol (>90%) after 4 h of incubation at
37 °C, whereas hardly any intact [3H]CO could be
detected in the medium (<3% of the initially cell-associated radiolabel) (not shown). In the same experimental set up, loading of
HepG2 cells with 125I-LDL (10 µg/ml) resulted in a
decrease of cellular 125I-apoB radioactivity with a
half-life of ~2 h. In contrast to apoE, virtually all radioactivity
released into the medium was trichloroacetic acid-soluble (>95%)
(data not shown), which is in full accordance with our previous
observations (28).
To evaluate whether the secreted apoE is still functional in that it
recombines with lipids, aliquots of the media were harvested after
3 h of incubation and subjected to density gradient
ultracentrifugation (Fig. 6). Whereas
both intact and degraded apoE were detected in the bottom fractions of
the tubes from media without acceptor (Fig. 6A), 45 and
50-60% of the trichloroacetic acid-precipitable radioactivity were
recovered in the emulsion and HDL fractions when the respective
acceptors were present in the media (Fig. 6, B and
C). The integrity of apoE on these particles was confirmed by the detection of a radiolabeled 34-kDa protein in these fractions after protein separation by non-reducing 4-20% gradient SDS-PAGE, followed by imaging (insets in Fig. 6, B and
C). Apparently, both emulsion particles and HDL can function
as acceptors of secreted apoE, albeit that HDL is more effective than
emulsion particles in stimulation of the total release of apoE under
the given conditions (Fig. 5).
Intrahepatic Processing of ApoE Emulsions in
Mice--
Subsequently, we investigated whether the finding that apoE
can escape intracellular degradation upon entry of the lysosomal route
is relevant for the intrahepatic metabolism of apoE emulsions in the
intact animal. We have previously shown that intravenous injection of
the protein-free [3H]CO-labeled emulsion (150 µg of TG)
into C57Bl/6KH mice results in the monophasic elimination of radiolabel
from the serum with a half-life of ~45 min. Concomitantly, a
progressively increasing LDLr-dependent liver uptake
reaching ~45% of the injected dose at 45 min after injection was
observed (18). As shown in Fig. 7,
preassociation of apoE with the [3H]CO-labeled emulsion
accelerated the serum clearance (t1/2 < 10 min) and
liver uptake (~50% at 20 min) of the emulsion. The initial rate of
serum clearance and liver uptake of the protein and lipid components of
the emulsion were essentially similar (Fig. 7). In contrast, injection
of an equal dose of 125I-apoE in a lipid-free state led to
the rapid elimination of 80% of the injected dose from the serum
within 2 min, with a high uptake by the liver (70% at 5 min after
injection).
In general, the intrahepatic degradation of endocytosed apolipoproteins
leads to rapid elimination of radiolabel from the liver and nonspecific
distribution over the body (58), which also appears to occur with
lipid-free apoE (Fig. 7). The hepatic uptake of lipid-associated apoE
(43.7 ± 2.3% of the dose at 20 min after injection) was not
coupled to rapid degradation, as 36.3 ± 2.1% was still present
within the liver at 60 min after injection. The stability of
lipid-associated apoE was also confirmed by a low level of degradation
products in the liver, reaching only 8.8 ± 0.2% of the recovered
radioactivity at 60 min after injection (Fig.
8). In contrast, the intrahepatic
hydrolysis of [3H]CO was rapid and efficient, with 50%
hydrolysis achieved at 45 min after injection.
Radioiodination of TG-rich lipoproteins results in the labeling of
various apolipoproteins (especially apoCs) that may all have a
different susceptibility to proteolysis, which hampers the
interpretation of the metabolic fate of apoE. In order to chase the
intracellular fate of apoE only, we thus decided to utilize VLDL-sized
TG-rich emulsions (44 ± 3 nm) that can be enriched with
radiolabeled apoE (4, 18). The metabolic behavior of these particles
in vivo is completely dependent on the presence of the LDLr,
as determined by kinetic studies on [3H]CO-labeled
emulsions in wild-type versus LDLr-deficient mice (18). As a
reference for LDLr-mediated processing of substrates, we used LDL,
which only contains a single copy of apoB that is labeled upon
radioiodination. Whereas radioiodinated apoE did contain some
disulfide-linked apoE homodimers, analysis of emulsion-bound 125I-apoE confirmed that the radioactivity was associated
with 34-kDa apoE only. It can thus be excluded that the results are
confounded by the presence of apoE homodimers (48).
The total binding values of the emulsions, calculated from the specific
radioactivities of either the [3H]CO-labeled and
125I-labeled emulsions, were similar (55.3 ± 3.4 and
51.5 ± 3.6 ng of protein/mg of cell protein, respectively), which
indicates that the apoE-emulsion particle binds to the cell as a unity, without preferential binding of either the lipid or protein moiety (Fig. 2). The binding of the emulsion to HepG2 cells appeared to be
largely mediated by the LDLr (~90% of the total binding), whereas
HSPG contributed for only a low extent (~10%) as determined after
treatment of the cells with heparinase. In accordance with previous
observations by Ji and Mahley (59), heparinase treatment inhibited the
binding of bLf to HepG2 cells by 45%, which demonstrates that HSPG had
been effectively removed. The finding that HSPG are hardly involved in
the binding of apoE emulsion particles is in agreement with our
previous observations that, as opposed to LPL, apoE is not essential
for the binding of The association and degradation of the emulsion by HepG2 cells appeared
to be dependent on the presence of apoE. The apoE-deficient emulsion
showed a low cellular association and degradation, which was not
substantially increased by the addition of 4 ± 1 molecules of
apoE per emulsion particle, obtained at a TG:apoE = 50:0.3 weight
ratio (not shown). In contrast, the addition of a physiologically relevant number of apoE molecules per particle (27 ± 2 at a 50:3 weight ratio), which is similar to the apoE content of rat VLDL (29 µg/mg of TG, corresponding to 26 molecules per particle) (61), resulted in a 5-fold increased cellular association of the emulsion CO
core. These findings correspond well with the previously reported stimulatory effect of apoE on LDLr-mediated uptake of TG-rich emulsions
by J774 macrophages (33).
After cellular uptake of LDL, both its protein and lipid constituents
follow the same lysosomal pathway (19, 20), although the initial rate
of CO hydrolysis appears to be faster than apoB degradation (Fig. 4).
This may be explained by the fact that full protein degradation
(leading to release of 125I-Tyr) requires multiple
enzymatic steps, whereas CO hydrolysis is achieved by a single
enzymatic cut. In addition, the optimal conditions for efficient
hydrolysis by cholesteryl esterases may be reached at an earlier stage
in the endosomal pathway than for proteases. Emulsion-derived apoE is
much more resistant to intracellular degradation as compared with
LDL-derived apoB, as evident from a later onset of protein degradation
(120 versus 30 min) and a much lower degradation rate (Fig.
4). These data are in full agreement with the observed relative
intracellular stability of apoE as compared with apoB after uptake by
human fibroblasts and mouse J774 macrophages (30).
After pulse labeling of HepG2 cells with apoE emulsions at 18 °C,
and subsequent incubation at 37 °C, a time-dependent
release of intact apoE into the medium (14% of the initially
endocytosed apoE) could be detected. The release of apoE was increased
to up to 20 and 26% in the presence of the protein-free emulsion or
HDL in the medium, respectively. The gradual reappearance of intact
125I-apoE in the medium cannot be explained by a slow
release of apoE that was incompletely washed from the cell surface
after incubation at 18 °C, since lipid-free 125I-apoE
that was bound to the cell surface after incubation for 3 h at
4 °C was effectively removed by heparin (770 units/ml).
Previous pulse-chase experiments with radioiodinated VLDL also resulted
in the appearance of intact protein in the medium (28). Our present
data rule out that secretion of intact particles occurs upon
endocytosis but rather show that apoE can selectively undergo
retroendocytosis. By contrast, most LDL-derived apoB was recovered in
the medium in a degraded state (>95%), which confirms previous
observations (28, 62). It is thus clear that retroendocytosis is a
unique feature of apoE as compared with apoB. In this study, we have
not examined the intracellular fate of the other apolipoprotein constituents of TG-rich lipoproteins. A recent paper suggests that
apoCs may also be released from cells upon internalization (54).
We observed that apoE, which is released from cells by
retroendocytosis, can recombine with both the protein-deficient
emulsion and HDL. Both the presence of the emulsion and HDL in the
medium stimulated retroendocytosis of apoE, without an effect on apoE degradation. In all cases, the rate of apoE retroendocytosis may even
be underestimated, since it is known that a considerable amount of
newly synthesized apoE remains associated with cells instead of being
secreted into the medium (56, 63). In addition, newly synthesized apoE
that binds to the cell surface may partially be proteolytically
degraded upon re-entry into the cell (57). It has been shown before
that the presence of serum or isolated lipoproteins may prevent the
degradation of re-endocytosed apoE by triggering the release of apoE
from HepG2 cells (57) and macrophages (55), but it is not clear from
these studies and our observations whether the applied concentration of
HDL in the medium can extract all of the secreted apoE from the cell
surface, especially since radiolabeled apoE must compete with
HepG2-derived apoE for the binding to HDL.
In an attempt to examine the physiological relevance of our in
vitro findings for the situation in vivo, we also
determined the intrahepatic handling of the apoE-enriched emulsion
after intravenous injection into mice. Indeed, it appeared that after simultaneous hepatic uptake of both the particle core (reflected by the
CO moiety) and the preassociated apoE (45-50% of the injected dose at
20 min after injection), only 10-20% of the apoE is degraded in
contrast to as much as 75% of the CO moiety at 1 h after
injection (Figs. 7 and 8). It is tempting to assume that in
vivo, intrahepatic apoE may also undergo retroendocytosis,
resulting in the release of apoE from hepatocytes with subsequent
attachment to HSPG in the space of Disse or circulating lipoproteins
such as HDL. However, this hypothesis is hard to establish conclusively
under the present experimental conditions.
The mechanism of apoE retroendocytosis remains an intriguing issue. In
theory, apoE may be shuttled through CURL as is the case for
transferrin, which returns to the cell membrane after having delivered
its iron load (64, 65). The fact that the recycling of transferrin in
HepG2 cells occurs with a half-time of less than 10 min (64), whereas
apoE is slowly and gradually released from these cells after pulse
labeling, suggests that other mechanisms should account for escaping
degradation. A recent paper (54) indeed showed by microscopic analysis
that incubation of fibroblasts with TG-rich lipoproteins and
transferrin results in the appearance of apoE and transferrin in
distinct endosomal vesicles. Alternatively, apoE may escape degradation
via reversible aggregation into multimeric complexes at low pH, as
suggested by Chen et al. (66), but this hypothesis evidently
requires further investigation.
In conclusion, we have shown that apoE is relatively resistant to
degradation after cellular uptake by hepatoma cells in vitro and hepatocytes in vivo and that retroendocytosis of apoE
occurs at least in vitro. It is already known that newly
synthesized apoE that is secreted and bound to cell surface HSPG can be
re-endocytosed upon the binding of lipoproteins (so called
secretion-recapture pathway) (40, 41, 57). Taking these data together,
it can be envisioned that apoE, after synthesis and secretion by the hepatocyte, can be recycled by the cell several times until final degradation occurs. It may also be possible that endocytosed apoE is
involved in the assembly and secretion of VLDL by hepatocytes (34, 35).
Provided that the stability of endocytosed apoE can also be
demonstrated in extrahepatic cells such as macrophages, our data may
also implicate a role of retroendocytosed apoE in reverse cholesterol
transport and regression of atherosclerosis.
We thank Dr. Tikva Vogel (Bio-Technology
General, Ltd., Rehovot, Israel) for generously supplying human
recombinant apoE. We thank Patrick H. C. van Berkel (Pharming
Technologies BV, Leiden, The Netherlands) for providing bovine
lactoferrin and Dr. Hendrik N. J. Schifferstein (Department of
Marketing and Marketing Research, Agricultural University, Wageningen,
The Netherlands) for statistical analysis.
*
This work was supported by the Netherlands Heart Foundation
Grants 95128 and 97067.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.
§
To whom correspondence should be addressed: Division of
Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, University of Leiden, Sylvius Laboratory, P.O. Box 9503, 2300 RA Leiden, The
Netherlands. Tel.: 31 71 5276051; Fax: 31 71 5276032; E-mail: p.rensen@lacdr.leidenuniv.nl.
The abbreviations used are:
apoE, apolipoprotein
E;
bLf, bovine lactoferrin;
BSA, bovine serum albumin;
CO, cholesteryl
oleate;
DMEM, Dulbecco's modified Eagle medium;
FCS, fetal calf serum;
HSPG, heparan sulfate proteoglycans;
LDLr, low density lipoprotein
receptor;
LPL, lipoprotein lipase;
PBS, phosphate-buffered saline;
TG, triglyceride;
PAGE, polyacrylamide gel electrophoresis;
EYPC, egg yolk
phosphatidylcholine;
VLDL, very low density lipoprotein;
HDL, high
density lipoprotein.
Apolipoprotein E Is Resistant to Intracellular Degradation
in Vitro and in Vivo
EVIDENCE FOR RETROENDOCYTOSIS*
§,,, and
Division of Biopharmaceutics,
Leiden/Amsterdam Center for Drug Research, University of Leiden,
Sylvius Laboratory, P. O. Box 9503, 2300 RA Leiden,
¶ TNO-Prevention and Health, Gaubius Laboratory, P O. Box 2215,
2301 CE Leiden, and the Departments of Cardiology and General
Internal Medicine, Leiden University Medical Center, P. O. Box 9600,
2300 RC Leiden, The Netherlands
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
120 min) than LDL-derived 125I-apoB (30 min). Incubation at 18 °C, which allows endosomal uptake but
prevents lysosomal degradation, with subsequent incubation at 37 °C
resulted in a time-dependent release of intact apoE from the cells (up to 14% of the endocytosed apoE at 4 h). The release of apoE was accelerated by the presence of protein-free emulsion (20%)
or high density lipoprotein (26%). Retroendocytosis of intact particles could be excluded since little intact
[3H]cholesteryl oleate was released (<3%). In contrast,
the degradation of LDL was complete with virtually no secretion of
intact apoB into the medium. The intracellular stability of apoE was
also demonstrated after hepatic uptake in C57Bl/6 mice. Intravenous injection of 125I-apoE and [3H]cholesteryl
oleate-labeled emulsions resulted in efficient LDLr-mediated uptake of
both components by the liver (45-50% of the injected dose after 20 min). At 1 h after injection, only 15-20% of the hepatic
125I-apoE was degraded, whereas 75% of the
[3H]cholesteryl oleate was hydrolyzed. From these data we
conclude that following LDLr-mediated internalization by liver cells,
apoE can escape degradation and can be resecreted. This sequence of events may allow apoE to participate in its hypothesized intracellular functions such as mediator of the post-lysosomal trafficking of lipids
and very low density lipoprotein assembly.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-VLDL, VLDL,
and (LPL-treated) VLDL as compared with LDL after equally rapid
endocytosis by macrophages (24-26), human fibroblasts (27), and HepG2
cells (28), respectively. Another difference between LDL and TG-rich
lipoproteins may be that their apolipoprotein components differ in
their susceptibility to intracellular degradation. Preliminary data on
radioiodinated apoE emulsions and LDL suggest that, in contrast to the
efficient degradation of apoB, the degradation of apoE may be retarded, whereas fluorescently labeled lipids in TG-rich particles and LDL
followed a similar intracellular route toward lysosomes (29, 30).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C. Bovine lactoferrin
(bLf) was generously provided by Dr. Patrick van Berkel, Pharming
Technologies BV, Leiden, The Netherlands.
[1
,2-3H]Cholesteryl oleate ([3H]CO)
and 125I (carrier-free) in NaOH were purchased from
Amersham Pharmacia Biotech. Triolein (99% pure) and egg yolk
phosphatidylcholine (EYPC; 98%) were from Fluka, Buchs, Switzerland.
L--Lysophosphatidylcholine (99%), cholesterol (>99%),
bovine serum albumin (BSA, fraction V), ethyl mercurithiosalicylate,
monensin, and heparinase I (EC 4.2.2.7) from Flavobacterium
heparinum were obtained from Sigma. Cholesteryl oleate (CO; 97%)
was from Janssen, Beersse, Belgium. Peroxidase type II (200 units/mg),
Precipath® L, and EDTA were from Roche Molecular Biochemicals. HEPES
was from Merck, and heparin (5000 units/ml) was from Leo Pharmaceutical
Products B.V., Weesp, The Netherlands. Multiwell cell culture dishes
were from Costar, Cambridge, MA. Dulbecco's modified Eagle's medium
(DMEM) and fetal calf serum (FCS) were obtained from Flow Laboratories,
Irvine, UK. All other chemicals were of analytical grade.
0.204 + (0.0560 × body mass (g)).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol (not shown). After radiolabeling of apoE, 96% of
the 125I-activity could be recovered at the position of
monomeric apoE (34 kDa). Elution of radiolabeled apoE on a Superdex®
200 column showed mainly radioiodinated tetramers (eluting in between
mouse IgG and BSA) in addition to monomers (eluting similarly as
ovalbumin) and a small amount of higher aggregates (eluting before IgG)
(Fig. 1B). Gel filtration of 125I-apoE using
SDS-containing eluent confirmed the presence of a small portion of
125I-labeled homodimers (eluting at a position close to
that of BSA) (Fig. 1C) that disappeared upon reduction with
-mercaptoethanol (not shown). In contrast, emulsion-bound
125I-apoE was only monomeric, which confirms earlier
observations that the interaction of apoE homodimers with lipidic
particles does not withstand ultracentrifugation conditions
(48).
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Fig. 1.
Electrophoresis and chromatography of
(radiolabeled) apoE. A, unlabeled (lane 1)
and 125I-labeled (lanes 2 and 3) apoE
(4 µg) were added to SDS incubation buffer without
-mercaptoethanol (non-reducing conditions) and subjected to 10%
SDS-PAGE. After electrophoresis, proteins were stained with Coomassie
Blue R-250 (lanes 1 and 2), and
125I activity was detected by autoradiography
(3). B and C, lipid-free (
) and
emulsion-bound (
) 125I-apoE (1.6 µg) were eluted on a
Superdex® 200 column using 50 mM NaPi, 0.15 M NaCl, pH 7.4, in the absence (B) or presence
(C) of 0.5% SDS, and fractions were analyzed for
radioactivity. Arrows indicate the void volume
(V0) and the elution positions of mouse IgG
(Mr 155,000), BSA (Mr
66,000), and ovalbumin (Mr 45,000).
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Fig. 2.
Binding of apoE emulsions to HepG2
cells. A, cells were treated (2 h at 37 °C) with
heparinase (0-5 units/ml), washed, and incubated (3 h at 4 °C) with
[3H]CO-labeled or 125I-labeled apoE-enriched
emulsions (previously incubated at TG:apoE = 50:3.0; 60 µg of
TG/ml) or 125I-bLf (2 µg/ml). B and
C, alternatively, non-pretreated cells were incubated (3 h
at 4 °C) with [3H]CO-labeled or
125I-labeled apoE-enriched emulsions (60 µg of TG/ml) in
the presence of increasing concentrations of unlabeled apoE-enriched
emulsions or LDL. A-C, after incubation, cells were washed,
lysed, and cell protein and cell-associated radioactivities were
determined. Data are expressed as percentage of binding in the absence
of treatment or competitor (55.3 ± 3.4 and 51.5 ± 3.6 ng of
protein per mg of cell protein for [3H]CO-labeled and
125I-labeled emulsions, respectively). Values are
means ± S.D. of triplicate incubations.
120 min) and was to a much lower
extent (19% at 4 h). The emulsion particles were taken up as
unity since the total uptake of both the CO and apoE moieties (11.6 nmol of CO and 5.3 µg of apoE per mg of cell protein at 4 h) is
proportional to the ratio of these components in the emulsion (1.9 nmol
of CO per µg of apoE). The involvement of apoE in the cellular uptake
of the emulsion was confirmed by a 5-fold increase in the total CO uptake as compared with the apoE-free emulsion (not shown).
View larger version (23K):
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Fig. 3.
Intracellular processing and apoE emulsions
in HepG2 cells. Cells were incubated at 37 °C with
[3H]CO-labeled (A) or 125I-labeled
(B) apoE-enriched emulsions (previously incubated at
TG:apoE = 50:3.0; 60 µg of TG/ml). At the indicated times, the
cells were washed and lysed, and cell protein was determined.
Subsequently, total lipids were extracted, and non-hydrolyzed ( ) and
hydrolyzed () [3H]CO were separated by thin layer
chromatography. Alternatively, cellular associated
125I-activity was measured (), and
125I-degradation products in the medium () were
determined by 10% trichloroacetic acid precipitation. Values are
means ± S.D. of triplicate incubations.
View larger version (19K):
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Fig. 4.
Degradation rates of apoE versus
apoB in HepG2 cells. Cells were incubated at 37 °C with
[3H]CO- or 125I-labeled apoE-enriched
emulsions (60 µg of TG/ml) or LDL (20 µg of protein/ml). At the
indicated times, non-hydrolyzed and hydrolyzed [3H]CO as
well as cell-associated and degraded 125I-apolipoprotein
were determined as described in the legend to Fig. 1. The
apolipoprotein degradation rates (fraction-degraded
125I-apolipoprotein/fraction-hydrolyzed
[3H]CO) were calculated. Values are means ± S.D. of
triplicate incubations.
View larger version (25K):
[in a new window]
Fig. 5.
Intracellular processing of endocytosed apoE
in HepG2 cells. Cells were incubated for 3 h at 18 °C with
125I-apoE emulsions (previously incubated at TG:apoE = 50:3.0; 60 µg of TG/ml), extensively washed in the presence of 770 units/ml heparin, and further incubated in the absence (A)
and presence of emulsion (0.50 mg of TG/ml) (B) or HDL (0.35 mg of protein/ml) (C) starting at t = 0. At
the indicated times, the amount of cell-associated 125I
radioactivity ( ), 125I-degradation products in the
medium (), and trichloroacetic acid-precipitable
125I-protein (
) in the medium were determined. Data are
expressed as percentage of initially cell-associated 125I
activity (41.2 ± 4.4 ng of apoE per mg of cell protein). Values
are means ± S.D. of triplicate incubations.
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Fig. 6.
Density gradient ultracentrifugation profiles
of intact and degraded 125I-apoE in media of HepG2
cells. Cells were incubated with 125I-apoE emulsions
in the absence (A) or presence of emulsion (B) or
HDL (C) as described in the legend to Fig. 5. At 180 min,
media were collected and subjected to density gradient
ultracentrifugation. The gradients were subdivided from top
(fraction 1) to bottom (fraction 24),
and 10% trichloroacetic acid precipitation on 400-µl aliquots was
performed to discriminate between degraded ( ) and intact ()
protein. Insets, emulsion-containing fractions
(B, fractions 1 and 2) and HDL-containing
fractions (C, fractions 13-17) were dialyzed and
subjected to non-reducing 4-20% SDS-PAGE, and radioactivity was
visualized by imaging.
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Fig. 7.
Liver uptake and serum decay of apoE
emulsions in mice. [3H]CO-labeled ( ) or
125I-apoE-labeled () apoE emulsions (150 µg of TG,
corresponding to 5 µg of apoE) or lipid-free 125I-apoE (5 µg) (), were injected into anesthetized mice. At the indicated
times, the liver uptake (left) and serum decay
(right) were determined. Liver values are corrected for
serum radioactivity. Values are means ± variation of two
experiments.
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Fig. 8.
Intrahepatic degradation of apoE emulsions in
mice. [3H]CO-labeled (left) or
125I-apoE-labeled (right) apoE emulsions (150 µg of TG) were injected into anesthetized mice. At the indicated
times, liver lobules were taken, immediately frozen in liquid
N2, and homogenized in ice-cold PBS. After total lipid
extraction, intact ( ) and hydrolyzed () [3H]CO were
separated by thin layer chromatography. Alternatively, intact
125I-apoE () was separated from degradation products
() by 10% trichloroacetic acid precipitation. Values are means ± variation of two experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-VLDL to HSPG (60).
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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