From the Departments of Pathology,
Medicine,
and ** Pharmacology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232
Received for publication, January 9, 2001, and in revised form, March 29, 2001
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ABSTRACT |
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We have investigated the recycling of apoE in
livers of apoE Apolipoprotein E (apoE)1
is unique among the plasma lipoprotein apoproteins because of its many
different functions in the metabolism of lipids and lipoproteins. Its
role as a ligand for the receptor-mediated endocytosis of lipoproteins
is well established (1-3). Additionally, apoE interacts with
lipoprotein lipase and hepatic lipase to modulate triglyceride
hydrolysis (4, 5). Even within the cell, apoE is known to serve
biologically relevant roles. Studies suggest that apoE directs the
intracellular routing of internalized remnant lipoproteins, with
smaller Wong (15) was among the first to suggest that apoE may be recirculated.
He reported that 62-66% of the intrahepatic apoE, compared with
7-10% of the intrahepatic apoB, was derived from plasma. This
suggested that internalized apoE may not undergo complete degradation
and may therefore follow a unique intracellular routing pathway. We
previously reported that a portion of the apoE component of
internalized lipoproteins is spared degradation and routed through the
Golgi apparatus (16). After injection of radioiodinated mouse
d < 1.019 g/ml lipoproteins into C57BL/6 mice,
radioactivity found in the Golgi apparatus-rich fractions from livers
of the recipient mice was almost exclusively due to apoE, although the
injected lipoproteins had less than 10% of their label associated with
apoE. The ratio of apoE/apoB48 in the Golgi fraction increased 10-fold
relative to serum, and only traces of apoB100 were detected.
Furthermore, the apoE recovered in the Golgi fraction was part of a
d 1.019-1.210 g/ml complex, indicating an association of
recycled apoE with newly formed lipoproteins. Quantitatively, similar
results were obtained when labeled VLDL was injected into mice
deficient in either apoE or the LDL receptor, indicating that the
phenomenon of apoE recycling is not influenced by the production of
endogenous apoE and is not dependent on the presence of LDL receptors.
Although our studies clearly demonstrated the presence of a mechanism
in vivo by which apoE escapes degradation, the appearance of
internalized apoE within the liver Golgi may have been influenced in
part by the bolus delivery of apoE in this model.
To address this issue and to explore hepatic apoE recycling in the
steady state, we have completed experiments utilizing an in
vivo model based on bone marrow transplantation. Reconstitution of
lethally irradiated apoE Mice--
ApoE-deficient (apoE Bone Marrow Transplantation (BMT)--
BMT was carried out as
described previously (17). One week before and 2 weeks after BMT, 100 mg/liter neomycin and 10 mg/liter polymyxin B sulfate (Sigma) were
added to the water. Bone marrow was collected from donor mice by
flushing femurs with RPMI 1640 media containing 2% fetal bovine serum
and 10 units/ml heparin (Sigma). Recipient mice were lethally
irradiated (9 gray), and 4 h later, 5 × 106 bone
marrow cells in 0.3 ml were transplanted by tail vein injection. Animals were used for studies from 6 to 12 weeks after transplantation.
Isolation of Golgi Apparatus-rich Fractions and Nascent
Lipoproteins--
Golgi apparatus-rich fractions were prepared as
described previously (18). Golgi fractions were isolated from 4-5 mice
(total liver 5-6 g). Typically we are able to isolate 200-300 µg of
Golgi protein from 5-6 g of liver. Nascent lipoproteins were released from the Golgi apparatus using sodium carbonate treatment (19) and
lipoprotein classes isolated by ultracentrifugation as described below.
Preparation and Culture of Mouse Hepatocytes--
Hepatocytes
were isolated from mouse livers as described by Horton et
al. (20). The mice were anesthetized using a ketamine/xylazine mixture, and a midline incision was made. The portal vein was cannulated with a 24-gauge plastic cannula, and the liver was perfused
with Ca2+/Mg2+-free Honks' Balanced
Salt Solution containing glucose (10 mM) and HEPES (10 mM) at a flow rate of 3 ml/min for 10 min. The perfusate was then switched to liver digest medium containing collagenase (Life
Technologies, Inc.), and perfusion was continued for another 10 min.
The liver was removed from the animal, placed in collagenase media in a
culture dish, minced gently with scissors, and incubated at 37 °C
for 4 min. The dissociated cells were dispersed by shaking followed by
filtration through 100-µm nylon cell strainers (Falcon cell strainer,
Becton Dickinson, Franklin Lakes, NJ). The liver capsule and dish were
rinsed with ~10-15 ml of low glucose Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) containing 1% bovine serum albumin,
0.8 mM oleate, 0.167 µg/ml insulin (4 milliunits/ml), 0.02 µg/ml dexamethasone, and 100 units of penicillin and 100 µg of
streptomycin/ml. The cells were pelleted by gravity sedimentation for 5 min at 4 °C. The media was aspirated, leaving 5 ml total volume, and
fresh media was added to 20 ml. The cells were resuspended, and
viability was assessed by trypan blue exclusion. The yield of
hepatocytes ranged from 3 × 106 to 6 × 106 cells/g of liver, and viability was consistently
greater than 85%. The cells were plated onto 60-mm mouse collagen
IV-coated dishes (BioCoat, Becton Dickinson) at a density of 1.2 × 106 viable cells/dish in 2 ml of the above medium. The
media used during the isolation, plating, washing, and incubations did
not contain serum to avoid introduction of exogenous apoE into the system. Media was collected at 4, 21, and 45 h after plating, and
any cells or cell fragments were pelleted by centrifugation. Cells were
recovered at the end of the experiment (45-h time point), washed in
phosphate-buffered saline, and frozen.
Isolation of Lipoproteins from Cell Cultures and Golgi
Fractions--
Lipoproteins were adsorbed from the media using
Liposorb (PHM-L Liposorb, Calbiochem-Novabiochem) per the
manufacturer's recommendations. Liposorb (0.1 g) was suspended in 1.5 ml of normal saline, and 5 µl was added to 2 ml of media. The samples
were gently mixed for 30 min, and the lipoproteins were pelleted. In
our laboratory this procedure has been shown to adsorb ~70% of the
apoE in hepatocyte media and 100% of apoE in isolated lipoprotein
fractions (data not shown). Furthermore, apoE is adsorbed from large
and small lipoproteins with equal efficiency. The adsorbed proteins
were solubilized with the lithium dodecyl sulfate-solubilizing buffer and separated on SDS gels as described below. Intracellular
lipoproteins were recovered by treating the cells with 0.1 M sodium carbonate for 1 h at 0 °C (19). The
membranes and cell debris were pelleted by centrifugation using a
Beckman Optima TLX ultracentrifuge (Beckman Coulter) and 120.2 rotor
(120,000 rpm × 30 min). The supernatant was dialyzed against
normal saline, and lipoproteins were adsorbed using Liposorb as
described above.
Lipoproteins classes were isolated from Golgi contents and hepatocyte
culture media by ultracentrifugation using the Beckman tabletop
ultracentrifuge. Golgi lipoprotein classes (d < 1.006 g/ml and d 1.006-1.210 g/ml) were isolated as described
previously using a 120.2 rotor (18). Lipoproteins from the culture
media were recovered using the 100.4 rotor. The d < 1.006 g/ml lipoproteins were isolated in 6 h at 100,000 rpm and
recovered using tube slicing (Beckman Centritube Slicer). The density
of the infranatant was raised to 1.210 g/ml using solid KBr, and the
d 1.006-1.210 g/ml fraction was isolated in 13 h at
100,000 rpm and recovered by tube slicing. The fractions were dialyzed
and lyophilized before separation of the apoproteins by SDS gel electrophoresis.
Metabolic Labeling of Mouse Hepatocytes--
Primary hepatocytes
were isolated and cultured for 16 h in low glucose Dulbecco's
modified Eagle's medium containing 1% bovine serum albumin, 0.8 mM oleate as described above. Fresh media was added, and
the cells were pulsed for 60 min with Promix L-35S in
vitro cell-labeling mix (100 µCi/ml, Amersham Pharmacia
Biotech). The media was removed, and the cells were washed twice with
phosphate-buffered saline and chased for 3, 6, and 12 h in the
same media. ApoE was immunoprecipitated from the media using an
anti-human polyclonal antibody (Biodesign International, Saco, ME) and
Pansorbin (Calbiochem-Novabiochem) as described previously (21). The
immunoprecipitates were solubilized and separated by SDS-PAGE as
described below. The gels were fixed with water/methanol/acetic acid
(10:10:1 v/v/v), dried, and exposed to a Cyclone SR screen for periods
up to 46 h. ApoE was visualized using Cyclone Storage Phosphor
System with OptiQuant software (Packard Instrument Co.).
SDS-Polyacrylamide Gel Electrophoresis and
Immunoblotting--
Proteins were separated using a NOVEX system
(Invitrogen, Carlsbad, CA) with NuPAGE bis-tris gels (4-12%
gradients). The samples were solubilized in NuPAGE lithium dodecyl
sulfate sample buffer, and gels were run using the MOPS-SDS running
buffer. The proteins were transferred to nitrocellulose membranes using
the NOVEX system with NuPAGE transfer buffer containing 10% methanol.
The membranes were blocked in 5% nonfat milk, incubated with primary
antibodies, washed extensively, and incubated with horseradish
peroxidase-conjugated secondary antibodies. Bands were visualized using
enhanced chemiluminescence (Amersham Pharmacia Biotech).
In Vivo Hepatic VLDL Triglyceride Production--
Hepatic VLDL
triglyceride production was measured using Triton WR1339. Mice were
fasted overnight, anesthetized with ketamine/xylazine mixture, and
injected via the retro-orbital plexus with Triton WR1339 (800 mg/kg of
body weight). Triton (Sigma) was dissolved in normal saline at a
concentration of 25 mg/dl. Preliminary studies in our laboratory
demonstrated a linear increase in VLDL triglyceride over a 6-h period
using this concentration of Triton, providing strong evidence that
plasma VLDL clearance is completely inhibited under these conditions.
Blood samples (~50 µl) were taken from the retro-orbital plexus
before injection (0 h) and 1, 2, 3, and 4 h after Triton
injection. Plasma triglycerides were measured using enzymatic assays
adapted to microtiter plates (Raichem, San Diego, CA). Slopes of the
lines were determined by GraphPad Prism (v. 3.01). Hepatic triglyceride
production rates were calculated from the slopes and presented as
µmol of triglyceride produced/kg/h assuming the plasma volume to be
33 µl/g (22).
ApoE Recycling in ApoE Ex Vivo Resecretion of ApoE--
To determine if the apoE that was
internalized was resecreted, primary hepatocytes were isolated from
C57BL/6 mice and apoE+/+
The time course of appearance of apoE in the media of the hepatocytes
from C57BL/6 mice and apoE+/+ Synthesis and Secretion of ApoE in Primary Cultures of Mouse
Hepatocytes--
The secretion of radiolabeled apoE from primary
hepatocytes from C57BL/6 mice and apoE+/+ Density Distribution of Resecreted ApoE--
In the media from
C57BL/6 hepatocytes, apoE was distributed approximately equally between
the d < 1.006 and d 1.006-1.210 g/ml
fractions, whereas in the hepatocytes from apoE+/+ Hepatic Triglyceride Production Rate--
Hepatic triglyceride
production rates using the Triton method were measured in C57BL/6,
apoE The present study explored the recycling of apoE in the livers of
apoE Our finding of apoE with nascent lipoproteins in Golgi apparatus-rich
fractions from apoE+/+ The resecretion of apoE from primary cultures of hepatocytes from
apoE+/+ Although Kupffer cells in our hepatocyte cultures may contribute to the
apoE recovered in the media, our data suggest that their contribution
is minimal. Immunocytochemical staining using antibody to MOMA-2, an
established marker for macrophages, indicates that less than 1-2% of
the cells in culture is of macrophage origin. Dawson et al.
(27) report that Kupffer cells in rat liver account for less than 1%
(0.7%) of the total liver apoE mRNA and that the level of apoE
mRNA in Kupffer cells was approximately one-third that of
hepatocytes. The total recovery of apoE in the media of the hepatocytes
from apoE+/+ The content of apoE with nascent hepatic Golgi VLDL from
apoE+/+ The physiologic relevance of apoE reutilization and resecretion is
unknown. Recycling may provide a mechanism whereby the impact of apoE
on intracellular and extracellular functions can be maximized.
Accumulating evidence points to a critical role for apoE in VLDL
assembly. Hepatic VLDL triglyceride production is decreased by nearly
50% in the apoE In conclusion, apoE internalized by the liver as a component of
lipoprotein particles escapes the degradative pathway, is routed to the
Golgi apparatus, and is subsequently secreted in quantitatively
significant proportions. Further studies are needed to identify the
functional correlates of this newly described cellular process.
/
mice transplanted
with wild type bone marrow (apoE+/+
apoE
/
), a model in which circulating apoE
is derived exclusively from macrophages. Nascent Golgi lipoproteins
were recovered from livers of apoE+/+
apoE
/
mice 8 weeks after transplantation.
ApoE was identified with nascent d < 1.006 and with
d 1.006-1.210 g/ml lipoproteins at a level ~6% that of
nascent lipoproteins from C57BL/6 mice. Hepatocytes from
apoE+/+
apoE
/
mice were
isolated and cultured in media free of exogenous apoE. ApoE was found
in the media primarily on the d < 1.006 g/ml
fraction, indicating a resecretion of internalized apoprotein.
Secretion of apoE from C57BL/6 hepatocytes was consistent with
constitutive production, whereas the majority of apoE secreted from
apoE+/+
apoE
/
hepatocytes
was recovered in the last 24 h of culture. This suggests that
release may be triggered by accumulation of an acceptor, such as very
low density lipoproteins, in the media. In agreement with the in
vivo data, total recovery of apoE from apoE+/+
apoE
/
hepatocytes was ~6% that of the
apoE recovered from C57BL/6 hepatocytes. Since plasma apoE levels in
the transplanted mice are ~10% of control levels, the findings
indicate that up to 60% of the internalized apoE may be reutilized
under physiologic conditions. These studies provide definitive evidence
for the sparing of apoE and its routing through the secretory pathway
and demonstrate that internalized apoE can be resecreted in a
quantitatively significant fashion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-migrating very low density lipoproteins (VLDL) particles
routed to the perinuclear region of the mouse macrophage in a fashion
similar to low density lipoproteins (LDL), whereas larger
-VLDL
remain closer to the plasma membrane (6, 7). Schwiegelshohn et
al. (8) report that apoE modulates intracellular lipid metabolism,
in particular the hydrolysis and utilization of triglyceride. ApoE is
also linked to and may be a regulator of cholesterol efflux from
macrophages (9-11), an effect with significant repercussions on
vascular health and the process of atherogenesis. Finally, increasing
evidence points to a role for apoE in hepatic lipoprotein assembly and the incorporation of triglycerides into newly forming VLDL (12-14). Due to its critical role in a number of biological processes within the
cell, apoE may follow unique pathways of secretion and internalization that maximize its impact on cellular functions.
/
mice with wild
type bone marrow results in a mouse model in which all apoE present in
the mouse is derived from macrophages. Our laboratory (17) has shown
that within 3 weeks of the introduction of normal macrophages into the
apoE
/
mouse, serum apoE levels reach 10%
of normal, resulting in complete normalization of plasma cholesterol
levels by virtue of an active hepatic clearance of remnant
lipoproteins. Since hepatocytes in this model do not produce endogenous
apoE but internalize apoE on remnant lipoproteins, this approach
provides an excellent model for investigating the routing of
internalized apoE through the secretory pathway via the Golgi apparatus
in the absence of endogenously produced apoE. The studies reported in
this manuscript demonstrate clearly that internalized apoE undergoes a
unique routing through the secretory pathway and is resecreted.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
)
mice on the C57BL/6 background were obtained from The Jackson
Laboratory (Bar Harbor, ME). A colony of C57BL/6J mice is established
in our animal facility. All mice were kept on a 12-h light/12-h 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 animal procedures were carried out in accordance with institutional
guidelines with approval from the Animal Care Committee of Vanderbilt University.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Mice Transplanted with Bone
Marrow from C57BL/6 Mice--
ApoE
/
mice
were transplanted with bone marrow from C57BL/6 mice
(apoE+/+
apoE
/
), and 8 weeks post-BMT, hepatic Golgi apparatus-rich fractions were isolated.
Previous studies in our laboratory show that the Golgi fractions are
enriched in galactosyl transferase with minimal contamination from
components of the endocytic compartment (18). Reconstitution of
apoE
/
mice with marrow from C57BL/6 mice
leads to the appearance of apoE on plasma lipoproteins, promoting
clearance of lipoprotein remnants and normalization of plasma
cholesterol (17). In C57BL/6 mice, apoE was easily detected in the
d < 1.006, d 1.006-1.210, and
d > 1.210 g/ml fractions (Fig.
1), and the relative distribution of apoE
among the three fractions as determined by densitometric scanning was
~55, 40, and 5%, respectively. ApoE was also found in the nascent
Golgi lipoproteins from the apoE+/+
apoE
/
mice (Fig. 1), with a distribution
that was very similar to that of control C57BL/6 preparations (~50%
in the d < 1.006 and 50% in the d
1.006-1.210 g/ml fractions). No apoE was detected in the
d > 1.210 g/ml fraction, perhaps reflecting the small
amount of total apoE recovered with the Golgi fractions. The apoE on nascent hepatic Golgi lipoproteins from apoE+/+
apoE
/
animals normalized to the amount of
apoB48 was ~6% that found with Golgi lipoproteins from control
animals
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Fig. 1.
Nascent hepatic Golgi lipoproteins from
C57BL/6 mice and apoE+/+ apoE
/
mice. ApoE
/
mice were transplanted
with marrow from C57BL/6 mice, and 8 weeks post-BMT, hepatic Golgi
apparatus-rich fractions were isolated and nascent lipoproteins
recovered. The apoproteins were separated by SDS-PAGE, blotted to
nitrocellulose, and probed for apoB and apoE.
apoE
/
mice. Fig.
2 presents a micrograph of the cultured
hepatocytes from C57BL/6 mice stained with hematoxylin and
eosin. The cultures were also immunostained for the presence of
Kupffer cells using the monocyte-macrophage marker MOMA-2. Cell
counts from numerous fields from three different preparations showed
less than 1-2% contamination of the hepatocyte cultures with Kupffer
cells. Fig. 3 shows the results of
experiments in which the media from the first two time points (4 and
21 h) were combined, and lipoproteins were adsorbed using
Liposorb. As can be seen, apoE is clearly present in the media from the
apoE+/+
apoE
/
mice. This
suggests that apoE, internalized in vivo, is routed to the
secretory pathway and is resecreted from the hepatocytes in
culture.
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Fig. 2.
Hepatocytes from apoE+/+
apoE
/
mice. Hepatocytes were isolated as described under "Experimental
Procedures," grown on coverslips coated with mouse type IV collagen,
fixed, and stained with hematoxylin and eosin (A)
or with antibody to MOMA-2 (B), a macrophage marker. Less
than 1-2% of the cells were stained with MOMA-2.
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Fig. 3.
Resecretion of apoE from hepatocytes from
apoE+/+ apoE
/
mice. ApoE
/
mice were transplanted
with bone marrow from C57BL/6 mice, and 6 weeks post-BMT, hepatocytes
were isolated as described under "Experimental Procedures."
Hepatocytes from nontransplanted C57BL/6 mice served as control. Media
from the 4 and 21 h time points was combined, and lipoproteins
were adsorbed using Liposorb. Apoproteins were separated by SDS-PAGE,
blotted to nitrocellulose, and probed for apoE.
apoE
/
mice is shown in Fig.
4A. ApoE was present in the
media from the C57BL/6 hepatocytes at each time point. The mass of apoE
appeared proportional to the time in culture. In addition, apoE was
found in the cells at the end of the experiment. ApoE was also observed in the media of the hepatocytes from apoE+/+
apoE
/
mice. At the early time points,
little apoE was detected, but at the later time points there appeared
to be a greater release of the apoprotein. There was little to no apoE
detected in the cells from the apoE+/+
apoE
/
mice at the end of the 45-h culture
period. Furthermore, the cells appeared healthy after the 45-h culture
period, with no evidence of massive cell death. In C57BL/6 hepatocytes,
~10% of the total apoE was secreted in the first 4 h with 40%
secreted in the next 17 h and 49% secreted in the last 24 h
(Fig. 4B). This suggests a constant production of apoE
throughout the culture period. In contrast, in hepatocytes from the
apoE
/
mice transplanted with C57BL/6
marrow, only 1.7% of the total secreted apoE was recovered in the
media after 4 h, whereas 15.2% appeared in the next 17 h,
and ~83% was recovered in the final 24 h of incubation (Fig.
4B). In two separate experiments the total apoE secreted
from the BMT hepatocytes over the 45-h period was 6.5% of the apoE
secreted from C57BL/6 hepatocytes over the same period.
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Fig. 4.
Time course of resecretion of
apoE from hepatocytes from apoE+/+ apoE
/
mice. Hepatocytes were isolated from
apoE+/+
apoE
/
mice and
cultured as described under "Experimental Procedures." Media was
collected at each time point, and cells were recovered at the end of
the experiment. Cellular and media lipoproteins were adsorbed using
Liposorb. Apoproteins were separated by SDS-PAGE, blotted to
nitrocellulose, and probed for apoB and apoE (A).
Hepatocytes from nontransplanted C57BL/6 mice served as control. Blots
were scanned, and the appearance of apoE in the media was
expressed as percent of total secreted apoE (B). Data
are expressed as mean ± S.D.; n = 3.
apoE
/
mice is shown in Fig.
5. Newly synthesized apoE was easily
detected in the media from C57BL/6 hepatocytes but was not detected in the hepatocyte cultures from apoE+/+
apoE
/
mice at any of the time points. As
expected, the recycling of unlabeled apoE was observed at all time
points (data not shown). These results demonstrate clearly that apoE in
the media of hepatocytes from apoE+/+
apoE
/
mice does not derive from
contaminating Kupffer cells but represents apoE internalized in
vivo and subsequently resecreted in culture.
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Fig. 5.
Synthesis and secretion of apoE in primary
cultures of mouse hepatocytes. ApoE /
mice were transplanted with bone marrow from C57BL/6 mice, and
25-day-post-BMT hepatocytes were isolated as described under
"Experimental Procedures." Hepatocytes from nontransplanted C57BL/6
mice served as control. The cells were cultured for 16 h in
Dulbecco's modified Eagle's medium supplemented with 0.8 mM oleate. The media was replaced with fresh media, and the
cells were labeled for 60 min with Promix L-35S in
vitro cell-labeling mix (100 µCi/ml). At the end of the pulse,
the media was removed, and the cells were washed twice with
phosphate-buffered saline and chased for 3, 6, and 12 h in the
same media. ApoE was immunoprecipitated from the media, the
immunoprecipitates were solubilized, and the proteins were separated by
SDS-PAGE. The gels were fixed in water/methanol/acetic acid (10:10:1
v/v/v), dried, and exposed to a Cyclone SR screen. ApoE was visualized
using Cyclone Storage Phosphor System. Lanes 1,
3, 5, C57BL/6; lanes 2, 4,
6, apoE+/+
apoE
/
.
apoE
/
mice, most of the apoE was found with
the d < 1.006 g/ml fraction (Fig.
6). This may reflect the affinity of apoE
for triglyceride-rich lipoproteins in the media that are
apoE-deficient, but it may also suggest that apoE-deficient
triglyceride-rich lipoproteins in the media stimulate apoE release from
the cells, or that recycling apoE induces assembly of triglyceride-rich
lipoproteins, a possibility that was investigated in the next set of
experiments.
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Fig. 6.
Ultracentrifugal distribution of apoE in
media from hepatocytes from apoE+/+
apoE
/
mice. Hepatocytes were isolated from apoE+/+
apoE
/
mice and cultured as described under
"Experimental Procedures." Media collected at the 45-h time point
was dialyzed against saline, and lipoproteins were isolated using the
Beckman Optima TLX ultracentrifuge. Apoproteins from each fraction were
separated by SDS-PAGE, blotted to nitrocellulose, and probed for apoB
and apoE. Hepatocytes from nontransplanted C57BL/6 mice served as
control.
/
mice, apoE+/+
apoE
/
mice, and
apoE
/
mice transplanted with
apoE
/
marrow. The production rates for all
groups were linear over the time period, with r values of
0.947-0.993 for the four groups. As has been shown previously (13,
14), hepatic triglyceride production rates in
apoE
/
mice were ~50% that found in
C57BL/6 mice (Fig. 7). Hepatic
triglyceride production rates in mice transplanted with marrow from
either apoE+/+ or apoE
/
mice
were similar and not significantly different from the rates found in
apoE
/
mice, suggesting that any effect of
recycling apoE on triglyceride production is not of the magnitude to
induce a bulk increase in lipoprotein accumulation.
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Fig. 7.
Hepatic triglyceride (TG)
production rates. Hepatic triglyceride production was measured
using the Triton method in C57BL/6 mice (C57BL/6),
apoE /
mice (E
/
),
apoE
/
mice transplanted with marrow from
C57BL/6 mice (E+/+
E
/
),
and apoE
/
mice transplanted with marrow
from apoE
/
mice
(E
/
E
/
). Triglyceride
secretion was linear over the 4-h period, and hepatic triglyceride
production rates were calculated from the slopes of the lines using
GraphPad Prism (version 3.01).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice transplanted with wild type bone
marrow. In this model all of the apoE derives from macrophages, and
plasma apoE levels are at steady state, with the production of apoE by
macrophages and its internalization and potential resecretion by the
liver at equilibrium. Therefore any apoE appearing in the secretory
pathway in the liver or recovered in the media of hepatocytes isolated from these mice is derived from internalized extrahepatic apoE. Using
the BMT model we have demonstrated the presence of apoE within nascent
hepatic Golgi lipoproteins, providing strong evidence that at least a
portion of internalized extrahepatic apoE is routed to the secretory
pathway. Additional studies using primary hepatocytes from the
apoE+/+
apoE
/
mice not only
confirmed the routing of internalized apoE to the secretory pathway but
also demonstrated the resecretion of internalized apoE. These results
coupled with our earlier in vivo studies (16) provide
overwhelming support for the hypothesis that a portion of apoE that is
internalized with lipoproteins is not completely degraded but is in
fact resecreted. The physiologic relevance of the reutilization and
recycling of apoE is unknown. Our studies show that introduction of
apoE via BMT does not affect hepatic triglyceride production rates, but
the lack of an effect may simply reflect suboptimal concentrations of
intracellular recycling apoE. Alternatively, the functional role of
recycling apoE may be linked to effects that are independent from or
more subtle than the bulk secretion of triglyceride-rich lipoproteins.
apoE
/
mice in the present study (Fig. 1)
provides definitive evidence for the routing of internalized apoE
through the secretory pathway under physiologic conditions. The finding
of apoE within the Golgi fractions was not due to contamination of the
preparations with elements of the endocytic compartment, as studies in
our laboratory have established the purity of the hepatic Golgi
fractions by this technique (18). Contamination of our fractions with
Golgi from Kupffer cells, a possible source of apoE, is not expected to
play a role here since engraftment of Kupffer cells in liver is less
than 20% at 8 weeks and only ~35% of total macrophages 6 months
after transplantation (23). In addition, it is unlikely that Kupffer
cell Golgi apparatus isolates with hepatocyte Golgi apparatus, as
Kupffer cells do not produce triglyceride-rich lipoproteins that
contribute to the buoyancy of the hepatocyte-derived organelle (24).
Therefore the appearance of apoE in the hepatic Golgi fractions of the
apoE+/+
apoE
/
mice
represents routing of internalized apoE through the secretory pathway.
apoE
/
mice provided
additional evidence for apoE recycling. In contrast to the constitutive
pattern of secretion of apoE from hepatocytes from C57BL/6 mice, the
majority of apoE recovered in the media of hepatocytes from
apoE+/+
apoE
/
mice was
released in the last 24 h (Fig. 4). This release was not caused by
cell death, as the cells from both control and transplanted animals
appeared healthy after the 45-h culture period. Rensen et
al. (25), studying the retroendocytosis of apoE from HepG2 cells,
report that the presence of high density lipoproteins or lipid emulsion
in the media resulted in a significantly increased rate of secretion of
intact protein compared with media with no acceptor. Heeren et
al. (26) also report that high density lipoprotein seemed to serve
as an extracellular acceptor for the resecretion of apoE from
fibroblasts. Although in our experiments the media did not contain an
exogenous acceptor to stimulate the release of apoE, the hepatocytes do
secrete apoE-deficient VLDL (Fig. 1), which may serve as acceptor for
and stimulator of apoE release from hepatocytes. A threshold
concentration of apoE-deficient VLDL, attainable only during the 24-h
culture period, may be required to trigger the release of apoE. In this
regard, it is important to note that apoE recovered in the media is
found mainly with the d < 1.006 g/ml lipoproteins
(Fig. 6).
apoE
/
mice is
~5-6% of apoE recovered in the media from C57BL/6 hepatocytes. Assuming the secretion of apoE is proportional to the apoE mRNA content of the Kupffer cells and hepatocytes, the number of Kupffer cells in our preparation cannot account for the mass of apoE found in
the media. In addition, the kinetics of appearance of apoE in the media
is not consistent with what might be predicted for a constitutively
secreted apoprotein as observed in the secretion of apoE from C57BL/6
hepatocytes, and as noted above, the engraftment of liver Kupffer cells
in mice after BMT is less than 20% of total tissue macrophages 8 weeks
after transplantation (23). Finally and perhaps most importantly, newly
synthesized apoE could not be detected in hepatocyte cultures from
apoE+/+
apoE
/
mice (Fig.
5). Therefore, based on the mass of apoE found in the media, the number
of cells identified as Kupffer cells using immunocytochemistry, the
secretion kinetics, the kinetics of engraftment of liver Kupffer cells,
and the absence of newly synthesized apoE in hepatocyte cultures from
apoE+/+
apoE
/
mice, we
conclude that the apoE recovered in the media does not derive from
Kupffer cells but represents recycled apoE.
apoE
/
mice was
~6% that found with control Golgi VLDL, and the mass of apoE
released from hepatocytes from apoE+/+
apoE
/
mice was ~6% that of apoE secreted
by hepatocytes from C57BL/6 mice. Since serum apoE levels in the
apoE+/+
apoE
/
mice are
~10% of control levels (17), the results suggest that as much as
60% of the internalized apoE is reutilized. Under normal conditions,
when the hepatocyte is faced with 10 times more apoE, the amount of
apoE that is recycled may change. Heeren et al. (26)
incubated radiolabeled apoE-containing triglyceride-rich lipoproteins
with Hep3b cells and fibroblasts and demonstrated that ~60% of the
labeled apoprotein internalized by the cell was released intact into
the medium after a 90-min chase. The released apoproteins were
primarily apoE and apoC. Rensen et al. (25) also report that
as much as 26% of the apoE taken up on triglyceride-rich emulsions by
HepG2 cells was released. Finally, Takahashi and Smith (28) estimate
that as much as 30% of apoE internalized by the murine macrophage RAW
264 cell line was resecreted. It seems clear that the process of apoE
sparing and reutilization may be variable among different cells but is
not simply limited to the liver and is quantitatively important.
/
mice compared with
C57BL/6 mice (13, 14), and the reconstitution of apoE expression via
adenoviral transfer with the human apoE gene markedly increases hepatic
VLDL triglyceride production (29). Our studies confirm that hepatic
triglyceride production rates are decreased in
apoE
/
mice compared with C57BL/6 mice (Fig.
7). However, introduction of apoE via BMT did not alter this rate,
suggesting that apoE is routed to a site in the secretory pathway
distal to VLDL assembly or triglyceride production. Alternatively,
introduction of apoE via BMT, an intervention that reconstitutes only
10% of the total serum apoE, may not provide intracellular
concentrations of apoE needed to affect hepatic triglyceride production
and VLDL secretion. Finally, it is plausible that recycling apoE may
have other effects, such as increasing remnant uptake through the
secretion-capture mechanism (30-32).
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL57984 and HL57986.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: Dept. of Pathology Vanderbilt University School of Medicine, Nashville, TN 37232-2561. Tel.: 615-343-2646; Fax: 615-343-7023; E-mail: larry.swift, sergio.fazio or macrae.linton@mcmail.vanderbilt.edu.
¶ Supported by a Vascular Biology Training Grant (National Institutes of Health Grant 5T32 HL07751 (NHLBI)).
Established Investigator of the American Heart
Association
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M100172200
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ABBREVIATIONS |
---|
The abbreviations used are: apo, apolipoprotein; LDL, low density lipoprotein; VLDL, very LDL; BMT, bone marrow transplantation; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
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