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J. Biol. Chem., Vol. 277, Issue 35, 31516-31525, August 30, 2002
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From the Canadian Institutes for Health Research Group in Molecular
and Cell Biology of Lipids, and the Department of Medicine, University
of Alberta, Edmonton, Alberta T6G 2S2, Canada
Received for publication, February 28, 2002, and in revised form, June 17, 2002
The assembly of very low density
lipoproteins in hepatocytes requires the microsomal
triacylglycerol transfer protein (MTP). This microsomal lumenal protein
transfers lipids, particularly triacylglycerols (TG), between membranes
in vitro and has been proposed to transfer TG to nascent
apolipoprotein (apo) B in vivo. We examined the role of MTP
in the assembly of apoB-containing lipoproteins in cultured murine
primary hepatocytes using an inhibitor of MTP. The MTP inhibitor
reduced TG secretion from hepatocytes by 85% and decreased the amount
of apoB100 in the microsomal lumen, as well as that secreted into the
medium, by 70 and 90%, respectively, whereas the secretion of apoB48
was only slightly decreased and the amount of lumenal apoB48 was
unaffected. However, apoB48-containing particles formed in the presence
of inhibitor were lipid-poor compared with those produced in the
absence of inhibitor. We also isolated a pool of apoB-free TG from the
microsomal lumen and showed that inhibition of MTP decreased the amount
of TG in this pool by ~45%. The pool of TG associated with apoB was
similarly reduced. However, inhibition of MTP did not directly block TG transfer from the apoB-independent TG pool to partially lipidated apoB
in the microsomal lumen. We conclude that MTP is required for TG
accumulation in the microsomal lumen and as a source of TG for assembly
with apoB, but normal levels of MTP are not required for transferring
the bulk of TG to apoB during VLDL assembly in murine hepatocytes.
During assembly of very low density lipoproteins
(VLDLs)1 in the liver,
triacylglycerol (TG) is concentrated within the hydrophobic core of
apolipoprotein (apo) B-containing lipoprotein particles. A microsomal
lumenal protein, the microsomal triacylglycerol transfer protein (MTP),
has been implicated in the acquisition of TG by nascent apoB for
assembly and secretion of VLDLs (reviewed in Refs. 1-4). Individuals
with the rare inherited disease abetalipoproteinemia have a defect in
the MTP gene and lack detectable MTP protein and MTP
lipid transfer activity (5). Despite a normal apoB gene,
plasma apoB is barely detectable in these patients. MTP has the ability
to transfer TG and other lipids, including cholesteryl esters,
diacylglycerols, and phospholipids, between membranes in
vitro (6) and has been proposed to transfer TG to nascent apoB-containing lipoproteins in vivo. This idea is
consistent with immunoprecipitation studies showing that apoB and MTP
interact physically at early stages of VLDL assembly (7-9). MTP is
expressed primarily in the liver and intestine as a soluble heterodimer with protein-disulfide isomerase (55 kDa) (10), a ubiquitous protein of
the endoplasmic reticulum (ER) lumen that catalyzes disulfide bond
formation during protein folding (11). The 97-kDa MTP subunit confers
all lipid transfer activity to the heterodimer (12). In in
vitro assays, the lipid transfer activity of MTP displays Ping
Pong Bi Bi kinetics implying that MTP transfers lipids between
membranes via a "shuttle" mechanism (13). The tissue and
subcellular location of MTP, and its preference for transferring
neutral lipids between membranes in vitro, have suggested that MTP participates in the loading of apoB with TG.
Specific inhibitors of the MTP lipid transfer activity have been
developed that lower plasma cholesterol levels by up to 80% in
rabbits, hamsters, and rats (14). Complete elimination of the
MTP gene in mice is embryonically lethal, probably because apoB-containing lipoproteins are required for transferring lipids from
the yolk sac to the developing embryo (15). However, viable mice were
generated in which the MTP gene was specifically inactivated in the liver (16, 17). In these mice, compared with wild-type mice,
plasma apoB100 levels were reduced by >90% but apoB48 levels were
reduced by only ~20% (16). In support of the hypothesis that MTP
provides TG for VLDL assembly, the majority of apoB48 in the plasma of
these MTP-deficient mice was present in lipoprotein particles that were
only partially lipidated (16). Moreover, apoB100 secretion from
cultured MTP MTP has been reported to play several roles in VLDL assembly: (i) as a
chaperone that mediates the translocation of newly synthesized apoB
across the ER membrane (20-22); (ii) in the co-translational lipidation of apoB during its translocation into the ER lumen (22-24);
(ii) in the transfer of the bulk of TG to poorly lipidated apoB in the
ER lumen (25, 26); and (iv) in the transfer of TG into the ER lumen for
assembly into VLDLs (16, 18). The majority of studies on the role of
MTP have been performed in cultured hepatoma cells, either HepG2 or
McArdle 7777 cells, that compared with primary hepatocytes,
inefficiently secrete VLDLs (21-23, 27-30). Moreover, in hepatoma
cells, the activities of several enzymes involved in lipid metabolism
are either absent or impaired. The function of MTP has also been
investigated in HeLa and COS cells that do not normally secrete
lipoproteins but in which apoB secretion was induced by transfection
with cDNAs encoding MTP and apoB variants (31-33). Some of the
conflicting data on the role of MTP in VLDL assembly might, therefore,
be related to the different model systems used. Only a few studies have
been performed on the function of MTP in primary hepatocytes (34-36).
Although the role of MTP has been investigated in liver-specific MTP
knock-out mice (16, 17), detailed studies were not reported on the
function of MTP in hepatocytes from these mice. Because many types of
genetically modified mice are now frequently used as models of human
lipoprotein metabolism it is important to understand the mechanism of
lipoprotein assembly in murine hepatocytes.
We have investigated the requirement of MTP for VLDL assembly and
secretion in murine primary hepatocytes. Our data show that secretion
of apoB100 is markedly more sensitive to inhibition of MTP than is
apoB48, and that when MTP is inhibited the apoB48 particles produced
are lipid-poor compared with those made in hepatocytes with normal MTP
activity. We have also isolated from the microsomal lumen a pool of TG
that is not associated with apoB and show that inhibition of MTP
reduces the amount of TG within this pool. However, inhibition of MTP
does not directly impair the transfer of TG from this pool to
lipid-poor apoB-containing particles.
Materials--
Dulbecco's modified Eagle's medium
(DMEM), Hanks' solution, collagenase, penicillin, streptomycin, and
fetal bovine serum were obtained from Invitrogen. Minimal
essential medium (methionine-free), fatty acid-free bovine serum
albumin, oleic acid, phenylmethylsulfonyl fluoride, leupeptin,
N-acetyl-Leu-Leu-norleucinal, lactacystin, the protease
inhibitor MG132, trypsin, soybean trypsin inhibitor, triolein standard
for thin-layer chromatography, digitonin, collagenase type I, insulin,
and cycloheximide were purchased from Sigma. Complete protease
inhibitor mixture tablets were from Roche Molecular Biochemicals
(Laval, PQ, Canada). [35S]Methionine,
[3H]oleic acid, protein A-Sepharose CL-4B, Amplify, and
rainbow protein molecular weight markers were from Amersham
Biosciences (Baie d'Urfe, PQ). Anti-human apolipoprotein B
antibodies were purchased from Roche Molecular Biochemicals. BIOCOAT
collagen-coated cell culture dishes and thin-layer chromatography
plates (Silica Gel G, 0.25 mm thickness) were from VWR (Mississauga,
Ontario, Canada). All chemicals used for SDS-polyacrylamide gel
electrophoresis were from Bio-Rad. The MTP inhibitor, BMS-197636 (a
generous gift from Dr. Haris Jamil, Bristol-Myers Squibb), was
dissolved in dimethyl sulfoxide. The final concentration of this
solvent in medium was 0.001%. C57Bl/6 mice and the
LDLR Isolation and Culture of Murine Primary Hepatocytes--
Adult
male C57/Bl6 mice that had been fed chow and water ad
libitum were anesthetized by intraperitoneal injection of Somnotol (0.022 ml/50 g body weight). A mid-line incision was made and Hanks'
EGTA solution containing 1 mg/ml insulin was perfused through the
portal vein until the liver was clear of blood. The upper and lower
vena cava were tied and the perfusion was continued with Hanks'
collagenase solution (100 units/ml) containing 1 mg/ml insulin until
the liver became soft (~3 min). The liver was removed, cut into
pieces, transferred to Hanks' collagenase solution, and mixed until
all clumps of tissue were removed. The hepatocytes were washed 3 times
in DMEM, then suspended in DMEM containing 10% fetal bovine serum.
Cells were plated on collagen-coated dishes (60 or 100 mm, 1 × 106 cells/ml). Cell viability (typically >90%) was
estimated by trypan blue exclusion. After hepatocytes had attached to
the dish (2 to 3 h), fresh DMEM containing 10% fetal bovine serum
was added and the cells were incubated overnight. The next morning, the medium was replaced with fresh serum-free medium containing 0.375 mM oleate and 10% bovine serum albumin.
Metabolic Labeling of Proteins and Lipids--
For experiments
in which proteins were labeled with [35S]methionine,
hepatocytes were washed twice with methionine-free minimal essential
medium containing 0.375 mM oleate and incubated
±MTP inhibitor in the same medium for 2 h to deplete the
intracellular methionine pool. Next, the cells were incubated with
methionine-free minimal essential medium containing
[35S]methionine (100 µCi/ml), 0.375 mM
oleate, and 10% bovine serum albumin in the presence or absence of MTP
inhibitor. For labeling of TG, hepatocytes were preincubated with DMEM
(no oleate) ±MTP inhibitor for 2 h followed by addition of
[3H]oleate (5 or 10 µCi/ml).
Isolation of Microsomal Lumenal Contents--
Cells were
collected then homogenized in buffer containing 250 mM
sucrose and 300 mM imidazole (pH 7.4) using a hand-operated Dounce homogenizer (37). Cell extracts were centrifuged for
10 min in a microcentrifuge at 14,000 rpm. Lumenal contents were released from microsomes in the supernatant by treatment with 0.1 M sodium carbonate (pH 11) for 25 min at 21 °C (38)
after which bovine serum albumin (5 mg/ml) was added. The samples were centrifuged at 34,000 rpm in a Beckman SW55 rotor for 90 min at 21 °C to separate microsomal membranes (pellet) from lumenal
contents (supernatant). Lumenal contents were adjusted to pH 7.4 by
addition of 10% acetic acid.
Immunoprecipitation of ApoB from Cells, Culture Medium, and
Lumenal Contents--
For immunoprecipitation of cellular apoB,
hepatocytes were washed with phosphate-buffered saline and then
homogenized in solubilization buffer containing Tris-HCl (0.63 M, pH 7.4), NaCl (0.75 M), EDTA (25 mM), phenylmethylsulfonyl fluoride (5 mM), and
Triton X-100 (5%, v/v). Cellular extracts were centrifuged in a
microcentrifuge at 14,000 rpm for 10 min, then the supernatant (1 ml)
was incubated overnight at 4 °C with anti-human apoB antibody (7.5 µl). Next, 45 mg of protein A-Sepharose was added and the sample was
mixed end-over-end for 2 h at 4 °C. The apoB-protein A complex
was pelleted by centrifugation for 2 min at 14,000 rpm. For
immunoprecipitation of secreted apoB, culture medium was centrifuged
for 2 min at 3,000 rpm to remove cell debris prior to
immunoprecipitation of apoB as described above. For immunoprecipitation
of apoB-containing lipoproteins from microsomal lumenal contents under
nondenaturing conditions, 1 ml of lumenal contents was incubated
overnight at 4 °C with 7.5 µl of anti-apoB antibody in the absence
of detergent, protein A was then added as described above.
Immunoprecipitated apoB was solubilized by boiling the
immunoprecipitate in buffer containing 6 mM Tris-HCl, 10%
glycerol, 2% SDS, and 5% Density Gradient Separation of Lipoproteins in Culture Medium and
Microsomal Lumenal Contents--
Lipoproteins in the microsomal lumen
or culture medium were separated according to density by
ultracentrifugation on a sucrose gradient. The sucrose concentration of
the samples (5 ml) was adjusted to 12.5% (w/v), the samples were then
placed on the top of a step gradient consisting of 2 ml of 49% sucrose
and 2 ml of 20% sucrose. The tube was filled with phosphate-buffered
saline. All solutions contained a mixture of protease inhibitors. The tubes were ultracentrifuged in a Beckman SW40 rotor at 35,000 rpm for
60 h at 12 °C (39). Eleven or 12 1-ml fractions with densities
ranging from 1.006 to 1.21 g/ml were sequentially removed from the top
of the tube. The density of each fraction was determined as the weight
of 1 ml.
Permeabilization of Hepatocytes with Digitonin--
Hepatocytes
were harvested and permeabilized by treatment with digitonin (75 µg/ml) in buffer containing 0.3 M sucrose, 0.1 M KCl, 2.5 mM MgCl2, 1 mM sodium-free EDTA, and 10 mM PIPES (pH 6.8)
for 5 min on ice. The digitonin concentration and time of incubation
required for permeabilization of the cells were established by
monitoring the release of the cytosolic protein, lactate dehydrogenase, from the cells (40). Under the conditions used, >85% of total lactate
dehydrogenase activity was released.
Trypsin Digestion of Proteins in Permeabilized Cells--
For
trypsin digestion of proteins exposed to the cytosol,
digitonin-permeabilized cells were washed with buffer containing 0.3 M sucrose, 0.1 M KCl, 2.5 mM
MgCl2, 1 mM sodium-free EDTA, and 10 mM PIPES (pH 6.8). Trypsin (100 µg/ml) was then added in the same buffer and the sample was incubated for 10 min on ice. Proteolysis was terminated by addition of soybean trypsin inhibitor (1 mg/ml), phenylmethylsulfonyl fluoride (1 mM), and leupeptin (0.1 mM) for 10 min on ice. Confirmation that trypsin had
degraded proteins exposed on the cytosolic surface of ER membranes was obtained by analysis of the trypsin sensitivity of two microsomal membrane proteins whose active sites are known to be exposed to the
cytosol:phosphatidylethanolamine N-methyltransferase (41) and CDP-choline:diacylglycerol cholinephosphotransferase (41).
Analysis of Radiolabeled TG Associated with ApoB in Culture
Medium, Lumenal Contents, and Cytosol--
Lipids were extracted by
the method of Folch et al. (42). Standard TG was added to
each sample and solvents were evaporated under a stream of nitrogen.
The lipids were separated by thin-layer chromatography in the solvent
system hexane:isopropyl ether:acetic acid, 60:40:4 (v/v/v). The band
corresponding to authentic TG was visualized by exposure to iodine vapor.
Isolation and Quantitation of Lumenal TG Not Associated with
ApoB--
Hepatocytes were harvested from one dish and homogenized in
1 ml of buffer containing 300 mM imidazole and 250 mM sucrose (pH 7.4) using a hand-held Dounce homogenizer.
The homogenate was centrifuged in a microrocentrifuge for 10 min at
14,000 rpm. KCl (final concentration of 0.5 M) was added to
the supernatant containing microsomes to release any cytosolic TG
associated with membranes. The microsomes were isolated by
centrifugation of the homogenate for 90 min at 34,000 rpm in a Beckman
SW55 rotor at 21 °C and then washed with phosphate-buffered saline.
Microsomal lumenal contents were released by sodium carbonate
extraction (38) and apoB was immunoprecipitated under nondenaturing
conditions, as described above. Immunoblotting confirmed that the
supernatant was devoid of apoB. Lipids were extracted from the
supernatant and TG was isolated by thin-layer chromatography in the
solvent system hexane:isopropyl ether:acetic acid, 60:40:4 (v/v/v). The band corresponding to authentic TG was visualized by exposure to iodine
vapor and scraped from the plate.
Other Methods--
The protein content of samples was determined
using the BCA protein assay kit (Pierce, Rockford, IL) with bovine
serum albumin as standard.
Secretion of Triacylglycerols from Murine Primary Hepatocytes Is
Decreased by the MTP Inhibitor, BMS-197636--
We investigated the
requirement of MTP for assembly of apoB-containing lipoproteins in
monolayer cultures of murine primary hepatocytes using the MTP
inhibitor BMS-197636. This inhibitor has previously been shown to be a
potent (IC50 = 36 nM) inhibitor of the in
vitro TG transfer activity of MTP (14). Nanomolar concentrations
of BMS-197636 have also been shown to decrease apoB secretion (within
10 min of incubation) from human HepG2 hepatoma cells (14), and TG
secretion from McArdle 7777 rat hepatoma cells (25). To establish the
effectiveness of this inhibitor in blocking TG secretion from murine
hepatocytes, we determined the concentration dependence of TG secretion
on the inhibitor. Hepatocytes were incubated for 2 h in the
presence of various concentrations of inhibitor (0-10
µM). [3H]Oleate (0.375 mM) was
then added for 4 h in medium containing the same concentration of
inhibitor. The MTP inhibitor reduced [3H]TG secretion in
a dose-dependent manner (Fig.
1). At an inhibitor concentration of 10 µM, TG secretion was decreased by 85%. We therefore used
this concentration of inhibitor for all experiments unless otherwise
noted. The concentration of inhibitor required to inhibit TG secretion
by 50% (~0.5 µM) was approximately an order of
magnitude greater than in McArdle hepatoma cells (25). The reason why
the inhibitor was less effective in primary murine hepatocytes than in
hepatoma cells is not clear but possible explanations include a less
efficient uptake of the drug by primary hepatocytes and/or a difference
in metabolism of the inhibitor in primary hepatocytes compared with
hepatoma cells. Control experiments were routinely performed to confirm
that secretion of radiolabeled TG was inhibited by ~85% in the
presence of 10 µM inhibitor.
The Amount of Newly Synthesized Lumenal ApoB100, but Not ApoB48,
Was Greatly Reduced by the MTP Inhibitor--
From previously reported
studies, the translocation of apoB100 into the ER lumen appears to be
more sensitive to inhibition of MTP activity than apoB48 (16, 34, 35).
We, therefore, determined whether or not inhibition of MTP reduced the
amount of apoB100 and apoB48 in the microsomal lumen of murine
hepatocytes. Synthesis of an apoB100 molecule takes ~10 min, and
~30 min later apoB100 is secreted (data not shown). Hepatocytes were
incubated for 2 h without or with MTP inhibitor, after which
[35S]methionine was added for 15 or 30 min to label apoB.
The cells were harvested, then permeabilized with digitonin under
conditions for which >85% of the cytosolic protein lactate
dehydrogenase was released from the cells. Next, trypsin was added to
digest proteins exposed to the cytosol. Using this experimental
protocol, apoB molecules that had translocated across the ER membrane,
and were either located completely within the lumen or were associated with the lumenal surface of the ER membrane, would be expected to be
protected from trypsin digestion (43). In control experiments we
confirmed that trypsin treatment digested the ER membrane proteins exposed to the cytosol. For example, under the same conditions the
activities of phosphatidylethanolamine N-methyltransferase and CDP-choline:diacylglycerol cholinephosphotransferase, whose active
sites are on the cytosolic side of the ER membrane (41), were reduced
by >90% (data not shown).
ApoB100 and apoB48 were immunoprecipitated from cellular material. Fig.
2 shows that after 15 min of labeling,
the amount of lumenal [35S]apoB48 was not significantly
altered by the MTP inhibitor, whereas the amount of radiolabeled
apoB100 was ~35% less in inhibitor-treated cells than in control
cells (Fig. 2). After 30 min of radiolabeling, the amount of
[35S]apoB100 that was protected from trypsin digestion
was ~85% less in inhibitor-treated cells than in control cells (Fig.
2). In contrast, [35S]apoB48 was equally well protected
from trypsin in inhibitor-treated and control cells.
Inhibition of MTP Impairs ApoB Lipidation--
To investigate
whether or not inhibition of MTP prevents addition of lipids to apoB,
hepatocytes were incubated ±MTP inhibitor for 2 h, then
[35S]methionine ± MTP inhibitor was added for 30 min to radiolabel apoB. Microsomes were isolated and lumenal contents
were released by treatment with sodium carbonate. Lumenal contents were
separated by ultracentrifugation into 11 fractions with densities
ranging from 1.21 to 1.006 g/ml, and apoB was immunoprecipitated from each fraction. Fig. 3 shows that in the
microsomal lumen of control hepatocytes apoB100 was primarily present
in fractions 10 and 11 that contain lipid-rich particles. However,
apoB100 was not detected in lumenal contents from inhibitor-treated
hepatocytes. It is noteworthy that in the previous experiment (Fig. 2)
some apoB100 was protected from trypsin and was present in the
microsomal lumen of inhibitor-treated hepatocytes. However, Fig. 3
shows that none of this lumenal apoB100 was present in buoyant
lipoprotein particles. A possible explanation for this apparent paradox
is that a portion of newly synthesized apoB100 is translocated across the ER membrane but remains membrane-associated and is not lipidated to
form lipoprotein particles.
In contrast to apoB100, the amount of apoB48 present in the lumen was
only slightly affected by the MTP inhibitor (Figs. 2 and 3). However,
the density distribution of lumenal apoB48 in inhibitor-treated cells
was clearly different from that in control cells (Fig. 3). Whereas in
control cells apoB48 was present in fractions with a wide range of
densities (i.e. in both lipid-rich and lipid-poor
lipoproteins), in cells treated with MTP inhibitor most apoB48 was
associated with lipid-poor particles (fractions 1-4).
To confirm that inhibition of MTP impaired apoB lipidation, we compared
the amount of microsomal lumenal TG associated with apoB in hepatocytes
incubated ±MTP inhibitor. Lipids of control and inhibitor-treated
hepatocytes were labeled with [3H]oleate for 30 min.
Microsomes were isolated in the presence of 0.5 M KCl, then
lumenal contents were released by treatment with sodium carbonate. ApoB
was immunoprecipitated from the lumenal contents under nondenaturing
conditions so that lipids remained associated with apoB (44). We
confirmed that immunoprecipitation of apoB was complete by
immunoblotting proteins remaining in the supernatant after the
immunoprecipitation using an anti-apoB antibody; no apoB was detected
in the supernatant (data not shown) indicating that the
immunoprecipitation was quantitative. ApoB-associated lipids were
extracted from the immunoprecipitate and TG was isolated. In lumenal
contents of inhibitor-treated cells, 40% less [3H]TG was
associated with apoB than in control cells (Fig.
4A, left-hand
side).
Inhibition of MTP Reduces the Amount of Lumenal TG Not Associated
with ApoB--
Ultrastructural analyses have suggested that the lumen
of the secretory pathway of enterocytes contains a pool of TG that is
not associated with apoB (18). Moreover, Raabe et al. (16) detected VLDL-sized particles within the secretory pathway of livers
from MTP+/+ mice, but noted that these "droplets" were
severely depleted in MTP
One concern in isolating a lumenal TG pool is that some TG from the
cytosolic storage pool might have been associated with the isolated
microsomes and consequently might have contaminated the lumenal TG
pool. To avoid this problem, we washed microsomes with 0.5 M KCl to remove any lipid droplets that might have adhered to the cytosolic surface of the membranes. Next, we released the lumenal contents and separated the lumenal contents from membranes by
ultracentrifugation. In a control experiment, cytosol was isolated from
hepatocytes that had been incubated for 30 min with
[3H]oleate (10 µCi/ml) to label TG, then this cytosolic
[3H]TG was mixed with microsomes that had been isolated
from unlabeled hepatocytes. We washed the microsomes with 0.5 M KCl to remove adhering cytosolic TG from the membranes
and reisolated the microsomes. Lumenal contents were released and TG
was extracted from the lumenal contents. Of the 32,000 dpm/dish of
cytosolic [3H]TG added, only 140 dpm of
[3H]TG/dish were associated with the lumenal contents. In
contrast, in a parallel experiment in which hepatocytes were incubated
for 30 min with 10 µCi/ml [3H]oleate, 3,500 dpm/dish
were present in TG in the microsomal lumen. Thus, less than 5% of the
TG in the isolated lumenal TG originated from cytosolic contamination.
Additional evidence that this lumenal TG pool did not originate from
contamination by cytosolic TG was provided by a control experiment in
which hepatocytes were pulse-labeled with [3H]oleate for
1 h, then incubated for 4 h with 0.375 mM
unlabeled oleate. Whereas the inhibitor decreased the lumenal apoB-free [3H]TG pool by ~45%, the amount of cytosolic
[3H]TG was increased by ~10% (data not shown).
Therefore, the finding that the labeling pattern of the lumenal TG pool
did not reflect that of the cytosolic TG pool provides further evidence
that the lumenal TG pool was not the result of contamination by
cytosolic TG.
Inhibition of MTP Does Not Disrupt TG Transfer to Partially
Lipidated ApoB--
We next determined the requirement of MTP for TG
transfer from the lumenal apoB-independent pool to apoB using the
pulse-chase protocol outlined in Fig.
5A. First, hepatocytes were
incubated with brefeldin A for 2 h to inhibit reversibly the
assembly of VLDLs and cause an accumulation of partially lipidated apoB
particles in the microsomal lumen (39). The hepatocytes were then
pulse-labeled with [3H]oleate for 30 min in the presence
of brefeldin A so that radiolabeled TG was transported into the ER
lumen but the assembly and secretion of fully lipidated apoB-containing
particles remained blocked by brefeldin A (39). We confirmed that under
these conditions of brefeldin A treatment, lumenal apoB was present
only in lipid-poor particles (data not shown), whereas in the absence
of brefeldin A the density distribution of lumenal apoB was as shown in
Fig. 3A (left panel). Fifteen minutes after
addition of radiolabel (15 min is required before the inhibitor becomes
fully active (25)) the MTP inhibitor was added to half the dishes. This
experimental protocol results in generation of a pool of lumenal
[3H]TG not associated with apoB, as well as a lumenal
pool of poorly lipidated apoB. After the 30-min radiolabeling period
the medium was removed and chase medium, lacking brefeldin A but
containing unlabeled oleate and cycloheximide ± MTP inhibitor,
was added for 4 h. Cycloheximide prevented the entry of any
additional newly synthesized apoB into the microsomal lumen (data not
shown). Upon removal of brefeldin A, VLDL assembly resumed from the
apoB and TG that had accumulated in the lumen. Culture medium was then subjected to density gradient ultracentrifugation to separate secreted
lipoproteins into 3 fractions: fraction I density was <1.02 g/ml;
fraction II density was 1.02-1.07 g/ml; fraction III density was
1.07-1.21 g/ml. The amount of [3H]TG in the fraction of
lowest density (fraction I) was the same in the presence and absence of
inhibitor (Fig. 5B). The amount of [3H]TG in
fraction II was only slightly reduced by inhibition of MTP. The amount
of [3H]TG associated with lipoproteins in the fraction of
the highest density (fraction III) was reduced by 32% by
inhibition of MTP (Fig. 5B). Because the MTP inhibitor was
not added until after TG and apoB had independently accumulated in the
ER lumen, this experiment demonstrates that inhibition of MTP does not
block the transfer of lumenal TG to partially lipidated apoB-containing lipoproteins for VLDL formation.
Secretion of ApoB48 Is Less Sensitive to Inhibition of MTP Than Is
Secretion of ApoB100--
We next compared the requirement of MTP for
the secretion of apoB48 and apoB100. Murine hepatocytes were incubated
±MTP inhibitor for 2 h after which [35S]methionine
was added ±inhibitor for 4 h. Fig.
6 shows that 1 µM MTP
inhibitor reduced the amount of secreted [35S]apoB100 by
~70%, whereas the amount of [35S]apoB48 secreted was
unaffected. When hepatocytes were exposed to a higher dose of inhibitor
(10 µM), the amount of [35S]apoB48 was
decreased by ~30% and apoB100 secretion was barely detectable. Thus,
apoB48 secretion was partially inhibited by a higher dose of MTP
inhibitor. However, the secretion of apoB100 was markedly more
sensitive to inhibition of MTP than was apoB48.
To determine whether lipidation of secreted apoB were reduced by
inhibition of MTP, hepatocytes were pulse labeled for 4 h with
[35S]methionine ± MTP inhibitor. Lipoproteins of
different densities were isolated from the medium by density gradient
ultracentrifugation and apoB was immunoprecipitated. ApoB100 was
secreted from control hepatocytes primarily as lipid-rich particles
(Fig. 7) but was not detected in denser
particles. As expected from Figs. 2, 3, and 6, almost no apoB100 was
secreted by inhibitor-treated hepatocytes. In contrast, apoB48 secreted
by control cells was associated with both lipid-rich and lipid-poor
particles, and the density distribution of apoB48 secreted from
inhibitor-treated hepatocytes was markedly different from that of
control cells (Fig. 7). The amount of apoB48 in lipid-rich particles
secreted by inhibitor-treated hepatocytes (fractions 9-12) was very
low compared with that of control cells. Moreover, the amount of apoB48
in higher density particles (fractions 1-5) was increased compared
with that from control cells (Fig. 7). These data show that apoB100
secretion was almost entirely abolished by the MTP inhibitor, whereas
the amount of apoB48 secreted was only slightly affected. However, the
apoB48 particles secreted in the presence of the inhibitor were
lipid-poor compared with those secreted by control hepatocytes.
Inhibition of ApoB100 Secretion by the MTP Inhibitor Is Independent
of the LDL Receptor--
Twisk and co-workers (45) have proposed that
the net output of apoB from hepatocytes is decreased by two different
mechanisms that involve the binding of apoB to the LDL receptor (LDLR).
First, because apoB and LDLRs both traverse the secretory pathway, apoB can bind to LDLRs within the secretory route and be targeted for degradation. Second, immediately upon exit from the cell, nascent apoB-containing lipoproteins can be captured by LDLRs on the cell surface, endocytosed, and targeted for lysosomal degradation. Because
apoB100 contains the principal LDLR-binding domain, which is absent
from apoB48, the secretion of apoB100 is influenced by the presence of
LDLRs to a much greater extent than is apoB48 (45).
In light of these findings we considered the possibility that the
greater sensitivity of apoB100, compared with apoB48, to inhibition of
MTP might be because of the presence of LDLRs. We reasoned that even if
a normal number of apoB100 particles had been formed in the presence of
MTP inhibitor, these particles might be defective (e.g.
lipid-poor) and therefore might be unstable in the aqueous environment
of the ER lumen. Consequently, these particles might bind to LDLRs,
either within the secretory pathway or immediately after secretion, and
be degraded. In contrast, because apoB48 lacks the LDLR-binding domain,
apoB48 particles would be much less likely to be removed by the LDLR.
As a result, the net secretion of apoB100 from MTP inhibitor-treated
cells would be reduced, whereas an almost normal number of apoB48
particles, albeit lipid-poor, would be secreted. If this sequence of
events occurred we would expect that (i) more apoB100 would be secreted from LDLR We have studied the role of MTP in the hepatic assembly of
apoB-containing lipoproteins in murine hepatocytes. Our experiments show that inhibition of MTP (as monitored by inhibition of TG secretion) abolished the secretion of apoB100, whereas the amount of
apoB48 secreted was unaffected, although the lipidation of apoB48 was
markedly diminished. Parallel differences were found in the amounts,
and degree of lipidation, of apoB100 and apoB48 in the microsomal
lumen. The difference in sensitivity of apoB100 and apoB48 to
inhibition of MTP was independent of the presence of LDLRs. We isolated
a microsomal lumenal pool of TG that was not associated with apoB and
showed that the size of this pool was ~3 times larger than the pool
of TG associated with apoB. Although the MTP inhibitor decreased by
40-45% the amount of both apoB-free and apoB-associated TG in the
microsomal lumen, inhibition of MTP did not impair the transfer of TG
to poorly lipidated apoB for VLDL formation.
Inhibition of MTP Differentially Impairs the Assembly of ApoB100-
and ApoB48-containing Lipoproteins--
Some of the apparently
conflicting reports on the role of MTP in lipoprotein assembly can
probably be ascribed to the different cell models used to study this
process. MTP has been proposed to act as a "chaperone" that assists
in translocating newly synthesized apoB across the ER membrane and into
the lumen (20, 21, 32). Our data demonstrate that when TG secretion is
inhibited by 85% the amount of apoB100 in the microsomal lumen is
reduced by ~90%, whereas the amount of apoB48 is unaffected. This
observation is consistent with studies in which the murine MTP gene had
been inactivated specifically in the liver. In hepatocytes from these mice apoB100 secretion was abolished, whereas apoB48 secretion was
reduced only slightly (16). In contrast, in another study using
hepatocytes from a different line of liver-specific MTP-deficient mice,
the secretion of both apoB100 and apoB48 was undetectable (17). The
reason for the different results obtained from these two lines of
genetically modified mice is not clear. Small amounts of apoB,
particularly apoB48 have been detected in the serum of individuals with
abetalipoproteinemia (20, 46).
The finding that some newly synthesized apoB100 was translocated across
the ER membrane and protected from trypsin digestion in the presence of
the inhibitor (Fig. 2), particularly after the 15-min labeling period,
might be explained by residual MTP activity. Alternatively, because no
buoyant apoB100-containing lipoproteins were detected in the lumen of
inhibitor-treated cells (Fig. 3), the protease-protected apoB100 might
represent apoB100 that is associated with the lumenal surface of the ER
membrane because the protease protection assay does not distinguish
between apoB that is associated with the lumenal leaflet of the
membrane and apoB that has been released into the lumen.
Our data using hepatocytes from LDLR MTP Is Required for Accumulation of Lumenal ApoB-free TG as Well as
for ApoB Lipidation--
To our knowledge, our study is the first
report of isolation of an apoB-free pool of TG from the microsomal
lumen of hepatocytes. We consider it unlikely that this pool of TG is
the result of contamination by the cytosolic TG pool for the following
reasons. (i) Before the lumenal contents were isolated, microsomes were thoroughly washed with 0.5 M KCl, which removes loosely
bound proteins and lipid droplets adhering to membranes. (ii) When
hepatocytes were incubated with the MTP inhibitor, the amount of
cytosolic [3H]TG was unaltered, whereas the amount of
apoB-free [3H]TG in the lumenal contents was decreased by
45%. Thus, the pattern of labeling of the lumenal TG did not reflect
that of cytosolic TG (3). When cytosolic [3H]TG was mixed
with unlabeled microsomes, the amount of [3H]TG in the
lumenal apoB-independent pool was only 4% of that found when this TG
pool was isolated from radiolabeled cells. Our isolation of the pool of
apoB-free TG from the microsomal lumen is consistent with an
ultrastructural analysis of mice that were unable to synthesize apoB in
the intestine (18). In these mice, chylomicron-sized, lipid-staining
particles (that did not contain apoB) accumulated in the ER lumen of
enterocytes. In another study (49), lipid-staining particles, the size
of VLDLs but lacking immunoreactive apoB, were detected in the lumen of
the rough ER of rat hepatocytes. From these studies, a model for VLDL assembly has been proposed in which a TG droplet in the rough ER lumen
fuses with a small apoB-containing particle at the junction of rough
and smooth ER (49).
A clue to the function of MTP is our observation that the
MTP inhibitor decreased by 45% the amount of microsomal lumenal TG not
associated with apoB. This finding, combined with our observation that
the MTP inhibitor reduced the amount of TG associated with apoB by
40%, supports the electron microscopy studies of Raabe et
al. (16) in which disruption of the MTP gene in liver
essentially eliminated VLDL-sized particles from the ER and Golgi
lumina. Thus, our data suggest that an important function of MTP is to mediate TG accumulation in the ER lumen in a pool not associated with
apoB. In light of published data (50-53), it seems likely that this TG
is derived primarily from the cytosolic TG pool.
Our experiments do not distinguish between the
apoB-independent TG being loosely associated with the lumenal surface
of the membrane (such a pool would have been released upon sodium
carbonate treatment) or floating freely in the lumen. Our laboratory
has previously concluded that VLDLs are completely assembled with their
full complement of lipid within the rough ER of rat hepatocytes (54).
Analysis of the apoB structure suggests that apoB contains a "lipid
pocket" that expands as lipids are added (55). Based on evidence that
MTP transfers TG between membranes via a shuttle mechanism (13),
one possibility is that a sequential addition of TG to apoB occurs on
the lumenal surface of the ER membrane. Our observation that apoB48 is
distributed among lipoprotein particles with a wide range of densities
in the microsomal lumen (Fig. 3) and in the medium (Fig. 7) is also
consistent with a sequential addition of TG to apoB. Alternatively, the
wide spectrum of densities of apoB48 particles might be the consequence
of lipid-poor apoB particles "fusing" with TG droplets of
different sizes.
The mechanism by which MTP regulates the supply of TG
within the ER lumen for lipoprotein assembly remains to be determined. Mobilization of cytosolic TG via a lipolysis re-esterification cycle is
thought to provide the majority (~70%) of TG that is assembled with
apoB (50, 51) but the enzymes involved in this lipolysis-esterification
cycle have not yet been fully characterized. A microsomal TG hydrolase
has been recently implicated in this TG lipolysis re-esterification
cycle (52, 53, 56). Interestingly, mice with targeted disruption of the
diacylglycerol acyltransferase-1 gene have normal plasma TG levels
(57), indicating that a different TG-synthesizing enzyme makes the TG
utilized for VLDL secretion. A candidate enzyme is diacylglycerol
acyltransferase-2, which is expressed primarily in liver and adipose.
The gene encoding this enzyme has recently been identified (58) but its
requirement for providing TG for VLDL secretion has not yet been established.
In conclusion, our data demonstrate that MTP is required for TG
accumulation within the ER lumen, as well as for apoB lipidation. However, in agreement with studies in hepatoma cells (2), MTP does not
appear to transfer the majority of TG to apoB. We, therefore, propose a
model (Fig. 9) for the role of MTP in
VLDL assembly that incorporates our new findings. When MTP is
inhibited, the accumulation of apoB-independent TG in the ER lumen is
compromised, resulting in the availability of less TG for assembly with
apoB. We speculate that the decreased lipidation of apoB induced by inhibition of MTP is a consequence of the decreased availability of TG
in the apoB-independent lumenal TG pool.
We thank Russ Watts for excellent technical
assistance and Dennis E. Vance for helpful comments on the manuscript.
*
This work was supported by an operating grant from the Heart
and Stroke Foundation of Alberta, Northwest Territories, and Nunavut.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.
Published, JBC Papers in Press, June 18, 2002, DOI 10.1074/jbc.M202015200,
The abbreviations used are:
VLDL, very low
density lipoprotein;
apo, apolipoprotein;
DMEM, Dulbecco's modified
Eagle's medium;
ER, endoplasmic reticulum;
LDLR, low density
lipoprotein receptor;
MTP, microsomal triacylglycerol transfer protein;
PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid);
TG, triacylglycerol.
Microsomal Triacylglycerol Transfer Protein Is Required for
Lumenal Accretion of Triacylglycerol Not Associated with ApoB, as
Well as for ApoB Lipidation*
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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/ hepatocytes was greatly reduced, whereas
apoB48 secretion was only slightly reduced compared with that in
MTP+/+ hepatocytes (16). Ultrastructural analyses revealed
that compared with MTP+/+ hepatocytes, MTP/
hepatocytes contained very few VLDL-sized particles within the ER and
Golgi lumina (16). In addition, in mice unable to synthesize apoB in
the intestine, chylomicron-sized, lipid particles (without apoB)
accumulated in the ER lumen of enterocytes (18) suggesting that ER
lumenal TG droplets might exist as a source of TG for assembly with
apoB. The importance of MTP for apoB secretion is underscored by the
demonstration that overexpression of MTP activity in mouse liver
increased plasma levels of apoB100 and -B48, as well as TG (19).
EXPERIMENTAL PROCEDURES
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DISCUSSION
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/ mice (stock number 00064) were purchased from
Jackson Laboratories, Bar Harbor, ME. All other chemicals and reagents
were from Fisher Scientific or Sigma.
-mercaptoethanol. The proteins were
separated by electrophoresis on 5% polyacrylamide gels in the presence
of 0.4% SDS. The proteins were fixed, incubated with Amplify, and
exposed to Kodak film at 
80 °C for 3-7 days. The amount of apoB
was quantitated by densitometric scanning of the gels.
RESULTS
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EXPERIMENTAL PROCEDURES
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Fig. 1.
Dose-dependent inhibition of TG
secretion by the MTP inhibitor BMS-197636. Murine hepatocytes were
incubated for 2 h with the indicated concentrations of MTP
inhibitor (MTPI), followed by addition of 5 µCi/ml
[3H]oleate (0.375 mM) for 4 h in the
presence of the same concentration of inhibitor. [3H]TG
was isolated from the culture medium. Data are average ± S.D. of
three independent experiments. One error bar is too small to
be visible.
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Fig. 2.
The amount of [35S]apoB100 in
the microsomal lumen is reduced by inhibition of MTP. Hepatocytes
were incubated with 0.375 mM oleate in the presence or
absence (control) of MTP inhibitor (MTPI) (10 µM) for 2 h, then labeled with 100 µCi/ml
[35S]methionine for either 15 or 30 min ± inhibitor. Next, the cells were permeabilized with digitonin, and
trypsin was added for 10 min at which time proteolysis was terminated
by addition of protease inhibitors. Cellular material was collected and
apoB was immunoprecipitated. Panel A, proteins from
duplicate samples were subjected to polyacrylamide gel electrophoresis
and analyzed by fluorography. Shown is one experiment representative of
three similar experiments. Panel B, intensities of
apoB100 and apoB48 bands were quantitated by densitometric scanning.
The amount of [35S]apoB in inhibitor-treated cells is
given as % of that in control cells. Data are averages ± S.D. of
three independent experiments. Open bars, control;
solid bars, + inhibitor.
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Fig. 3.
Inhibition of MTP decreases apoB
lipidation. Hepatocytes were incubated with or without (control)
MTP inhibitor (MTPI) (10 µM) for 2 h and
then labeled for 30 min with [35S]methionine (100 µCi/ml) ± inhibitor. Microsomes were isolated and lumenal
contents were released by sodium carbonate extraction. Lumenal contents
were separated from membranes by centrifugation then applied to a
sucrose gradient and ultracentrifuged for 60 h yielding 11 fractions of densities ranging from 1.006 (fraction 1, containing
lipid-rich lipoproteins) to 1.21 g/ml (fraction 11, containing
lipid-poor lipoproteins). Panel A, apoB was
immunoprecipitated from each fraction and analyzed by
polyacrylamide gel electrophoresis and fluorography. One representative
experiment of three similar experiments is shown. Panel
B, quantitation of intensities of bands corresponding to
apoB48 and apoB100. Data are given as % of total apoB in lumenal
contents and are averages ± S.D. of three independent
experiments. Some error bars are obscured by symbols.
Open circles, inhibitor; closed circles,
+inhibitor; open squares, density of fraction, g/ml.
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Fig. 4.
Amounts of apoB-associated and apoB-free
[3H]TG in the microsomal lumen are reduced by inhibition
of MTP. Hepatocytes were incubated with or without MTP inhibitor
(10 µM) for 2 h followed by addition of
[3H]oleate (10 µCi/ml) ± inhibitor for 30 min.
Cells were homogenized and then microsomes were isolated in the
presence of 0.5 M KCl to remove adhering cytosolic TG.
Microsomal lumenal contents were released by sodium carbonate
extraction and separated from membranes by centrifugation.
ApoB-containing lipoproteins were immunoprecipitated from lumenal
contents under nondenaturing conditions. Lipids were extracted from the
immunoprecipitate and the [3H]TG content was measured
(apoB-associated TG). Lipids were also extracted from the supernatant
(devoid of apoB) and the apoB-free [3H]TG content was
measured. Panel A, the amount of [3H]TG
is expressed as a % of that in cells incubated without inhibitor. Data
are averages ± S.D. of three experiments each performed in
duplicate. Open bars, no inhibitor; filled bars,
+inhibitor. Panel B, the amount of microsomal lumenal
[3H]TG associated with, and independent of, apoB was
determined in the absence of MTP inhibitor. Data are from one
experiment representative of three similar experiments performed in
duplicate.
/ mice. These studies suggested
that MTP might play a role in transferring TG into the ER lumen for
VLDL assembly. Although to our knowledge this lumenal pool of TG had
not yet been isolated or characterized biochemically, the existence of
such a pool of apoB-independent TG has frequently been invoked as the
basis for models of VLDL assembly. We, therefore, attempted to isolate
TG that was not associated with apoB from the microsomal lumen of
hepatocytes. We also asked the question: does inhibition of MTP
decrease the amount of TG in this lumenal TG pool? Hepatocytes were
incubated ±MTP inhibitor for 2 h, then [3H]oleate
was added for 30 min to label TG. Next, cells were homogenized and 0.5 M KCl was added to release cytosolic TG adhering to
microsomal membranes. Microsomes were isolated and apoB was
quantitatively immunoprecipitated from the lumenal contents under
nondenaturing conditions. Lipids were extracted from the
immunoprecipitate and from the supernatant remaining after the
immunoprecipitation. The amount of lumenal [3H]TG not
associated with apoB was ~3-fold greater than that associated with
apoB (Fig. 4B). The MTP inhibitor decreased the amount of apoB-free TG in the microsomal lumen by ~45% (Fig. 4A,
right-hand side), indicating that MTP participates in the
accumulation of this pool of TG. The amount of [3H]TG
associated with apoB in the lumen was similarly reduced by ~40%
(Fig. 4A). The MTP inhibitor reduced the amount of lumenal apoB-free [3H]TG in a dose-dependent manner
with maximum reduction being achieved with an inhibitor concentration
of 10 µM. The secretion of [3H]TG was
similarly inhibited in a dose-dependent fashion (Fig. 1)
although the maximum inhibition of [3H]TG secretion was
greater than for the decrease in the lumenal pool of apoB-free
[3H]TG (Fig. 4).
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Fig. 5.
Inhibition of MTP does not decrease TG
transfer to partially lipidated apoB. Hepatocytes were incubated
for 2 h with brefeldin A (BFA, 10 µM),
after which [3H]oleate (10 µCi/ml) was added for 30 min
in the presence of BFA. Fifteen min after addition of the radiolabel,
MTP inhibitor (MTPI, 10 µM) was added to half
the dishes. At the end of the 30-min labeling period, fresh medium
containing 0.375 mM unlabeled oleate, 10 µM
cycloheximide (CHX, to inhibit apoB synthesis), ±MTP
inhibitor (10 µM) was added and the incubation was
continued for an additional 4 h. Panel A, summary
of the time course of the pulse-chase experiment. Panel
B, culture medium was collected, applied to a sucrose density
gradient, and ultracentrifuged for 60 h. Samples of 3 densities
were collected: fraction I density, <1.02 g/ml; fraction II density,
1.02-1.07 g/ml; fraction III density, 1.07-1.21 g/ml. Lipids were
extracted from each fraction and TG was isolated. Data are
averages ± S.D. of three independent experiments. Open
bars, no inhibitor; closed bars,
+inhibitor.
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Fig. 6.
The MTP inhibitor more potently decreases the
secretion of apoB100, than apoB48. Hepatocytes were incubated with
or without (control) MTP inhibitor (MTPI) (1 or
10 µM) for 2 h, then labeled for 4 h with 100 µCi/ml [35S]methionine. ApoB was immunoprecipitated
from culture medium and then subjected to polyacrylamide gel
electrophoresis and fluorography. Panel A,
polyacrylamide gel electrophoresis of duplicate samples. Panel
B, quantitation of apoB by densitometric scanning of gels.
The amount of apoB is given as % of that secreted by cells incubated
without MTP inhibitor. For experiments with 1 µM
inhibitor, data are averages ± S.D. of three independent
experiments; for experiments with 10 µM inhibitor, data
are averages of two experiments for which values did not differ by more
than 11%. Open bars, no inhibitor; closed
bars, +inhibitor.
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Fig. 7.
ApoB100 secretion is abolished by inhibition
of MTP and apoB48 is secreted only on partially lipidated
particles. Hepatocytes were incubated with or without (control)
MTP inhibitor (MTPI) (10 µM) for 2 h and
then labeled for 4 h with [35S]methionine (100 µCi/ml). Culture medium was applied to a sucrose density gradient and
ultracentrifuged for 60 h. Twelve fractions were collected with
densities ranging from 1.006 (fraction number 1, lipid-rich particles)
to 1.21 g/ml (fraction number 12, lipid-poor particles). Panel
A, apoB was immunoprecipitated and analyzed by polyacrylamide
gel electrophoresis and fluorography. Panel B,
intensities of bands corresponding to apoB48 and apoB100 were
quantitated by densitometric scanning. Data are from one experiment
representative of three similar experiments. Open circles,
no inhibitor; closed symbols, +inhibitor; open
squares, density of fraction, g/ml.
/ cells than from LDLR+/+ cells,
and (ii) inhibition of MTP would not reduce apoB100 secretion from
LDLR/ hepatocytes if the binding of apoB100 to the LDLR
occurred prior to the action of MTP. To test this hypothesis, we
incubated hepatocytes from LDLR/ and
LDLR+/+ mice for 2 h ±MTP inhibitor then radiolabeled
the proteins for 4 h with [35S]methionine ± MTP inhibitor. Fig. 8 (top
panel) shows that approximately twice as much apoB100 was secreted
from LDLR/ hepatocytes as from LDLR+/+
hepatocytes, whereas the amount of apoB48 secreted from
LDLR+/+ and LDLR/ hepatocytes was
approximately the same, consistent with the observations of Twisk
et al. (45). Moreover, the MTP inhibitor reduced only slightly apoB48 secretion from both types of hepatocytes (Fig. 8,
lower panel), whereas the secretion of apoB100 from both
LDLR/ and LDLR+/+ hepatocytes was reduced
by ~75%. Thus, the differential effect of the MTP inhibitor on the
secretion of apoB100 and apoB48 cannot be explained by pre-secretory
binding of poorly lipidated apoB100 particles to LDLRs.
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Fig. 8.
Differential sensitivity of secretion of
apoB100 and apoB48 to inhibition of MTP is independent of the
LDLR. Hepatocytes were isolated from LDLR / and
LDLR+/+ mice and incubated for 2 h ±MTP inhibitor
(MTPI, 10 µM). Next, proteins were labeled for
4 h with 100 µCi/ml [35S]methionine ± MTP
inhibitor, medium was collected, and apoB was immunoprecipitated. ApoB
was analyzed by polyacrylamide gel electrophoresis and fluorography and
the relative amounts of apoB100 (upper panel) and apoB48
(lower panel) were compared by densitometric scanning of the
gels. Data are averages ± S.D. from duplicate samples of three
independent experiments.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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/ and
LDLR+/+ mice show that the preferential reduction in
secretion of apoB100, compared with apoB48, upon inhibition of MTP
cannot be attributed to LDLRs. An alternative explanation might be that
apoB100 is preferentially degraded either during, or shortly after, its
translocation into the lumen because apoB that is misfolded and/or
underlipidated is rapidly degraded by the ubiquitin-proteasome pathway
(47, 48). We speculate that because of its large size, apoB100 requires more lipids to attain a stable conformation than does the smaller apoB48. Thus, if lipid availability were reduced by the MTP inhibitor, apoB100 might preferentially be targeted for degradation. We attempted, unsuccessfully, to test this hypothesis by incubating hepatocytes with
the proteasome inhibitors lactacystin,
N-acetyl-Leu-Leu-norleucinal, or MG132. However, although
these inhibitors have been successfully used in other cell types, the
viability of the murine hepatocytes was compromised by equivalent doses
of the inhibitors (data not shown).
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Fig. 9.
Proposed model for assembly of
apoB-containing lipoproteins. As newly synthesized apoB
translocates across the ER membrane, a small lipid-poor apoB-containing
particle (small gray circle surrounded by solid
line) is formed by addition of small amounts of lipid. A
requirement of MTP for this process has been proposed. When
insufficient lipid is available for apoB to assume a stable
conformation, poorly lipidated particles are degraded in the cytosol by
the proteasome and/or by lumenal protease(s). A VLDL particle is formed
by addition of more TG in an MTP-independent process. MTP is required
for acquisition of apoB-free TG in the lumen, probably from the
cytosolic TG pool. This source of lumenal TG is used for assembly into
VLDLs but MTP is not directly required for the transfer. Although VLDL
assembly is shown as occurring within the aqueous lumen of the ER,
alternatively, VLDLs might be completely assembled on the inner surface
of the ER membrane.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: 332 Heritage Medical
Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2,
Canada. Tel.: 780-492-7250; Fax: 780-492-3383 E-mail: jean.vance@ualberta.ca.
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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