© 1999 American Heart Association, Inc.
Original Contributions |
Effects of 
-Tocotrienol on ApoB Synthesis, Degradation, and Secretion in HepG2 Cells
From the Division of Medical Technology (A.T., Q.W.), University of Hawaii at Manoa, Honolulu; Palm Oil Research Institute of Malaysia (A.G.), Kuala Lumpur; and Department of Laboratory Medicine, Hospital for Sick Children, University of Toronto (K.A.), Canada.
Correspondence to A. Theriault, Division of Medical Technology, University of Hawaii at Manoa, 1960 East-West Rd, Bio C-206, Honolulu, HI 96822. E-mail andret{at}hawaii.edu
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Abstract |
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Abstract—
-Tocotrienol
(-T3), a naturally occurring analog of tocopherol
(vitamin E), has been shown to have a
hypocholesterolemic effect in animals and humans.
Unlike tocopherol, it has also been shown to reduce plasma
apoB levels in hypercholesterolemic subjects. The aim
of this study was to define the mechanism of action of -T3 on
hepatic modulation of apoB production using cultured HepG2
cells as the model system. HepG2 cells preincubated with -T3 were
initially shown to inhibit the rate of incorporation of
[14C]acetate into cholesterol in a
concentration- and time-dependent manner, with a maximum 86±3%
inhibition at 50 µmol/L observed within 6 hours. -T3, on the
other hand, had no significant effect on the uptake of
[14C]glycerol into pools of cellular
triacylglycerol and phospholipid relative to
untreated control. The rate of apoB synthesis and secretion was then
studied by an [35S]methionine pulse-labeling experiment
and quantified by immunoprecipitating apoB on chasing up to 3 hours. An
average reduction of 24±3% in labeled apoB in the media was apparent
with -T3 despite a 60±2% increase in apoB synthesis. Fractionation
of secreted apoB revealed a relatively denser lipoprotein particle,
suggesting a less stable particle. Using a
digitonin-permeabilized HepG2 cell system, the effects
of -T3 on apoB translocation and degradation in the endoplasmic
reticulum were further investigated. The generation of a specific
N-terminal 70-kDa proteolytic fragment proved to be a sensitive measure
of the rate of apoB translocation and degradation. The abundance of
this fragment increased significantly in -T3-treated cells relative
to untreated control cells (50±21%) after 2 hours of chase. In
addition, the presence of -T3 resulted in an average decrease of
64±8% in intact apoB. Taken together, the data suggest that -T3
stimulates apoB degradation possibly as the result of decreased apoB
translocation into the endoplasmic reticulum lumen. It is speculated
that the lack of cholesterol availability reduces the
number of secreted apoB-containing lipoprotein particles by limiting
translocation of apoB into the endoplasmic reticulum lumen.
Key Words: apoB • tocotrienol • tocopherol • HMG-CoA reductase inhibitor • degradation
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Introduction |
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Apolipoprotein B-100 (apoB), the major structural component of VLDL and LDL, is an important risk factor for coronary artery disease (CAD).1 Many patients with CAD have been found to have abnormally high production rate of VLDL- and LDL-apoB.2 Therefore, overproduction of apoB-Lp by the human liver is an important contributing factor in the development of CAD.
Numerous studies have shown that hepatic production apoB-Lp can be regulated by diet, hormones, and drugs.3 4 The specific mechanism involved in the regulation of hepatic apoB-Lp secretion and faulty overproduction appears to be at the cotranslational and posttranslational levels, as nascent apoB molecules are either secreted or degraded intracellularly.5 6 7 Evidence suggests that translocation efficiency across the endoplasmic reticulum (ER) is a key determining factor of apoB-Lp secretion.8 It is well documented that this point of regulation determines the amount of nascent apoB that enters the secretory pathway and how much is degraded. ApoB, which is efficiently translocated across the ER membrane, assembles into a lipoprotein particle and is secreted through the secretory pathway. On the other hand, any apoB that is not translocated across the ER membrane fails to assemble into a lipoprotein particle and is diverted into a degradative pathway.9 10 11 An ER-localized proteolytic system has been found to be responsible for degrading apoB,10 as both an ER luminal cysteine protease and a cytosolic ubiquitin-proteosome pathway have been implicated in the intracellular degradation of apoB.12 13 It is generally accepted that incomplete translocation results in a significant amount of apoB exposed to the cytosolic side of the ER membrane with the N-terminal end of apoB partially translocated into the ER lumen.14 15 This membrane-bound apoB is suggested to come into contact with components of the ubiquitin-proteosome pathway, releasing an N-terminal 70-kDa apoB fragment into the ER lumen.12 This N-terminal fragment is apparently produced from partially translocated apoB and has been used as an indirect indicator of inefficient translocation in a number of studies.15 16
It is generally believed that the supply or availability of lipid components in the ER plays an important role in regulating the assembly and secretion of apoB-Lp. Both dietary and hormonal factors known to influence lipid availability have been shown to modulate the rate of apoB translocation, degradation, and secretion in cultured hepatocytes (reviewed in Reference 1717 ). Oleic acid, for example, was shown to stimulate apoB secretion through an inhibition of protein degradation.18 The protection by this fatty acid appeared to be the result of increased triacylglycerol (TAG) synthesis. Under these conditions, the rate of apoB translocation is thought to accelerate, and apoB is sorted for assembly into a lipoprotein particle destined for secretion.19 20 On the other hand, insulin was shown to enhance apoB degradation, possibly by preventing lipid interaction with apoB, rendering the partially translocated apoB susceptible to specific protease(s).21 22 Thus, it is widely accepted that lipid availability is obligatory for the assembly of apoB-Lp. Without lipids, apoB remains unprotected and is degraded. Despite the many factors known to regulate apoB-Lp secretion, there are still other factors that remain unknown.
One such factor is tocotrienol, a naturally occurring analog of
tocopherol (vitamin E) found mainly in cereal grains and
palm oil. Tocotrienols differ from tocopherol by possessing
double bonds in the phytyl tail. They are further divided into isomers
(
, ß, , and 
) on the basis of the degree and placement of
the methyl substitution around the chromanol ring.23
Unlike tocopherol, tocotrienol has been shown to have an
intrinsic hypocholesterolemic activity in animals and
man.24 25 The favorable cholesterol-lowering
profile of tocotrienols was attributed mainly to their downregulation
of the rate-limiting enzyme of the cholesterol biosynthetic
pathway, HMG-CoA reductase (HMGR).26 In vitro models later
identified -tocotrienol (-T3) and -T3 to be more potent than
-T3 in suppressing cholesterol
biosynthesis.23 When assessed in
hypercholesterolemic human subjects, Palmvitee (Palm
Oil Research Institute of Malaysia), a tocotrienol-rich fraction
(TRF) of palm oil, and purified -T3 were shown to mediate decreases
in plasma total cholesterol, LDL-cholesterol,
and apoB levels.27 Similar results were shown with a novel
TRF from rice bran oil with also a reduction in the thrombogenic Lp(a)
plasma levels.28
The beneficial effects of -T3 on plasma LDL-cholesterol
and apoB levels deserve further investigation. Indeed, -T3 was shown
to upregulate LDL receptor in mammalian cells and may be implicated in
part for the reduction of apoB-Lp in vivo.26 However, the
production rate of apoB-Lp may also be affected by -T3.
Numerous studies have suggested that HMGR inhibitors can
also reduce apoB secretion by limiting the availability of lipids
(reviewed in Reference 2929 ). The purpose of our study was to investigate
the effects of -T3 on the secretion of apoB using the human hepatoma
cell line, HepG2, as the model system. This cell line has been used
extensively to study hepatic apoB-Lp secretion.30 31 In
our experiments, we used both intact and
semipermeabilized HepG2 cells32 to study
cellular processes such as synthesis, ER translocation, degradation,
and secretion of apoB.
In this article, we have shown that -T3 inhibited the secretion of
apoB despite a stimulation in apoB synthesis. The discrepancy is
accounted for by a net increase in the intracellular degradation of
apoB in the ER, which prevented apoB from being secreted. These
findings suggest that the decreased plasma LDL-cholesterol
and apoB levels mediated by -T3 in vivo may be partly the result of
decreased apoB secretion.
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Methods |
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Materials
-T3 was provided by Palm Oil Research Institute of Malaysia,
Kuala Lumpur. Stock solutions of -T3 were freshly prepared in 100%
DMSO and preserved at -25°C for no longer than 3 weeks. Immediately
before use, the stock solution was diluted in culture medium to give a
final DMSO concentration of 0.1% (vol/vol). Experiments were performed
in subdued light. The preparation was found to be pure by HPLC (data
not shown).
HepG2 cells (HB 8065) were obtained from American Type Culture
Collection. Cell culture media, FBS (certified grade),
antibiotic-antimycotic mixture, and immunoprecipitin (10% wt/vol) were
from Life Technologies Inc. Culture dishes and flasks were obtained
from Corning Costar Corp. Digitonin (50% purity),
lipoprotein-deficient FBS (LDS), - and -tocopherol,
rabbit anti-goat IgG, goat IgG, protease inhibitors, and
other common laboratory reagents were from Sigma Chemical Co.
Electrophoresis reagents were from Bio-Rad Laboratories.
L-[35S]methionine (1175 Ci/mmol),
[1-14C]acetic acid, sodium salt (40 to 60
mCi/mmol), [1,3-14C]glycerol (>40 mCi/mmol),
ENHANCE and Reflection autoradiography films were
purchased from NEN Life Science Products. Monospecific goat
anti-human apoB antiserum and affinity-purified apoB polyclonal
antibody were obtained from Medix-Biotech Inc. Plastic-backed
thin-layer chromatography (TLC) plates (silica gel 60)
were from Alltech Associates Inc.
Growth of HepG2 Cells
Monolayer cell cultures were maintained in RPMI-1640 medium with
10% FBS at 37°C with 5% CO2 and 95% air and
subcultured in 35- or 60-mm-diameter dishes to about 80% confluence.
Once confluence was reached, cells were first supplemented with 7% LDS
in RPMI-1640 (LDS-RPMI) for 16 hours to induce
cholesterologenesis.26 After induction, cells
were treated with -T3 in LDS-RPMI. Untreated control cells received
0.1% (vol/vol) DMSO without -T3.
Analysis of Cellular and Secreted Lipids
To measure the rates of cholesterol and
cholesterol ester (CE) synthesis and secretion, treated and
untreated cells were labeled with 5 µCi/mL
[14C]acetate for 2 or 6 hours. TAG and
phospholipids synthesis and secretion, on the other hand, were labeled
with [14C]glycerol for 2 or 6 hours. After
labeling, the medium was collected and the cells were washed twice with
cold PBS. Cellular and media lipids were then extracted with
hexane-isopropanol (3:2, vol/vol) as described by Goldstein et
al.33 The organic solvent was evaporated, resuspended in
hexane, and spotted on a TLC plate. Neutral and polar lipids were
separated using a 2-solvent system. Plates were first developed in
chloroform-methanol-acetic acid-formic acid-water (70:30:12:4:2) and
then developed in ether-diethylether-glacial acetic acid. (90:10:1) The
TLC plates were dried, the lipids were visualized with
I2 vapor, and zones corresponding to the lipid
standard were cut, mixed in scintillation cocktail, and counted on a
Packard Tri-Carb model 1500 liquid scintillation counter. After lipid
extraction, cell proteins were digested in 1 mL of 0.1 mol/L NaOH and
measured as described below.
Pulse-Chase Experiments
Treated and untreated HepG2 cells were preincubated in
methionine-free RPMI for 15 minutes and pulsed with an
[35S]methionine labeling medium (100 µCi/mL
of [35S]methionine in methionine-free RPMI, 7%
LDS, with and without -T3) for 15 minutes. After the short
pulse, the cells were washed with Hanks' balanced salt-solution (HBSS)
and chased in 7% LDS-RPMI-1640 supplemented with 5 mmol/L
methionine, with and without -T3. At various chase times, duplicate
35-mm dishes were harvested and cells were lysed in solubilization
buffer (PBS containing 1% NP40, 1% deoxycholate, 5 mmol/L EDTA,
1 mmol/L EGTA, 2 mmol/L PMSF, 100 kallikrein-inactivating
units/mL aprotinin, 0.1 mmol/L leupeptin, 5 µmol/L ALLN
[N-acetyl-leucyl-leucyl-norleucinal]). The lysates were
centrifuged for 5 minutes in a microfuge (7500g),
and the supernatants were collected for immunoprecipitation. Media
collected at each time point were spun as above to remove any cell
debris and mixed with a protease inhibitor cocktail (2
mmol/L PMSF, 100 kallikrein-inactivating units/mL aprotinin, 0.1
mmol/L leupeptin, 5 µmol/L ALLN, final concentration) before
immunoprecipitation.
Lipoprotein Fractionation
Cells were treated and pulse-chased similarly as above. The
culture medium was collected after 3 hours of chase, adjusted to 10%
sucrose, and separated by sucrose gradient
ultracentrifugation as previously
described.34 Centrifugation was carried
out at 35 000 rpm at 12°C for 65 hours in a SW41 rotor. Gradients
were fractionated into 1-mL fractions, and the density and apoB were
measured in each fractions. All solutions contained the protease
inhibitor cocktail as above.
Degradation Assay
The degradation assay was carried out essentially as
described.32 Treated and untreated HepG2 cultures grown in
35-mm dishes were depleted of methionine by incubation in
methionine-free RPMI for 15 minutes. HepG2 cells were pulse-chased and
made semipermeable as follows. Cells were incubated in
[35S]methionine labeling medium (100 µCi/mL
of [35S]methionine in methionine-free RPMI, 7%
LDS, with and without -T3) for 20 minutes, washed in HBSS 2 times,
and chased in 7% LDS-RPMI-1640 supplemented with 5 mmol/L
methionine, with and without -T3, for 10 minutes (to elongate
labeled nascent apoB into full-length apoB). After extensive washing,
the cells were incubated in cytoskeletal (CSK) buffer (300 mmol/L
sucrose, 100 mmol/L KCl, 2.5 mmol/L
MgCl2, 1 mmol/L sodium-free EDTA, 10
mmol/L PIPES, pH 6.8) containing 40 µmol/L of digitonin, with
and without -T3, for 10 minutes.
Digitonin-permeabilized cells were then washed 2 times
in CSK buffer and were immediately used for the degradation studies.
Semipermeable cells were incubated in a CSK buffer with and without
-T3 at 37°C for 2 hours. At the beginning and at the 2-hour
interval, duplicate dishes were harvested by removing the CSK treatment
buffer, followed by washing with CSK buffer and solubilizing the cells
in solubilization buffer (as described above). The cell extracts were
then centrifuged in a microfuge at 9000g for 10
minutes at 4°C, and the supernatants were subjected to
immunoprecipitation.
Immunoprecipitation
Solubilized cell extracts, media, and fractions collected from
sucrose gradients were diluted with PBS and 5x C buffer (0.25 mol/L
Tris, pH 7.4, 0.75 mol/L NaCl, 0.025 mol/L EDTA, 5% Triton X-100) and
were immunoprecipitated by the addition of an excess amount of goat
anti-human apoB antiserum. Immunoprecipitation was allowed to proceed
overnight at 4°C. After incubation, excess amount of rabbit anti-goat
IgG was added, and the mixture was further incubated for 1 hour at
4°C. Immunoprecipitin (10% wt/vol) was added, and the incubation
continued for 1 hour on a shaker at room temperature. The
immunoprecipitin-antibody complex was then separated by
centrifugation and washed extensively with an
immunoprecipitation wash buffer (0.14 mol/L NaCl 0.02 mol/L sodium
dihydrogen phosphate, pH 7.5). Finally, the immunoprecipitates were
prepared for SDS-PAGE by suspending and boiling in 60 µL of
electrophoresis sample buffer.
In the degradation assay, a preimmune step was used to eliminate nonspecific binding. Samples were precleared once with goat serum/immunoprecipitin before immunoprecipitation with an affinity-purified apoB polyclonal antibody.
SDS-PAGE and Fluorography
SDS-PAGE was performed essentially as described.35
Gels were composed of 3% (wt/vol) stacking and 6% or 7% (wt/vol)
resolving gels. Electrophoresis was at 60 V for 16 hours. The gels were
fixed, stained, and fluorographed by incubating in ENHANCE. The gels
were dried, exposed to an autoradiography film and
incubated at -80°C for 1 to 3 days. Radiolabeled proteins visualized
on the fluorographs were quantified by cutting the corresponding band
from the dried gel, mixing in a scintillation cocktail, and counting on
a Packard Tri-Carb model 1500 liquid scintillation counter.
TCA Precipitation
The incorporation of [35S]methionine
into total cell protein was determined by trichloroacetic acid (TCA)
precipitation. An aliquot of the sample was spotted on GF/C filters,
and the filters were washed twice in 10% (wt/vol) TCA at 60°C for 5
minutes and in absolute ethanol for 2 minutes. Radioactivity was
measured on a Packard Tri-Carb model 1500 liquid scintillation counter
after placing the filters in 5 mL of scintillation cocktail.
Other Methods
Cell protein content was measured according to
Bradford36 using BSA as the standard. The activity of
lactate dehydrogenase was measured on a Beckman Synchron CX4 automated
analyzer using a modification of the method of Gay et
al.37
Statistical Analysis
Data were normalized to the amount of cellular protein.
Statistical differences were analyzed by using paired
Student's t test with the level of significance set at
P<0.05.
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Results |
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-T3, but not Tocopherol, Inhibits
Cholesterol Synthesis in a Dose- and Time-Dependent
MannerInitial studies were performed to determine an optimal concentration of
-T3 that would inhibit de novo
cholesterol synthesis without altering cell viability. As
shown in Figure 1
, -T3 added in
various concentrations to the culture medium for 6 hours decreased the
rate of incorporation of [14C]acetate into
cellular cholesterol in a dose-dependent manner. Percent
inhibition was 42±2% at 2 µmol/L, 54±4% at 10 µmol/L,
71±3% at 25 µmol/L, 86±3% at 50 µmol/L, and 93±2%
at 75 µmol/L. A preliminary experiment showed -T3 was
significantly more active in suppressing cholesterol
synthesis when cells were incubated with LDS than without LDS (data not
shown). For this reason, we chose to pretreat our cells with LDS to
maximize the effects of -T3 on cholesterol synthesis.
From the results obtained, the optimal inhibition was observed at
50 µmol/L. At this concentration, there was no significant
release of lactate dehydrogenase into the media (data not shown).
Furthermore, TCA-precipitable radioactivity from cells and medium
incubated with 50 µmol/L -T3 increased slightly (see below),
indicating that the vitamin did not have any deleterious effect on cell
metabolism. The decrease in total cholesterol
was accompanied by a decrease in both free cholesterol and
esterified cholesterol, which reached 85±2% and 80±3%
of control, respectively. In all further experiments, 50 µmol/L
-T3 was used as the optimal concentration.
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Time-course studies of HepG2 cells preincubated with 50 µmol/L
-T3 were also performed similarly as described above. In these
studies, cells were incubated and labeled with a
[14C]acetate labeling medium with and without
-T3 for a total of 2 hours, 6 hours, and 18 hours. The
inhibitory effect of -T3 on newly synthesized
cholesterol synthesis was found to be time-dependent with
an optimal inhibition observed within 6 hours (data not shown). In all
further experiments, a 6-hour treatment was used.
In further support of the specific effect of -T3 on
cholesterol synthesis, -tocopherol and
-tocopherol added at 50 µmol/L for 6 hours were
studied for its effect on de novo cholesterol synthesis. As
expected, the incorporation of [14C]acetate
into cholesterol remained essentially the same as the
untreated control (P>0.05, n=4) (Table 1). Similarly, combination of -T3 and
-tocopherol were also examined to test for possible
additive effects. When assayed together, -tocopherol did
not increase the activity of -T3. To the contrary, there was a
slight decrease in cholesterol synthesis, but it did not
reach statistical significance when compared with the effects of -T3
treatment alone.
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To evaluate the effects of -T3 on the synthesis of TAG and
phospholipids, HepG2 cells were labeled with
[14C]glycerol with and without 50 µmol/L
-T3 for a total of 6 hours. As summarized in Figure 2, -T3 did not have an
inhibitory effect on the accumulation of
triglyceride and phospholipids in the cell
(P>0.05, n=4).
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-T3 Also Inhibits Cholesterol Secretion
Secretion of lipids into the culture media was further
analyzed in cells treated with and without 50 µmol/L
-T3. Results showed a similar response as that observed
intracellularly. -T3 significantly inhibited the secretion of free
cholesterol and CE by 77±4% and 88±3%, respectively
(P<0.05, n=4). No appreciable effects on the secretion of
TAG with -T3 were observed (P>0.05, n=4); however,
phospholipid secretion was slightly elevated (15±3%;
P<0.05, n=4). Together, the data suggest that the
T3-induced inhibition of cholesterol synthesis resulted
in concomitant decrease in its secretion (summarized in Figure 2
).
-T3 Stimulates ApoB Synthesis, but Inhibits Its
Secretion
An initial metabolic pulse-chase labeling experiment
was performed to assess the postsynthetic fate of nascent apoB grown in
untreated control and -T3-treated cell cultures. Cells were pulsed
with [35S]methionine for 10 minutes, and chased
with excess cold methionine medium with and without -T3 for up to
180 minutes. Aliquots in the intracellular and extracellular fractions
were collected at various times and analyzed. A preliminary
experiment showed a 20 minutes' delay in reaching peak incorporation
of [35S]methionine into full-length apoB in
both control and treated cells (data not shown). Figure 3A is the fluorograph with the
corresponding line graph (Figure 3B) showing the amount of apoB
synthesized and secreted by HepG2 cells in the presence and absence of
-T3 of a typical pulse-chase experiment (reproducible in 2 other
experiments). A 60% increase in the incorporation of
[35S]methionine into immunoprecipitable apoB
was apparent with -T3 at the 20-minute chase time
(representing peak incorporation). When the radioactivity
was further chased, a gradual reduction in the intracellular labeled
apoB was noted with a simultaneous increase in secreted
labeled apoB. In the presence of -T3, a significant reduction in
apoB secretion was noted (26%) when compared with the untreated
control cells at the 180-minute chase time (representing
peak secretion). Table 2 shows the amount
of radioactive apoB depleted during the chase and the estimated amounts
of apoB that were lost (by a process of degradation). ApoB content was
calculated by slicing and counting the apoB bands from the dried gel
and expressed as number of disintegrations per minute per milligram of
cell protein. ApoB secreted as a percentage of intracellular peak was
60% for control and 28% for -T3-treated cells for 180 minutes.
ApoB recovery in cells plus medium was 74% for control cells and 41%
for -T3-treated cells at the 180-minute chase time. The data suggest
that -T3 increased the proportion of labeled apoB that was lost
(degraded) from 26% to 59% and decreased the proportion of apoB that
was secreted from 60% to 28%. Interestingly, most of the degradation
took place within the first hour (28%).
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As a control, the postsynthetic fate of nascent albumin (Figure 4A) and total protein synthesis and
secretion were also examined during the chase. Results from 3
independent experiments are summarized in Figure 4B. Measurement
of albumin was performed by immunoprecipitation as was apoB,
and total protein was performed by TCA precipitation. A
consistent increase in synthesis of both albumin
(14±4%) and total protein (13±7%) were observed at the beginning of
the chase. The increase in synthesis corresponded with a similar
increase in albumin (15±3%) and total protein (12±1%)
secretion at the end of the chase. As shown in Figure 4B, the
effect of -T3 on apoB synthesis and secretion was found to be
specific.
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-T3-Treated HepG2 Cells Secrete Denser ApoB-Containing
Lipoproteins
The effect of -T3 on the density of secreted lipoprotein
particles in HepG2 cells was also investigated. As depicted in Figure 5, cells incubated with -T3 secreted
apoB-Lp with a density that was relatively denser than untreated
control cells (fractions 2 to 5 represent HDL apoB-Lp, whereas
fractions 6 to 12 represent the lower-density apoB-Lp). This
appears to suggest that -T3 may interfere with the assembly and
secretory processes of the apoB-Lp particle, resulting in a less stable
particle. Although -T3 stimulated apoB secretion, the amount of apoB
recovered in -T3-treated cells was slightly lower compared with
untreated controls (data not shown).
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-T3 Stimulates ApoB Degradation by Decreasing ER
Translocation
The role of -T3 on apoB translocation and degradation was
investigated using semipermeable HepG2 cells. This system has been
shown to be specific for studying apoB degradation in the ER as
generation of an N-terminal 70-kDa proteolytic fragment proved to be a
sensitive measure of the rate of apoB degradation.32 38 We
further used this fragment as an indicator of incomplete translocation.
In our assay, apoB translocation and degradation were monitored during
a 120-minute chase in CSK buffer supplemented with and without 50
µmol/L of -T3. Figure 6 shows intact apoB (550 kDa) and its
major 70-kDa apoB fragment immunoprecipitated at the beginning (0
minutes) and at the 120-minute chase time.
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As shown in Figure 6, degradation
of the 550-kDa intact apoB was observed in
permeabilized cells with and without treatment.
Interestingly, the appearance of the 70-kDa degradation fragment at the
120-minute chase was found to coincide with the loss of the intact apoB
band. We then analyzed the data by measuring the level of
intact apoB remaining at the 120-minute chase time and expressed it as
a percentage of the amount at time 0. Percent intact apoB remaining was
67.4% in control cells and 32.6% in -T3-treated cells. When
expressed as a percentage of treated cells over control, a 52%
increase in apoB degradation was noted (average was found to be
64±8%, from 3 independent experiments performed in duplicate, versus
control; P<0.05). Interestingly, the lower level of intact
apoB remaining under -T3 treatment corresponded with an increased
abundance of the 70-kDa fragment at the 120-minute chase. The fragment
increased significantly in -T3-treated cells, relative to untreated
control cells, by nearly 2-fold. When taking the increase in intact
apoB synthesis into account, an increase of 79% in the 70-kDa fragment
was found (average was found to be 50±21%, from 3 independent
experiments performed in duplicate, versus control;
P<0.05). Taken together, the data suggest that -T3
stimulated apoB degradation in the ER by decreasing apoB translocation
into the ER lumen.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Factors that control the rate of apoB secretion by the liver have been of great importance in view of the role of apoB in atherogenesis. The purpose of this study was to investigate the effects of a dietary factor,
-T3, on apoB secretion. The isolation of tocotrienols from
palm oil and the making of synthetic derivatives have led to a new
potential drug to treat
hypercholesterolemia.23 39 This
drug mimics other HMGR inhibitors; however, unlike HMGR
inhibitors, -T3 is active against free radicals and is
thought to inhibit LDL oxidation.39 40 Furthermore, in
vitro studies have shown that tocotrienols exhibited greater
antioxidant activity and antitumor activity than
tocopherols.41 42 The beneficial effects of
tocotrienols in cardiovascular disease and cancer are
becoming more apparent.
In our study, we first examined the effects of -T3 on lipid
synthesis and secretion in HepG2 cells. Our results confirmed that
-T3, unlike - and -tocopherol, is able to inhibit
cholesterol synthesis in a dose- and time-dependent
fashion. These results compare well with those of Parker et
al.26 In their studies with -T3-treated HepG2 cells,
they observed an inhibition in cholesterol synthesis of
nearly 80% within 4 hours at a concentration of 30 µmol/L. In
elucidating the mechanism of action, they found that the suppression
was because of a posttranslational process involving an accelerated
rate in the intracellular degradation of HMGR. These authors, however,
did not investigate whether -T3 was exerting its effect primarily
through CE or free, unesterified cholesterol. Our report
showed that -T3 exerted an equal inhibitory effect on
both esterified and free cholesterol synthesis.
Furthermore, our results indicated a similar decrease in free
cholesterol and CE accumulation in the media. On the other
hand, the synthesis and secretion of TAG was not significantly altered
by -T3. As for phospholipids, only secretion showed a slight
increase with -T3, whereas synthesis remained essentially
unchanged.
To further examine whether lipid availability is associated with apoB
secretion, we investigated the effects of -T3 on apoB synthesis,
degradation, and secretion with pulse-chase experiments using intact
HepG2 cells. As shown, -T3 significantly reduced apoB secretion
despite an excess amount of apoB in the cultured cell. It is postulated
that the lack of lipid availability, primarily in esterified
cholesterol, in -T3-treated cells, prevented lipid
interaction with nascent apoB. Without lipids, it is thought that apoB
molecules are unable to translocate across the ER membrane and are
degraded intracellularly by the ubiquitin-proteosome pathway located on
the cytosolic side of the ER membrane (reviewed in Reference 4343 ).
Interestingly, degradation was found to be rapid in -T3-treated
cells relative to the untreated control cells, as nearly all of nascent
apoB was degraded within the first hour of chase. In turn, apoB
secretion was found to be inhibited by an average of 24±3% at the
3-hour chase time despite a 60±2% increase in apoB synthesis at the
beginning of the chase (from 3 independent experiments performed in
duplicate). Furthermore, -T3-treated HepG2 cells secreted denser
apoB-Lp, suggesting that lipoprotein assembly may be hampered. Dense
HDL-like apoB-Lp particles have been shown to be more unstable because
they undergo a higher level of intracellular degradation than LDL-like
particles.34 Together, the results implicate a beneficial
role for -T3 in the regulation of apoB secretion and illustrate the
importance of lipid availability in the secretion of apoB-Lp.
Simultaneously, we analyzed the
metabolic fate of labeled albumin and total protein
in both the intracellular and extracellular fractions. The synthesis
and secretion of albumin and the total amount of newly
synthesized protein were only slightly affected by -T3. Both the
synthesis and secretion increased slightly indicating that the response
with apoB was specific. The increase in protein synthesis, including
apoB, is interesting and may reflect a global transcriptional effect.
This is compatible with the mechanisms of action of
tocopherols, which can influence RNA polymerase activity by
interacting with tocopherol-binding protein,44
a response similar to vitamin A and the retinol-binding protein.
The site of apoB degradation has been of great interest. In HepG2
cells, the major site of apoB degradation is the ER.10 14
A cytosolic ubiquitin-proteosome pathway associated with the ER
membrane has been largely implicated in the intracellular degradation
of apoB in HepG2 cells.12 However, Adeli and
coworkers13 further suggested that an ER luminal cysteine
protease is also involved in a second degradative pathway crucial in
controlling the rate of apoB secretion. To further elucidate the role
of -T3 on apoB degradation, a specific ER degradation assay using
semipermeabilized cells was used to examine its effect
on apoB degradation. This assay has been described previously and has
been used to study factors that modulate the rate of apoB translocation
and degradation.45 It uses digitonin, which selectively
permeabilizes the plasma membrane leaving the ER
morphologically intact and functional. These
semipermeabilized cells have the unique ability to
allow cellular processes such as ER translocation, folding, and
assembly of secretory proteins to occur as they would in the intact
cell. Interestingly, the slower rate of apoB degradation in this system
allows for the detection of a specific N-terminal 70-kDa proteolytic
fragment.32 According to the current hypothesis of the
apoB secretory pathway, generation of this fragment apparently results
from inefficient apoB translocation across the ER membrane. Therefore,
the appearance of the 70-kDa fragment was used in our study as a marker
of apoB translocation and degradation. As shown in our report, the
greater abundance of this fragment in -T3-treated cells at the
2-hour chase time suggested an increase in apoB degradation. Supporting
this hypothesis, the appearance of the 70-kDa fragment correlated with
less intact apoB remaining under -T3 treatment compared with the
untreated control. Together, the present results are
consistent with the pulse-chase and fractionation studies and
provide further evidence that -T3 stimulates apoB degradation,
possibly as the result of decreased translocation across the ER
membrane.
Today, much controversy still exits as to which lipid is the primary
constituent in the assembly of apoB-Lp. Both TAG and CE have been
suggested as the lipids that primarily regulate apoB translocation and
degradation; however, attempts to distinguish which of these lipids
play a more important role have yielded conflicting results. In the
current study, we demonstrate that CE had the major regulatory role in
the secretion of apoB. This agrees well with the work of Cianflone et
al46 and Musanti et al,47 who
demonstrated that when an ACAT (acyl CoA:cholesterol
acyltransferase) inhibitor, a potent suppressor of
cholesterol esterification, was added to HepG2 cells, the
secretion of apoB-Lp dropped significantly. Additionally,
pravastatin, a potent HMGR inhibitor, was shown
to reduce apoB secretion significantly when supplemented in cultured
rabbit hepatocytes.48 The addition of
pravastatin was shown to reduce apoB secretion by
accelerating the intracellular degradation of apoB. This was coincident
with a decrease in CE synthesis while free cholesterol and
TAG synthesis remained unchanged. Similarly, simvastatin
significantly reduced apoB secretion in HepG2 cells and paralleled
changes in cholesterol synthesis.49 On the
other hand, not all studies saw a requirement for CE synthesis in the
regulation of apoB secretion. In a series of experiments in HepG2
cells, Wu et al50 demonstrated that changes in cellular CE
content did not affect apoB secretion, whereas changes in TAG synthesis
were always associated with changes in apoB secretion. It was suggested
that the availability of newly synthesized TAG was the most important
factor to regulate apoB secretion. The data are in accord with previous
studies in which various ACAT and HMGR inhibitors had no
effect on apoB secretion.51 52 53 Clearly, there are
discrepancies. A recent study by Cartwright et al54 argued
that different pools of CE may be differentially modified by the
various inhibitors used to alter CE, which may explain the
discrepancies. Hence, it is plausible that -T3 affected CE
availability early in the assembly process of apoB-Lp and that this
particular pool of CE behaved as the primary constituent in regulating
apoB secretion.
The failure of some hypercholesterolemic subjects to
respond to -T3 have prompted investigators to evaluate the
preparation and standardization of tocotrienol for human tests. Most
human studies used a TRF of palm oil as their preparation. The
composition of TRF is highly variable, and the
tocopherol content can vary from 15% to
44%.55 It was observed that preparations with 20% or
more tocopherol can attenuate the
cholesterol-suppressive action of -T3 and may be
responsible for conflicting reports on responses to TRF in human
subjects.27 Our results (Table 1), as well as those
of others,23 indicated that tocopherol did not
significantly decrease the activity of -T3 in HepG2 cells.
Therefore, it appears that the attenuation observed in vivo may be
related to transport systems in the blood. Qureshi et
al55 hypothesized that there might be a preferential
transport of -tocopherol in serum lipoproteins.
In all, the data in this report indicate that -T3 influences apoB
secretion by both cotranslational and posttranslational processes
involving a decreased rate of apoB translocation and accelerated
degradation of apoB. This activity correlated with a decrease in free
and esterified cholesterol. Taken together, the information
indicates an association between the suppression of hepatic
cholesterol synthesis and apoB secretion, and the observed
lowering of apoB and LDL-cholesterol levels in animal
models.
|
Acknowledgments |
|---|
This work was supported by a Grant-in-Aid from the American Heart Association-Hawaii Affiliate (HIGS-06-97).
Received December 29, 1997; accepted September 8, 1998.
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References |
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