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INTRODUCTION |
The peroxisome proliferator-activated receptor
(PPAR)1 
has a central
role in the regulation of lipid metabolism, and unsaturated long-chain
fatty acids (LCFAs) are among the natural ligands for this
receptor (1). PPAR is also activated by so-called peroxisome proliferators, a group of compounds that includes the lipid-lowering fibrates (1, 2). The activation of PPAR results in important and
diverse effects on lipid metabolism, such as an increase in the
transcription of genes involved in mitochondrial and peroxisomal 
-oxidation, and the regulation of the expression of apolipoprotein (apo) A-I, apoC-III (2), and PPAR itself (3). Moreover, PPAR
regulates the transcription of LFABP, and the LFABP gene promotor has a
PPAR-responsive element (2, 4). It has been shown that both fibrates
and unsaturated LCFAs increase the gene expression and production of
LFABP (5-8).
LFABP is an abundant cytosolic protein present in the liver and
intestine. The protein binds LCFAs and their CoA-esters in a reversible
manner (9-11) through two high affinity binding sites (9-11). LFABP
serves as an intracellular acceptor of LCFAs, which can enhance both
the cellular uptake and the intracellular diffusion of these fatty
acids (Refs. 12-16; for a review, see Refs. 9-11 and 17). This
function of LFABP may be reflected in the observations that the protein
stimulates enzyme activities and processes that depend on fatty acids
(9, 18, 19), including the biosynthesis of phospholipids and
triglycerides (14, 20).
LFABP binds the peroxisome proliferators (10) and seems to participate
in the trafficking of PPAR ligands, such as LCFAs, to the nucleus
(21). Thus, LFABP may have an important role in the regulation of the
ligand-dependent transactivation of PPAR via a direct
protein-protein interaction with PPAR in the nucleus (22). In
summary, the available results indicate that LFABP has a role in a
fatty acid-driven regulation of the transcription of PPAR-regulated
genes. Because LCFA has a key role in the biological activities of the
two proteins, it is possible that PPAR and LFABP have important
roles in the regulation of the assembly and secretion of
apoB-containing lipoproteins, processes that are highly dependent on LCFAs.
The process involved in the assembly of these lipoproteins has recently
been reviewed (23, 24). It consists of two major steps, the first of
which is a co-translational lipidation of apoB (25). This lipidation is
followed by the uptake of the major amount of triglycerides in a second
step of assembly (26). The process is highly dependent on lipid
biosynthesis and the availability of fatty acids (for review, see Refs.
23 and 24). It is also well known that the regulation of apoB
production involves a variation in the co-translational or
posttranslational degradation of the protein (for reviews with
references, see Refs. 23, 24, and 27). Thus, it has recently been
demonstrated that apoB undergoes degradation at three levels: (i)
co-translationally, most likely from the translocon, a process that
involves proteasomes (28, 29); (ii) posttranslationally, by a hitherto
unknown pathway (28); and (iii) via the LDL receptor (28, 30).
The size of the assembled very low density lipoprotein (VLDL) has been
demonstrated to be of importance for the size, turnover, and
atherogenicity of the LDLs formed during the intravascular degradation
of VLDL. Factors that influence the size of the assembled VLDL could
therefore be of great importance for the atherogenicity of LDL. It has
previously been shown that insulin resistance and type 2 diabetes give
rise to large VLDLs that are converted to small dense LDLs with a
half-life of 5 days (see Ref. 31 for review). The role of PPAR for
the size of apoB-containing lipoproteins has not been elucidated in
detail; however, it has been demonstrated that PPAR agonists induce
a dramatic decrease in the apoB-48-containing VLDL particles secreted
from rat hepatocytes. Instead, apoB-48 is secreted on particles that
band in the HDL density range (32).
We have used primary rat hepatocytes as well as the rat hepatoma cell
line McA-RH7777 to investigate the importance of PPAR activation and
LFABP for the assembly and secretion of the apoB-containing lipoproteins.
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EXPERIMENTAL PROCEDURES |
Materials--
Eagle's minimum essential medium, nonessential
amino acids, glutamine, penicillin, and streptomycin were obtained from
BioWhittaker. Eagle's minimum essential medium without methionine and
Williams E medium with glutamax were obtained from Invitrogen. Matrigel was purchased from Collaborative Research Medical Products (Bedford, MA). Insulin (Actrapid®) was from Novo Nordisk A/S, dexamethasone and
clofibrate were from Sigma, and WY 14,643 was from Chemsyn Co. (Lenaxa,
KS). Fetal calf serum was purchased from JRH Biosciences, and rabbit
immunoglobulin was from DAKO. Lactacystin and Pansorbin® were from
Calbiochem. Methionine, sodium pyruvate, disodium carbonate, phenylmethylsulfonyl fluoride, pepstatin A, and leupeptin were from
Sigma. Trasylol® (aprotinin) was obtained from Bayer
Leverkusen. N-Acetyl-Leu-Leu-norleucinal was from Roche
Molecular Biochemicals. Amplify®,
[9,10(n)-3H]palmitic acid,
[35S]methionine-cysteine mix, and Rainbow colored protein
molecular weight markers were from Amersham Biosciences, and Ready
Safe® was from Beckman (Fullerton, CA). GF/F glass
microfiber filter was obtained from Whatman. Geneticin was from
Duchefa. The BCA kit was purchased from Pierce. AG1-X18 ion-exchange
resin was from Bio-Rad.
Cell Culture and Metabolic Labeling of McA-RH7777
Cells--
McA-RH7777 cells were cultured in Eagle's minimum
essential medium containing 20% fetal calf serum, 1.6 mM
glutamate, 1.6 mM sodium pyruvate, 140 mg/ml streptomycin,
140 IU/ml penicillin, and 60 mg/ml nonessential amino acids in 5%
CO2 at 37 °C, as described previously (26). The cells
were split twice weekly and fed every day. The cells were pulse-labeled
with [35S]methionine-cysteine mix and chased in culture
medium supplemented with 10 mM methionine (26) as indicated
in the text.
Gradient ultracentrifugation of apoB-containing lipoproteins secreted
into the medium was carried out as described previously (26). Cytosol
was recovered as the 100,000 × g supernatant after homogenization of cells (33) or liver (34).
Cell Culture and Metabolic Labeling of Primary Rat
Hepatocytes--
Hepatocytes were prepared by a nonrecirculating
collagenase perfusion through the portal vein of 200-300-g normal
female Sprague-Dawley rats as described previously (35). For
measurements of mRNA, the cells were seeded at a density of
~170,000 cells/cm2 in plastic 100-mm dishes. For
measurements of total secretion of apoB-48 and apoB-100, 6-cm dishes
were used. The dishes were coated with laminin-rich Matrigel, and the
cells were plated during the first 16-18 h in Williams E medium
supplemented as described previously (35). After 16-18 h of culture,
the culture medium was changed to a medium with a lower level of
insulin (3 nM) with 1 nM dexamethasone.
The medium was changed every day, and the cells were treated for 4 days
with WY 14,643 or clofibrate dissolved in Me2SO (0.15%
v/v). The cells were pulse-labeled with
[35S]methionine-cysteine mix and chased in culture medium
supplemented with 10 mM methionine (36) as indicated
in the text. In some experiments (as indicated in the text), the cells
were preincubated with Eagle's minimum essential medium without
methionine and cysteine before they were pulse-labeled with the
[35S]methionine-cysteine mix (Promix) in the methionine-
and cysteine-free medium (36).
Gradient ultracentrifugation of apoB-containing lipoproteins secreted
into the medium was carried out as described previously (36).
Transfection of McA-RH7777 Cells with LFABP--
McA-RH7777
cells were transfected with pCIN4, which is a derivative of pCIN1 (37)
containing the full-length rat LFABP gene. This construct was a kind
gift of Dr N. Bass. Expression of LFABP is driven by the human
cytomegalovirus intermediate early promotor. The day before
transfection, the cells were seeded at ~4.4 × 105
cells/9.6-cm2 culture well. Transfection was performed as
recommended by the manufacturer (DOSPER Liposomal Transfection Reagent;
Roche Molecular Biochemicals). The cells were maintained in culture
medium containing 800 µg/ml Geneticin. Approximately 3-4 weeks after
transfection, single colonies were picked and maintained in selective
medium. Geneticin was omitted from the culture medium 2 weeks before
experiments were started.
Oxidation of Palmitic Acid--
The methods described in Refs.
38 and 39 were used. Briefly, cells were incubated with
[9,10(n)-3H]palmitic acid at 37 °C for 0, 30, 60, and 120 min, respectively. Conditioned medium containing the
3H2O generated from the palmitic acid by
-oxidation and oxidative phosphorylation was collected and applied
to an AG1-X18 ion-exchange column (38, 39). The radioactive water
eluted with the unretained fraction was collected, and the
radioactivity was determined.
Apolipoprotein B Biosynthesis and Secretion--
ApoB was
immunoprecipitated from cells and medium by methods described
previously (26). Electrophoresis in SDS-polyacrylamide gels,
autoradiography, and determination of the radioactivity in proteins
separated in the gels were carried out as described previously (40).
DNA was determined as described in Ref. 41.
Permeabilization of McA-RH7777 Cells--
To establish the
method of permeabilization in McA-RH7777 cells, previously published
protocols were used (42, 43). Confluent McA-RH7777 cultures were
incubated with methionine-free modified Eagle's medium for 2 h.
Thereafter, the cells were pulse-labeled (30 min) with
[35S]methionine-cysteine and chased for 30 min in culture
medium supplemented with 10 mM methionine to get fully
elongated, labeled apoB-100. The cells were then washed and incubated
in CSK buffer (0.3 M sucrose, 0.1 M KCl, 2.5 mM MgCl2, 1 mM sodium-free EDTA, and 10 mM PIPES, pH 6.8) containing different amounts of
digitonin. Digitonin-treated cells were washed three times in CSK
buffer and chased for 60 min in the presence of cytosol, an
ATP-regenerating system, fetal calf serum (FCS), and 360 µM oleic acid. Cytosol to supplement the permeabilized
cells was prepared as described previously (42). The ATP-regenerating
system has been described in detail previously (42).
To establish the system for McA-RH7777 cells, we permeabilized them
with different concentrations of digitonin (0-25 µg/ml) for 5 or 10 min and followed (i) the release of lactate dehydrogenase, (ii) the
ability of the cells to adhere to the culture dish, and (iii) the
ability of the cells to secrete apoB. Our results demonstrated that the
largest release of lactate dehydrogenase that allowed the cell to stick
to the surface and secrete apoB was obtained when the cells were
treated with 12.5 µg/ml digitonin for 10 min at room temperature. We
therefore used these conditions.
The secretion of VLDL from the permeabilized cells was dependent on
FCS, as is the case in intact cells. We observed that this effect was
highly dependent on supplementation with cytosol (Fig.
1). Thus, when the permeabilized cells
were incubated with cytosol alone, small amounts of apoB-100-VLDL were
secreted into the medium. The addition of FCS without cytosol gave rise
to a small increase in the secretion of apoB-100-VLDL, whereas a 4- to
5-fold increase in the secretion was obtained when both cytosol and FCS
were added to the permeabilized cells.
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Fig. 1.
FCS together with cytosol
(ATP/Cytosol/FCS) induces a higher secretion of
apoB-100 from permeabilized McA-RH7777 cells than cytosol
(ATP/Cytosol) or FCS (ATP/FCS)
alone. McA-RH7777 cells were preincubated in methionine-free
medium for 2 h, labeled with
[35S]methionine-cysteine mix for 30 min, and chased for
30 min. The cells were then permeabilized with 12.5 µg/ml digitonin
for 10 min and chased for 60 min in the presence of oleic acid as
follows: ATP/Cytosol, CSK buffer, cytosol, and the
ATP-regenerating system; ATP/Cytosol/FCS, CSK buffer,
cytosol, 20% FCS, and the ATP-regenerating system; and
ATP/FCS, CSK buffer, 20% FCS, and the ATP-regenerating
system. The medium was collected after the 60-min chase and subjected
to gradient ultracentrifugation. ApoB was recovered from each fraction
by immunoprecipitation and SDS-PAGE. The figure shows the sum of the
apoB-100 radioactivity in the VLDL and LDL/IDL density regions. The
values are means ± S.D. (n = 3 culture dishes).
*, p < 0.05 versus cells incubated with
ATP/Cytosol; #, p < 0.05 versus cells
incubated with ATP/Cytosol/FCS (one-way ANOVA followed by Bonferroni's
test).
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We also investigated the importance of oleic acid for apoB secretion
from these cells. The results (Fig. 2)
demonstrated that in order to obtain maximal secretion of
apoB-100-VLDL, the cells had to be preincubated with oleic acid for
2 h before the permeabilization. Moreover, oleic acid had to be
present during the 60-min chase (Fig. 2).
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Fig. 2.
Addition of oleic acid (OA)
to the cytosol induces a higher secretion of apoB-100 from
permeabilized McA-RH7777 cells if the cells are preincubated with oleic
acid for 2 h before the permeabilization. The cells were
preincubated (for 2 h) in the absence ( OA) or
presence (+OA) of 360 µM oleic acid for 2 h (as indicated in the figure) before labeling and permeabilization.
The cells were pulse-labeled, chased, and permeabilized as described in
the Fig. 1 legend. The permeabilized cells were then chased for 1 h with CSK buffer, ATP-regenerating system, 20% fetal calf serum, and
cytosol with (+OA) or without (OA) 360 µM oleic acid. The secretion of apoB-100 was determined
as described in the Fig. 1 legend. The values are means ± S.D.
(n = 3 culture dishes). *, p < 0.05 versus the cells with no oleic acid during the 1-h chase
period (Student's t test).
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As a precaution, we subjected the culture medium to gradient
ultracentrifugation (26) in the experiments carried out on permeabilized cells. The total apoB-100 radioactivity was then calculated as the sum of apoB-100 radioactivity in the LDL and VLDL
density ranges. The reason for this precaution is the large pool of
apoB-100 that is associated with the microsomes. Detergent treatment
may damage these membranes and solubilize this pool and release it into
the medium, where it can be measured as a true secretion. Because this
form of apoB floats in the HDL density range upon gradient
ultracentrifugation (44), and because very low levels of apoB-100 are
present in the HDL density range in normal conditioned medium (26),
gradient ultracentrifugation could be used to detect such a possible
side effect of the permeabilization. However, gradient
ultracentrifugation demonstrated that the major amount of apoB was
present in the VLDL density range, with small amounts floating as LDL
(data not shown). Thus, we have not obtained evidence of any major
release of the microsomal pool into the medium in the experiments that
were performed.
Only low levels of apoB-100-VLDL were detected in the microsomes (data
not shown), indicating that the permeabilized McA-RH7777 cells release
the predominant amount of secretable lipoproteins into the culture medium.
Thus, in summary, the following basic protocol was used in the studies
with permeabilized cells. The cells were preincubated for 2 h with
360 µM oleic acid. They were then pulse-labeled for 30 min, chased for 30 min, and permeabilized. The permeabilized cells were
chased for 60 min with the ATP-regenerating system, cytosol, oleic
acid, and FCS. ApoB-100 was recovered form the VLDL and LDL density
range of the conditioned medium.
Other Methods Used for Protein Analysis--
Immunoblot was
carried out as described previously (34) using a polyclonal antibody to
LFABP (35) raised against recombinant LFABP that was kindly supplied by
Dr. D. Cistola (Washington University School of Medicine, St. Louis,
MO). Trichloroacetic acid precipitation of proteins was carried
out by adding 600 µl of 10% trichloroacetic acid to a 100-µl
sample. The mixture was chilled to 5 °C for 30 min, and an
additional milliliter of 5% trichloroacetic acid was added. The
mixture was then filtered through a GF/F glass microfiber filter. Ready
Safe® was added to the filter, and the radioactivity was
determined. The protein concentration was determined using the BCA kit (Pierce).
Gel Ribonuclease Protection Assays--
Total RNA was prepared
by the method described in Ref. 45. PPAR and apoB mRNA levels
were determined with gel ribonuclease protection assays as described
previously (8, 36) using biotin-labeled probes (PPAR) or
32P-labeled probes (apoB) and the RPA III kit (Ambion). A
126-bp fragment of -actin cDNA (Ambion) and an 80-bp fragment of
18 S (Ambion) were used as internal controls. The amounts of PPAR and apoB mRNA were expressed as the ratio between these bands and
the -actin mRNA or ribosomal 18 S band.
Solution Hybridization--
LFABP mRNA was measured by a
solution hybridization assay as described previously (35). The signal
was compared with a standard curve, which was obtained by hybridization
of in vitro-transcribed LFABP mRNA. The results are
expressed as amol LFABP/µg RNA.
Analysis of ApoB mRNA Editing--
The extent of apoB
mRNA editing was determined by primer extension analysis as
described previously (36).
Statistics--
Values are expressed as mean ± S.D.
Comparisons between means were made by analysis of variance (ANOVA).
The ANOVA was followed by Bonferroni's post hoc test to do
pairwise comparisons between all groups or by Dunnet's test to compare
untransfected cells with the transfected clones. Student's
t test was used to do pairwise comparisons. The values were
transformed to logarithms when appropriate.
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RESULTS |
PPAR Agonists Increase ApoB Secretion, Decrease the Biosynthesis
of Triglycerides, and Redistribute the Secreted ApoB from VLDL to More
Dense Lipoproteins--
Treatment of primary hepatocytes with the
PPAR agonist WY 14,643 stimulated the secretion of apoB-100 (Fig.
3A), whereas there was no
effect on the secretion of apoB-48 (Fig. 3B). This experiment was repeated five times with cells from different liver perfusions, and all gave the same result. The mean increase in the
secretion of apoB-100 from the WY 14,643-treated cells (compared with
controls) was 2.2 ± 0.8 times (mean ± S.D. of six
experiments; p < 0.05, Student's t test).
No change in the secretion of apoB-48 was observed in the six
experiments. In these and the following experiments, we used 50 µM WY 14,643. The choice of concentration of the agonist
was based on the observation that a plateau level in the rate of
secretion of apoB-100 was reached at 50 µM WY 14,643 and
that no further increase in the secretion of apoB-100 was observed when
a WY 14,643 concentration of 200 µM was used. Treatment of the cells with clofibrate gave rise to an increase in the secretion of apoB-100 of the same magnitude as that seen for WY 14,643 (data not
shown). Moreover, clofibrate doubled the secretion of apoB-100 in rat
hepatoma McA-RH7777 cells (Fig. 3C). These results
demonstrate that PPAR agonists can induce an increased secretion of
apoB-100, but not of apoB-48.
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Fig. 3.
The effect of WY 14,643 (A
and B) and clofibrate (C) on
the secretion of apoB-100 (A) and apoB-48
(B) from primary hepatocytes and the secretion of
apoB-100 from McA-RH7777 cells (C). Primary
hepatocytes and McA-RH7777 cells were cultured as described under
"Experimental Procedures." The primary hepatocytes were cultured in
the absence or presence of 50 µM WY 14,643 for 4 days,
whereas McA-RH7777 cells were grown in the absence or presence of 1 mM clofibrate for 4 days. Both primary hepatocytes and
McA-RH7777 cells were labeled for 2 h with a
[35S]methionine-cysteine mix and then chased for 4 h. The medium was collected, apoB-100 and apoB-48 were isolated by
immunoprecipitation and SDS-PAGE, and the radioactivity was determined.
Each observation is the mean of three cell culture dishes. The
radioactivity was related to the DNA content of the culture dishes.
Values are means ± S.D. *, p < 0.05 versus the group given no WY 14,643 or no clofibrate
(Student's t test).
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Incubation of the primary hepatocytes with WY 14,643 significantly reduced the biosynthesis and secretion of
triglycerides, as judged from the decreased incorporation of
[3H]palmitic acid into triglycerides in the cells and
culture medium (Table I). This
observation is in agreement with previously published results (46). WY
14,643 treatment of primary hepatocytes tended to increase the
oxidation of palmitic acid (Table I).
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Table I
Effect of WY 14,643 on triglyceride labeling in cells and medium,
-oxidation, apoB mRNA expression, and apoB mRNA editing
in primary cultures of rat hepatocytes
After plating the cells for 16 h in 16 nM insulin, the
cells were cultured for 4 days in the presence of 3 nM
insulin and 1 nM dexamethasone with or without 50 µM WY 14,643 (dissolved in Me2SO; 0.15%, v/v).
The triglyceride labeling in cells and medium was determined after
labeling the cells with a [9,10(n)-3H]palmitic
acid reaction mixture for 60 min. The triglycerides were recovered by
thin layer chromatography, and the radioactivity was determined. The
rate of -oxidation was determined after labeling cells with
[9,10(n)-3H]palmitic acid (110 µM
with a total radioactivity of 8 µCi/ml culture medium) for 0, 30, 60, and 120 min. After each period, the radioactive water was isolated from
the conditioned medium by ion-exchange chromatography, and the
radioactivity was measured. The rate of production of radioactive water
(dpm/(min and µg DNA)) was determined from the slope of the linear
curves describing the production of radioactive water (from
[3H]palmitic acid). The radioactivity was related to the DNA
content of the culture dishes. ApoB mRNA was quantified using a
radioactive gel ribonuclease protection assay. The intensity value of
the apoB band was divided by the intensity value of the ribosomal RNA
18 S band. Editing of apoB mRNA was measured with primer extension
analysis and expressed as a percentage of edited apoB mRNA
(apoB-48/(apoB-100 + apoB-48)). Each observation is the mean of
three culture dishes obtained from one rat liver, except for
measurement of apoB mRNA editing, which is based on three different
liver perfusions with three to four culture dishes in each group.
Values are means ± S.D.
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The results presented above could indicate that the PPAR agonists
induced a change in the density of the apoB-containing particles that
were secreted from the cells. We therefore studied the effect of WY
14,643 on the density of the apoB-containing lipoproteins that were
secreted. In control cells, the major amount of apoB-100 appeared on
VLDL particles in the medium, whereas a smaller amount could be found
on more dense particles, mainly banding in the IDL/LDL density region
(36, 47). VLDL and these more dense lipoproteins were isolated from WY
14,643-treated and control cells, and the apoB-100 radioactivity was
determined. Treatment with WY 14,643 gave rise to a decrease in the
relative amount of apoB-100 that appeared in the VLDL density range,
whereas there was a pronounced increase in the proportion of apoB-100 that was associated with the IDL/LDL density region (Fig.
4A). The observation of a
shift from VLDL to more dense particles is in agreement with the
increased production of apoB-100 and the decreased biosynthesis of
triglycerides that was induced by the PPAR agonist. Thus, the
PPAR agonist gave rise to less buoyant apoB-100-containing
lipoproteins.
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Fig. 4.
The effect of WY 14,643 on the proportion of
apoB-100 (A)- and apoB-48
(B)-containing lipoproteins secreted as VLDL and more
dense lipoproteins from rat hepatocytes. Primary rat hepatocytes,
isolated and cultured as described under "Experimental Procedures,"
were cultured in the absence or presence of 50 µM WY
14,643 for 4 days. The cells were then labeled with
[35S]methionine-cysteine mix for 2 h. The culture
medium was changed, and the cells were chased for 4 h. The
conditioned medium was recovered and subjected to gradient
ultracentrifugation using gradient 2 described in Ref. 36. This
gradient separated VLDL from the more dense apoB-containing
lipoproteins. The upper 3 ml containing VLDL was removed from the
denser fractions in the infranate. ApoB was isolated from these two
fractions as described in the Fig. 1 legend, and the radioactivity was
determined. The radioactivity was related to the DNA content of the
culture dishes. Each observation is the mean of three different liver
perfusions with three culture dishes in each group. The results are
given as the percentage of total radiolabeled apoB secreted to the
medium. Values are means ± S.D. *, p < 0.05 versus the group given no WY 14,643 (Student's t
test).
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ApoB-48 appears on VLDL particles as well as on particles that band in
the HDL density range (36, 47). The relative amount of apoB-48 in VLDL
decreased significantly in the cells that had been treated with 50 µM WY 14,643 (Fig. 4B). On the contrary, there
was an increase in the secretion of apoB-48 on more dense particles
(i.e. in the HDL density range). These observations are in
agreement with our previous observation that the assembly and secretion
of apoB-48-VLDL are highly dependent on the availability of fatty acids
and the rate of triglyceride biosynthesis (26). Moreover, they confirm
the results of other authors (32).
PPAR Agonists Increase the Production of ApoB-100 by Influencing
the Intracellular Posttranslational Degradation of the Protein--
WY
14,643 treatment had no effect on the level of apoB mRNA or on the
editing of apoB mRNA (Table I). These results indicate that the
increased secretion of apoB-100 reflects a decreased co-translational
and/or posttranslational degradation.
Next, we included lactacystin, a proteasome inhibitor, in pulse-chase
studies of control and WY 14,643-treated primary rat hepatocytes.
Lactacystin had no effect on the accumulation of radiolabeled apoB-100
in control cells (Fig. 5A),
indicating that the co-translational proteasomal degradation has little
importance in primary rat hepatocytes. Incubation with WY 14,643 induced a 2-fold increase in the amount of radiolabeled apoB-100 as
early as after the 15-min pulse period (Fig. 5A). Incubation
with the PPAR agonist also induced a change in the sensitivity to
lactacystin. Thus, there was a significant increase in the accumulation
of radiolabeled apoB-100 when the WY 14,643-treated cells were
incubated with lactacystin (Fig. 5A). This finding indicates
that treatment with WY 14,643 not only protected apoB-100 from
degradation but also made apoB-100 more sensitive to proteasomal
degradation. One obvious reason is the PPAR agonist-induced decrease
in the rate of triglyceride biosynthesis. A decrease in the lipidation of apoB-100 has been shown to increase the co-translational,
proteasome-dependent degradation of apoB-100 (28, 29, 48,
49). The WY 14,643-induced increase in the amount of radiolabeled
apoB-100 remained after the 15-min chase, but the lactacystin effect
was gone (Fig. 5B).
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Fig. 5.
The effect of WY 14,643 and lactacystin on
the recovery of radiolabeled intracellular apoB-100 (A, B,
E, and F) and apoB-48 (C
and D) after different pulse times (A, C,
E, and F) and a 15-min pulse followed by a
15-min chase (B and D). Rat
hepatocytes were recovered and cultured as described under
"Experimental Procedures." The cells were cultured in the absence
() or presence (+) of WY 14,643 (WY) for 4 days before the
experiment and during the pulse and chase periods. All cells were
preincubated for 60 min in the absence of methionine and cysteine with
or without 10 mM lactacystin (as indicated in the figure).
The cells were pulse-labeled with
[35S]methionine-cysteine mix for 15 min (AD)
and chased for 0 (A and C) or 15 min
(B and D). In another perfusion experiment, cells
were pulsed for 2 or 5 min (E and F). After the
pulse or pulse and chase periods, the cells were harvested, apoB was
recovered by immunoprecipitation and SDS-PAGE, and the radioactivity in
apoB-100 and apoB-48 was determined. A representative autoradiogram
showing the effect of WY 14,643 on intracellular apoB-100 and nascent
polypeptides after a 5-min pulse period is shown in F. The
radioactivity was related to the DNA content of the culture dishes. The
values are means ± S.D. of four culture dishes in each group.
*, p < 0.05 versus untreated cells; #,
p < 0.05 versus WY 14,643-treated cells
(one-way ANOVA followed by Bonferroni's test (AD) or
Student's t test (E)).
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There was no effect of WY 14,643 or lactacystin on the accumulation of
apoB-48 (Fig. 5, C and D) or albumin (data not
shown) after the 15-min pulse or the 15-min pulse followed by a 15-min chase. These results indicate that the PPAR agonist had a different influence on the intracellular turnover of apoB-48 and apoB-100, which
in turn indicates that the intracellular processing of the two proteins differs.
The protective effect of WY 14,643 on apoB-100 was seen long before it
was secreted. Thus, PPAR influences a pre-secretory degradation. To
further address the question of the localization of the proteolysis
that was inhibited by PPAR agonists, we carried out experiments with
shorter pulse periods (2 and 5 min) and followed the accumulation of
radiolabeled apoB-100 in normal and WY 14,643-treated cells. The
results indicate that the WY 14,643-induced increase in accumulation of
apoB-100 that was seen already after a short pulse (Fig. 5E)
remained during the intracellular transport and secretion of the
protein. This indicates that the effect of WY 14,643 occurs very early
in the secretory pathway. This conclusion was further supported by the
observation that there was an increased protection of longer apoB-100
nascent polypeptides in the WY 14,643-treated cells (Fig.
5F). We used a phosphorimager to estimate the amount of
radiolabeled apoB-100 nascent chains with a size of ~300 kDa and
longer. The results demonstrated that treatment with WY 14,643 gave
rise to a significant increase in these nascent chains compared with
control cells (64,769 ± 7156 versus 37,462 ± 5066 Storm units; mean ± S.D.; n = 3;
p < 0.05, Student's t test). Thus, the
increase in the apoB-100 nascent chains that was induced by WY 14,643 was in the same order as the increase in full-length apoB-100 seen after the treatment with the agonist. This finding indicates that the
major effect of PPAR was to inhibit the co-translational degradation
of apoB-100. However, we cannot exclude the possibility that the
agonist also has a minor influence on early posttranslational degradation. The nascent chains were identified as described previously (25, 50). It could be noticed that the nascent chains have the
appearance that has been previously described for apoB-100 (25,
50).
In the next set of experiments, we investigated whether the same
protection of apoB-100 by a PPAR agonist could be seen in McA-RH7777
cells. The results (Fig. 6) demonstrated
that clofibrate induced an increase in the intracellular amount of
apoB-100 seen after the 15-min labeling period and also after the 15- and 30-min chases. However, one difference was observed; whereas in the
primary rat cells, the ratio between agonist-treated and control cells remained almost constant between 15-min pulse and 15-min pulse plus
15-min chase, this ratio increased with time in the McA-RH7777 cells
(Fig. 6, inset). This finding may reflect different levels of posttranslational degradation in the two cell types.
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Fig. 6.
The effect of clofibrate ( ) on the
recovery of intracellular apoB-100 compared with untreated McA-RH7777
cells  . The inset shows the ratio
between the radiolabeled apoB-100 in clofibrate-treated and
control cells as a function of the chase period. Cells were cultured in
the absence or presence of clofibrate (1 mM) for 4 days.
They were then pulse-labeled for 15 min with
[35S]methionine-cysteine mix and chased in the presence
of cold methionine for periods of 0, 15, or 30 min. After each chase
period, apoB-100 was recovered from the cells and from the medium by
immunoprecipitation and SDS-PAGE, and the radioactivity was determined.
Before the 30-min chase, no apoB was present in the medium. A small
secretion (corresponding to <5% of the intracellular pool) was seen
after the 30-min chase. There were three culture dishes in each group.
Results are given as means ± S.D. *, p < 0.05 versus untreated cells (Student's t test).
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Effect of LFABP on the Intracellular Pools and Secretion of
ApoB--
As discussed above, LFABP seems to be important for the
transcription factor activity of PPAR (22). Moreover, both PPAR itself and LFABP are among the genes that are regulated by PPAR (3,
5-8). We confirmed these observations by showing that PPAR agonists
(clofibrate and WY 14,643) could induce an increase in the expression
of PPAR mRNA as well as gene expression and biosynthesis of
LFABP in both primary rat hepatocytes and McA-RH7777 cells (data not shown).
Thus, LFABP is one of the genes that could be of importance for the
influence of PPAR on the assembly of apoB-containing lipoproteins.
We therefore investigated the effect of an increased expression of
LFABP on the biosynthesis and secretion of apoB-100 and apoB-48, the
biosynthesis of triglycerides, and -oxidation of palmitic acid.
Cytosol from the McA-RH7777 cells contained a much lower amount of
LFABP than cytosol from rat hepatocytes, as revealed by immunoblots
(Fig. 7A). It would therefore
be possible to substantially increase the levels of LFABP in the
McA-RH7777 cells without exceeding the physiological levels seen in
normal rat liver. The McA-RH7777 cells were stably transfected with
LFABP. Four clones expressing LFABP were selected, all of which had an
increased expression of LFABP compared with the nontransfected cells
(Fig. 7B).
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Fig. 7.
Immunoblot using a polyclonal antibody to
LFABP of (A) a dilution series of cytosol from
McA-RH7777 cells or rat liver and (B) untransfected
McA-RH7777 cells (Normal) and McA-RH7777 cells
transfected with LFABP (2, 4, 5, and
10). Cytosol was prepared as described under
"Experimental Procedures." The indicated amount (A) of
cytosol was electrophoresed in SDS-polyacrylamide (15%) gels. In
B, 50 µg of cytosol from normal and transfected cells was
electrophoresed under the same conditions. The gels were immunoblotted
against a polyclonal antibody to LFABP, and the bound antibodies were
detected by horseradish peroxidase and chemiluminescence.
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The cells were labeled for 2 h and chased for 3 h, and both
apoB-100 and apoB-48 were recovered from the medium, and the
radioactivity was determined. We used the apoB radioactivity obtained
after the 2-h pulse as an estimate of the intracellular pool of apoB. The results (Fig. 8) indicate that the
size of the intracellular pool (Fig. 8, A and C)
and the secretion (Fig. 8, B and D) of apoB-100
(Fig. 8, A and B) and apoB-48 (Fig. 8,
C and D) were higher in the four clones than in
the nontransfected McA-RH7777 cells.
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Fig. 8.
The intracellular pool (A
and C) and secretion (B and
D) of apoB-100 (A and
B) and apoB-48 (C and
D) from untransfected McA-RH7777 cells
(N) and the different clones (2, 4, 5, and 10). McA-RH7777 cells were labeled with
[35S]methionine-cysteine mix for 2 h. Cells and
medium were collected, apoB-100 and apoB-48 were recovered from the
cells (A and C) by immunoprecipitation and
SDS-PAGE, and the radioactivity was determined. To recover apoB from
the medium, cells were labeled for 2 h and then chased for 3 h. ApoB-100 and apoB-48 were recovered from the conditioned medium as
described in the Fig. 1 legend. Each group consisted of four culture
dishes. Values are given as means ± S.D. *, p < 0.05 versus the untransfected (N) control
group (one-way ANOVA followed by Dunnet's test).
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We also measured the intracellular pools and secretion of total
proteins (i.e. trichloroacetic acid-precipitable
radioactivity) in the cells and in the medium during the 2-h incubation
with labeled methionine-cysteine. The total protein labeling in the cells and the medium showed some variation between the clones (data not
shown). Because of this variation, we related the intracellular pool
size and the secretion of apoB to the size of the intracellular pool
and the secretion of total (trichloroacetic acid-precipitable) proteins. The results (data not shown) demonstrated that the
significant differences in apoB-100 and apoB-48 secretion between the
four clones and the nontransfected cells remained. These results
indicate that LFABP transfection induces an increase in the secretion
of apoB-100 and apoB-48.
Effects of LFABP on the Biosynthesis and Secretion of
Triglycerides--
To estimate the rate of biosynthesis and
secretion of triglycerides, the control cells and clones 2, 4, 5, and
10 were incubated with [3H]palmitic acid for 60 min, and
the accumulation of radioactive triglycerides was measured in the cells
and culture medium. The results (Table
II) indicate that the clones had a
decreased biosynthesis of triglycerides; however, it reached
significance only in clones 4 and 5. On the contrary, all four clones
showed a highly significant decrease in the rate of triglyceride
secretion (Table II). Because there was an increase in apoB secretion,
our results indicate that apoB-containing lipoproteins with less
triglycerides/apoB protein were formed when LFABP was overexpressed.
However, using gradient ultracentrifugation analysis, we could not
detect any change in the distribution of apoB-100 lipoproteins compared
with normal cells. Thus, the major amount of apoB-100 was still
secreted on VLDL particles, with smaller amounts present in the IDL/LDL density range (data not shown).
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Table II
Triglyceride labeling in cells and medium and -oxidation in
LFABP-transfected McA-RH7777 cells
Normal McA-RH7777 cells as well as clones 2, 4, 5 and 10 were incubated
with [9,10(n)3H]palmitic acid for 60 min to
measure accumulation of labeled triglycerides in the cells and in the
medium. The triglycerides were recovered by thin layer chromatography,
and the radioactivity was determined. The rate of -oxidation was
determined after labeling normal and transfected cells with
[9,10(n)-3H]palmitic acid (110 µM
with a total radioactivity of 8 µCi/ml culture medium) for 0, 30, 60, and 120 min. After each period, the radioactive water was isolated from
the conditioned medium by ion-exchange chromatography, and the
radioactivity was measured. The radioactivity was related to the
protein content of the culture dishes. Values are given as means ± S.D.
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We also investigated the effect of the transfection on the rate of
-oxidation. The results (Table II) demonstrate a significantly increased rate of -oxidation in clones 5 and 10. However, the rate
of -oxidation for clones 2 and 4 did not differ significantly from
that for the nontransfected McA-RH7777 cells.
LFABP Increases the Expression of PPAR mRNA--
Because
LFABP has been suggested to be of importance for the transactivating
activity of PPAR, it is possible that the increased levels of LFABP
are reflected in an increased activation of the receptor, which in turn
may lead to an increased activation of genes that are transactivated by
PPAR. To address this possibility, we investigated whether the
increase in LFABP gave rise to an increased gene expression of PPAR.
PPAR mRNA was expressed in the control cells (Fig.
9), and all the investigated clones had increased levels of PPAR mRNA.
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Fig. 9.
Determination of PPAR
mRNA by a gel ribonuclease protection assay in untransfected
McA-RH7777 normal cells (N) and in clones 2, 4, 5, and
10. Total RNA was prepared, and 20 µg of RNA was hybridized with
biotin-labeled rat PPAR and -actin antisense probes. Protected
fragments were separated on denaturing polyacrylamide 6%
Tris-borate-EDTA-urea gels. The protected fragments were
transferred from the gels to Bright star-plusTM membranes
by a semi-dry transfer system. The detection was carried out using
Bright starTM BioDetectTM Kit, and the
chemiluminescence was detected. The intensity value for the PPAR
band was divided by the intensity value for the -actin band. The
amounts of the transcripts are expressed as the ratio between these two
bands. There were four culture dishes in each group. Values are given
as means ± S.D. *, p < 0.05 versus
the untransfected (N) control group (one-way ANOVA followed
by Dunnet's test).
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These results may indicate that the overexpression of LFABP influences
the secretion of apoB-100 through the increased expression of PPAR.
To further address this question, we used permeabilized cells to
investigate whether or not the increased secretion of apoB was due to
the induction of a cytosolic factor that directly influenced apoB-100
secretion. In these experiments (Fig.
10), either normal or
clofibrate-treated cells (both were preincubated with oleic acid) were
labeled for 30 min and chased for 30 min to obtain labeled, full-length
apoB-100. The cells were then permeabilized and chased in the presence
of cytosol from normal (with low amounts of LFABP) or
clofibrate-treated cells. Treatment with clofibrate gave rise to a
10-fold increase in the production of LFABP in McA-RH7777 cells (data
not shown). When clofibrate-treated permeabilized cells were chased in
the presence of cytosol from normal cells (Fig. 10,
Clof./Normal), there was a 2-fold increase in the secretion of apoB compared with that observed in nontreated permeabilized cells
that were chased in the presence of cytosol from nontreated cells (Fig.
10, Normal/Normal). There was no significant difference in
the rate of secretion of apoB-100 between clofibrate-treated permeabilized cells incubated with cytosol from clofibrate-treated cells (Fig. 10, Clof./Clof.) and those cells incubated with
cytosol from untreated cells (Fig. 10, Clof./Normal). These
results argue against a direct effect of a PPAR agonist-induced
cytosolic factor, such as LFABP, on the secretion of apoB.
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Fig. 10.
The apoB secretion from permeabilized
McA-RH7777 cells that had been pretreated (Cell
treatment) with (Clof.) or
without clofibrate (Normal). The cells were
chased in the presence of cytosol (Source of cytosol) from
untreated (Normal) and clofibrate-treated (Clof.)
McA-RH7777 cells. McA-RH7777 cells were incubated (Cell
treatment) for 4 days in the absence or presence of 1 mM clofibrate. The cells were then pretreated,
pulse-labeled, and chased as described in the Fig. 1 legend. After
permeabilization, the cells were chased in cytosol (Source of
cytosol) from untreated cells (Normal) or cells that
had been treated with clofibrate (Clof.). After the chase
period, the medium was recovered and subjected to gradient
ultracentrifugation, and apoB was recovered from the gradient by
immunoprecipitation and SDS-PAGE. The major amount of apoB was present
in the VLDL fraction, with a small amount present in the LDL density
range. To calculate the secretion of apoB-100, the radioactive proteins
present in these two density fractions were combined. The values are
means ± S.D. (n = 3 culture dishes). *,
p < 0.05 versus the
Normal/Normal group (one-way ANOVA followed by Bonferroni's
test).
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DISCUSSION |
The results presented in this study indicate that the activation
of PPAR by known agonists increased the secretion of apoB-100, but
not that of apoB-48. Our observations that neither the expression of
apoB mRNA nor the editing of this mRNA was changed when primary hepatocytes were treated with PPAR agonists indicate that the increased production of apoB-100 reflects a decreased degradation of
the protein rather than an increased biosynthesis. It is well known
that co-translational or posttranslational degradation has an
important role in the regulation of apoB-100 production (for reviews
with references, see Refs. 23, 24, and 27), and apoB seems to undergo
degradation at three levels: (i) co-translational or early
posttranslational degradation involving proteasomes (this degradation
has been referred to as endoplasmic reticulum-associated degradation)
(28, 29); (ii) posttranslational degradation by a hitherto unknown
pathway referred to as post-endoplasmic reticulum presecretory
proteolysis (28); and (iii) postsecretory degradation via the LDL
receptor (28, 30).
Our results demonstrate that the PPAR agonists decreased
intracellular degradation because the accumulation of pulse-labeled apoB-100 was seen long before the protein had been secreted. The activation of PPAR had a complex influence on the intracellular degradation of apoB-100. Because the PPAR agonists decreased the
rate of triglyceride biosynthesis, these agonists gave rise to
an increase in the proteasomal degradation of apoB-100 (28, 29, 48,
49). This conclusion is based on the finding of an increased recovery
of apoB-100 when PPAR agonist-treated cells were incubated with the
proteasome inhibitor lactacystin. This effect of lactacystin was only
seen after the pulse, when apoB-100 was still in or close to the
translocon, and disappeared when apoB-100 had moved away from the site
of synthesis after a 15-min chase. This is in agreement with the
proposed involvement of proteasomes in the co-translational or early
posttranslational degradation of apoB-100. Our results also indicate
that the extra protein saved by the inhibition of the proteasomes was
lost during the chase. Thus, the protein that escapes proteasomal
degradation due to treatment with lactacystin will be removed by
another mechanism during the transfer through the secretory pathway.
Interestingly, the PPAR agonist treatment did not result in
lactacystin sensitivity for apoB-48. This difference between apoB-100
and apoB-48 may reflect a difference in the need for co-translational lipidation.
Based on the failure of lactacystin to influence the recovery of apoB
in control cells, we conclude that there is virtually no proteasomal
degradation of apoB-100 or apoB-48 in primary hepatocytes. This is in
contrast to the observations in commonly used hepatoma cell lines (28,
48) and could indicate that co-translational degradation is of no
importance in normal hepatocytes. However, the observation that the
PPAR agonist induced an increase in proteasome-dependent
degradation of apoB-100 (by inhibiting the rate of biosynthesis of
triglycerides) indicates that proteasomal co-translational degradation
is functional and also participates in the regulation of production of
apoB-100 in normal hepatocytes.
The major effect of the PPAR agonists was a decreased intracellular
degradation of apoB-100, an effect that was not seen for apoB-48 or
albumin. Thus, there is a fundamental difference in the intracellular
turnover of apoB-100 and apoB-48. The increase in apoB-100 was already
seen after a short pulse when the protein was close to the translocon,
indicating that the influence of the PPAR agonist on the secretion
of apoB-100 occurs early during the intracellular transfer,
i.e. during the co-translational period and/or the very
early posttranslational period. The observation of a significant
(1.7-fold) increase in the longer apoB-100 nascent chains in the
agonist-treated cells supports this conclusion and indicates that a
significant part of the effect of the PPAR agonist could be
explained by an inhibition of the co-translational degradation of
apoB-100. Together, our results indicate that the effect of the PPAR
agonist on the co-translational degradation and perhaps also on a very
early posttranslational degradation explains the increased secretion of
apoB-100 that is induced by the PPAR agonists.
The results presented in this study also indicate that PPAR is of
importance for regulation of the type of apoB-containing particles that
are assembled and secreted. In the case of apoB-48, the secretion of
VLDL virtually disappeared; instead, there was an increase in the
secretion of the dense apoB-48-containing particles. This finding
confirms results by other authors (32) and agrees well with our
previous results, indicating that the assembly of apoB-48-VLDL is
highly dependent on the availability of fatty acids and the
biosynthesis of triglycerides (26, 36, 47). Because the production of
apoB-48 is not influenced, the decrease in the rate of triglyceride
biosynthesis most likely explains the change in density of the secreted particles.
The secretion of apoB-100-VLDL also decreased; instead, there was a
significant increase in the secretion of particles banding in the IDL
and LDL density ranges. This change in density of the particles could
be explained by the increased production of apoB-100 and the decrease
in both the biosynthesis and secretion of triglycerides. Thus, the
activation of PPAR gave rise to more apoB-100 particles with less
triglycerides and higher density, in turn indicating that the size and
triglyceride load of the VLDL particles can be regulated by PPAR.
Our observations may therefore give some insight into the mechanism of
the hypolipidemic action of fibrates. Thus, the well known lowering of
the plasma triglycerides could be explained, at least in part, by the
decreased secretion of triglycerides. The cholesterol-lowering and
antiatherogenic effect may be explained by the change toward a
secretion of smaller, less triglyceride-rich apoB-containing
lipoproteins. It has been shown that smaller, less triglyceride-rich
VLDLs promote the formation of less atherogenic LDL particles with
shorter half-lives than those generated when large triglyceride-rich
VLDLs are secreted (see Ref. 31 for review).
It has been shown previously that PPAR agonists induce the
transcription of LFABP, and this was confirmed in the present study.
LFABP could influence the assembly and secretion of apoB-100-containing lipoproteins in at least two ways. First, LFABP is an acceptor of
long-chain fatty acids, which enhances their uptake and intracellular transport (12, 13, 51, 52), and the assembly and secretion of apoB have
been demonstrated to be highly dependent on fatty acids. Secondly,
LFABP may enhance the transactivating activity of PPAR (22), which
in turn could influence the assembly and secretion of apoB-100. We
observed that the expression of LFABP gave rise to a 2- to 3-fold
increase in the intracellular pool and the secretion of apoB-100. The
magnitude of this increase is in the order of that seen after treatment
of hepatoma cells with oleic acids (Refs. 26 and 33; see Ref. 24 for
review with references), a well-known inducer of apoB secretion in such cells. However, this increase in the apoB-100 secretion is also of the
same magnitude as that seen after treatment of the McA-RH7777 cells
(and primary rat hepatocytes) with PPAR agonists. In contrast to
what could be expected from an increased supply of fatty acids and from
the observations in in vitro studies (14, 20),
overexpression of LFABP gave rise to a decrease in the secretion of
triglycerides. Thus, in this way, the effect of the overexpression of
LFABP is reminiscent of the changes induced by the PPAR agonists,
and, indeed, we observed that an increase in the intracellular levels of LFABP gave rise to an increase in the expression of PPAR. One
explanation for this observation is the above-mentioned
LFABP-dependent increase in the transacting activity of
PPAR (22). Such an increase may lead to an increased expression of
PPAR itself because this gene is activated by PPAR (3). Our
observation may therefore suggest a mechanism that amplifies the effect
of a PPAR agonist on the assembly and secretion of apoB-containing
lipoproteins. Thus, the ligand increases the expression of LFABP, which
enhances the transacting activity of PPAR. This effect gives rise to
an increase in the transcription of PPAR itself, which influences the genes involved in inhibition of the co-translational and/or early
posttranslational degradation of apoB-100 and the biosynthesis of triglycerides.
The observation that overexpression of LFABP failed to induce the
change in density of the secreted apoB-100 particles that was seen in
the agonist-treated primary rat hepatocytes may argue against the
hypothesis that LFABP exerts its influence on VLDL assembly by
increasing PPAR levels. One reason could be that neither the
decrease in triglyceride biosynthesis nor the decrease in secretion of
triglycerides was as large as that seen in the agonist-treated cells.
This finding may reflect a lack of a strong PPAR agonist in these
experiments. The addition of a PPAR agonist would have obscured the
effects of the overexpression of LFABP.
The expression of LFABP in the McA-RH7777 cells resulted in an
increased secretion of apoB-48. This was not observed in the PPAR
agonist-treated primary hepatocytes. When evaluating this result, it
should be kept in mind that there were differences in the regulation of
secretion of apoB-48 between primary cells and McA-RH7777 cells,
differences that were not seen for apoB-100. Thus, apoB-48 is not
secreted as VLDL from the McA-RH7777 cells unless they are cultured in
the presence of oleic acid (data not shown) (26). The primary cells, on
the other hand, also secrete apoB-48 on VLDL particles in the absence
of oleic acid (36, 47). Thus, under the conditions needed to
investigate the effect of overexpression of LFABP, the second step in
the assembly of apoB-48-VLDL was not active. Moreover, clofibrate
induced an increase in secretion of apoB-48 in McA-RH7777 cells (data
not shown), whereas such an increase was not observed in primary cells.
We have therefore confined the comparison between the primary cells and
McA-RH7777 cells to apoB-100. However, due to the differences discussed
above, we cannot exclude the possibility that LFABP may influence the
assembly of apoB-containing lipoproteins in ways other than increasing
the PPAR activity. On the other hand, the experiments in the
permeabilized cells with LFABP-enriched cytosol (cytosol from
clofibrate-treated cells) may argue against a direct effect of LFABP or
other cytosolic factors induced by the PPAR agonist on the assembly
of apoB-100-containing lipoproteins.
In summary, PPAR and LFABP may cooperate in the regulation of the
amount and density of apoB-100 lipoproteins that are secreted from
hepatocytes. This cooperation leads to protection of apoB-100 against
co-translational and/or early posttranslational degradation, in turn
resulting in increased secretion of apoB-100. At the same time, the
activation of PPAR gives rise to a decrease in the biosynthesis of
triglycerides. Together, these effects lead to an increased secretion
of apoB-100 on denser particles with a decreased load of triglycerides.
Agonists on the Intracellular Turnover and Secretion of
Apolipoprotein (Apo) B-100 and ApoB-48*
§,
,
TOP
ABSTRACT
INTRODUCTION
