© 1997 American Heart Association, Inc.
Articles |
Assembly and Secretion of VLDL in Nondifferentiated Caco-2 Cells Stably Transfected With Human Recombinant ApoB48 cDNA
From the Departments of Pathology (J.L., A.B., M.M.H.) and Biochemistry
(Z.Z., M.M.H.), Allegheny University of the Health Sciences,
MCP
Hahnemann School of Medicine, Philadelphia, Pa, and Bristol-Myers
Squibb (H.J.), Pharmaceutical Research Institute, Princeton, NJ.
Correspondence to M. Mahmood Hussain, PhD, Allegheny University of the Health Sciences, MCPHahnemann School of Medicine, 2900 Queen Ln, Philadelphia, PA 19129. E-mail hussain{at}auhs.edu
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Abstract Intestinal cells secrete apoB48-containing very low density lipoproteins (VLDLs) and chylomicrons for the transport of biliary and dietary lipids. The molecular mechanisms regulating the assembly of intestinal lipoproteins are not known due to a lack of reliable and specific cell culture models. Caco-2 (a human colon carcinoma) cells have been used to study intestinal lipid metabolism. These cells have been shown to secrete both apoB100- and apoB48-containing triglyceride (TG)-rich lipoproteins only after differentiation into enterocyte-like cells. To study lipoprotein assembly in nondifferentiated Caco-2 cells, we stably expressed human recombinant apoB48 cDNA under the control of a constitutive cytomegalovirus promoter. Pulse-chase analysis revealed that the majority (>50%) of apoB48 synthesized was degraded intracellularly in the presence or absence of oleic acid. Transfected nondifferentiated cells secreted lipoproteins with flotation densities similar to those of plasma HDL or LDL when cultured in serum-free or serum-containing media, respectively. Incubation of cells with media containing serum and oleic acid resulted in the secretion of VLDL-like particles. Secretion of VLDL was inhibited (>80%) by triacsin C due to >60% inhibition of oleate-induced TG synthesis. However, inhibition of cholesteryl ester synthesis by 70% with an acyl coenzyme A:cholesterol acyltransferase inhibitor did not affect VLDL secretion. Efficient assembly of lipoproteins usually requires the microsomal TG transfer protein (MTP). The presence of MTP in transfected Caco-2 cells was investigated by measuring TG transfer activity in microsomal fractions. Microsomal fractions had 0.2% TG transfer activity per hour per microgram of protein, which corresponds to 30% to 60% of the MTP activity present in liver-derived cells. To determine whether MTP activity was required for lipoprotein assembly, transfected cells were incubated in the presence of the MTP inhibitor CP-10,447. This compound completely abolished the secretion of apoB. These data show that the transfected cell lines secrete lipoproteins of different densities under different culture conditions and that the assembly of larger VLDL particles requires active TG synthesis and MTP activity. Thus, in nondifferentiated Caco-2 cells, the amount of apoB secreted and not the MTP activity is the limiting factor for lipoprotein assembly.
Key Words: intestine • chylomicrons • triglycerides • apolipoprotein B • microsomal triglyceride transfer protein
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Introduction |
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Enterocytes are highly differentiated cells specialized for the absorption of nutrients and arise from nondifferentiated crypt cells that do not assemble and secrete lipoproteins (reviewed in Reference 11 ). These cells assemble biliary and dietary lipids into apoB48-containing VLDL and chylomicrons for transport into the blood (for some reviews, see References 2 through 62 3 4 5 6 ). Besides apoB48, the apolipoprotein composition of both lipoprotein types is similar, but they differ in their flotation densities, size, and electrophoretic mobility.7 Intestinal VLDLs are secreted in both the fasting and postprandial state, whereas chylomicron secretion is mainly a postprandial phenomenon.8 For these reasons, it has been proposed that the major function of chylomicrons is to transport dietary fat, whereas intestinal VLDLs provide a mechanism for the reabsorption of endogenous (biliary) lipids.5 8
Attempts to study intestinal lipoprotein assembly have been frustrating because enterocytes cannot be maintained in culture for long times. Caco-2 (human colon carcinoma) cells have been used to study intestinal lipid metabolism (for reviews, see References 2, 3, and 92 3 9 through 12). These cells exist in either the nondifferentiated or differentiated state but secrete lipoproteins only after differentiation. During differentiation, Caco-2 cells cease to proliferate after reaching confluence. They become transformed into enterocyte-like cells, polarize into apical and basolateral surfaces, and secrete most of the lipoproteins from the basolateral side. These cells secrete predominantly apoB100-containing lipoproteins with a flotation density similar to that of human plasma LDL and HDL. Secretion of larger lipoproteins by these cells has been documented after OA supplementation.13 14 15 16 The factors that induce lipoprotein assembly during differentiation and reasons for the lack of lipoprotein assembly in nondifferentiated Caco-2 cells are not known.
It has been shown that nondifferentiated cells do not express apolipoproteins probably because they lack a transcription factor required for apolipoprotein gene expression.17 Is apoB the only limiting factor for the assembly of buoyant, TG-rich lipoproteins? Can these cells assemble larger lipoproteins if apoB expression is induced? From studies in abetalipoproteinemia patients, it is known that MTP is required for the assembly of intestinal lipoproteins by enterocytes.18 Do nondifferentiated Caco-2 cells express MTP? On the basis of studies with nonintestinal and nonhepatic cells, we hypothesized that nondifferentiated Caco-2 cells do not express apoB and MTP and would behave like other nonintestinal and nonhepatic cells. To test this hypothesis, we expressed human recombinant apoB48 cDNA under the control of a cytomegalovirus promoter in nondifferentiated Caco-2 cells and studied the expression of MTP in these cells. Furthermore, their abilities to assemble larger lipoproteins and some of the requirements for the core expansion of primordial lipoproteins were evaluated.
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Methods |
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Materials
The apoB monoclonal antibody 1D1 was obtained from the Ottawa Heart Institute, Ottawa, Canada. Human apoA-I monoclonal antibodies were gifts from Dr G. Castro (Pasteur Institute, Lille, France). Sheep anti-human apoB and apoA-I polyclonal antibodies were purchased from Boehringer Mannheim. Rabbit anti-sheep IgG tagged with alkaline phosphatase was from Cappel. The geneticin analogue G418 and antibiotic-antimycotic mixture were obtained from GIBCO/BRL. Triacsin C was purchased from Biomol Research Laboratories. The ACAT inhibitor compound CD113,818 [(-)-N-(2,4-bis(methylthio)-6-methylpyridin-3-yl)-(hexylthio)decanoic amide] and MTP inhibitor CP-10,447 were provided by Pfizer Central Research. OA, fatty acid–free BSA, and Cab-o-sil (fumed silica) were obtained from Sigma Chemical Co. Centriprep concentrators (30-kDa cutoff) were from Amicon.
Human Recombinant ApoB48 cDNA Expression Vector
The expression plasmid pB48 was derived from pB53L-L containing
an engineered Mlu I restriction site at
nucleotide 7011.19 20 The expression plasmid
is similar to the one described before20 except that this
vector does not contain a neomycin resistance gene. The neomycin
resistance gene was provided by cotransfection with a pSV2-Neo plasmid.
In this plasmid, expression of apoB48 was under the control of a
cytomegalovirus promoter. Thus, apoB48 synthesis was independent of its
own cis-control elements and of apo B mRNA editing activity.
Cell Cultures
Caco-2 cells were obtained from the American Type Culture
Collection (Rockville, Md); grown in DMEM containing high glucose, 20%
fetal bovine serum, and a 1% antibiotic-antimycotic mixture; and
subcultured before they reached confluence. Cells (
25% confluent)
were transfected by incubation with pSV2-Neo (1 µg) with or without
pB48 (10 to 20 µg) at 35°C in a humidified chamber containing 3%
CO2.21 22 Individual colonies
resistant to 400 µg of G418 per milliliter were amplified and
assayed for apoB48 secretion. Positive clones were subcultured in the
same medium containing 200 µg of G418 per milliliter. For
experiments, the transfected Caco-2 cells were plated in
75-mm2 flasks at a split ratio of four and grown in the
absence of G418. The next day the cells were washed, and experiments
were performed within the next 3 days. All experiments were performed
using the MH-1 clone, and some experiments were repeated with the JL-9
and JL-4 clones.
Density Gradient Ultracentrifugation
KBr (1.5 g) was added to the conditioned medium (3 mL) to obtain
a density of 1.3 g/mL. This was sequentially overlaid with 2 mL
each of 1.21, 1.063, 1.019, and 1.006 g/mL density solutions by
using the Auto Density Flow (Buchler Instruments) and
ultracentrifuged (40 hours, 40 000 rpm, at 4°C). Fractions
(0.5 mL) were collected from the top of the gradient and used for
measuring apoB and the refractive index (Bausch and Lomb
Refractometer). The densities corresponding to VLDL/IDL (VLDL,
d<1.02 g/mL), LDL (d=1.02 to 1.063
g/mL), and HDL (d=1.063 to 1.21 g/mL) are
indicated on different graphs.
Immunoblot Analysis of Secreted ApoB
Proteins from conditioned medium were adsorbed to Cab-o-sil,
desorbed in the sample buffer, separated on a polyacrylamide
gel by electrophoresis, transferred to nitrocellulose, and visualized
by reaction with the monoclonal antibody 1D1 (specific for human apoB;
see below), followed by anti-mouse IgG and chemiluminescence reagents
(DuPont/NEN).
Measurement of ApoB and ApoA-I
ApoB was quantified by using a sandwich ELISA20
with monoclonal antibody 1D1 that recognizes an epitope in the
N-terminus (amino acids 474 to 539) of human
apoB23 24 and thus interacts with all apoB polypeptides
>apoB13. To measure apoA-I, plates were coated with a 1:1000 diluted
mixture of three monoclonal antibodies (A05, A17, and A44) and
incubated in succession with HDL3 (0 to 15 ng/well),
sheep polyclonal anti–apoA-I serum (Boehringer Mannheim),
alkaline phosphatase–labeled rabbit anti-sheep IgG (Cappel), and
p-nitrophenyl phosphate (1 mg/mL in 10
mmol/L ethanolamine, 0.5 mmol/L MgCl2,
pH 9.5). The plates were washed three times between different
incubations. The absorbance at 405 nm was determined by using a
Dynatech MRX microplate reader (Dynatech Labs).
Lipid Analyses
Cells were labeled with 5 µCi of [3H]glycerol
for 6 hours. Lipids were extracted from cells or medium by using
isopropanol or chloroform/methanol (2:1, vol/vol),
respectively, and separated by thin-layer
chromatography on LK5D silica gel (Whatman) with
petroleum ether/ethyl ether/acetic acid (90:10:1,
vol/vol/vol). After visualization with I2
vapor, lipid bands corresponding to TGs and phospholipids were scraped
off, extracted with chloroform/methanol (2:1, vol/vol), dried
in a scintillation vial under N2, and counted after adding
3 mL of scintillation cocktail.
Other Analyses
Cell monolayers were washed, scraped, and
homogenized on ice by using a Polytron microprobe at
setting 2 for 20 seconds. Sucrase activity, a marker of intestinal cell
differentiation, was determined as described by
Dahlqvist,25 using a glucose measuring kit from Sigma. MTP
activity was determined by measuring the transfer of
[14C]TGs between small unilamellar vesicles as described
previously.26 Protein content was
determined27 using the Coomassie Plus reagent according to
the manufacturer's protocol (Pierce Chemical Co) with BSA as a
standard. Data were analyzed by Student's t test
(Primer of Bio-statistics, McGraw-Hill Co, New York, NY).
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Results |
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Expression of Human Recombinant ApoB48 in Nondifferentiated Caco-2 Cells
To express human recombinant apoB48 in nondifferentiated Caco-2 cells, we used a heterologous, constitutive, cytomegalovirus promoter (Fig 1A
). It is known that apoB48 is a
translational product of a posttranscriptionally edited mRNA
transcribed from the apob gene (for reviews, see References
2 and 282 28 through 33). The need for mRNA editing was eliminated by
mutating cytosine6666 to a thymidine, which
introduces a stop codon at nucleotide 2153. The plasmid was
stably transfected into Caco-2 cells, and nondifferentiated cells were
analyzed for secretion of human apoB48 by Western blot
analysis (Fig 1B). Of the five clones, only one (No. 1, MH-1)
secreted human apoB48. Subsequently in independent transfections, two
more clones (JL-9 and JL-4) secreting apoB48 were obtained (Table 1). The amount of apoB secreted by these
cells was twofold to sixfold higher than the amounts secreted by
nontransfected or Neo-transfected cells. The amount of apoA-I secreted
was not affected by the stable expression of human recombinant apoB48
(data not shown).
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Experiments were performed to show that apoB48 expression does not induce differentiation. Differentiation of transfected and nontransfected cells (2x105 per 60-mm2 dish) was monitored in parallel by measuring changes in cellular protein and sucrase activity at different days after cell plating. The amount of cell protein increased until day 10 of subculturing and then remained constant, indicating cessation of proliferation and induction of differentiation in both cell lines. Sucrase activity was first detectable at day 6 and later increased progressively to a maximum by day 20 in both transfected and nontransfected cells. These studies indicated that the induction of differentiation starts around day 6 in these cells but that induction was not affected by apoB48 expression.
Intracellular Degradation and Secretion of Apolipoproteins by
Transfected Cells
Intracellular degradation and secretion of different
apolipoproteins by the stably transfected nondifferentiated Caco-2
cells were studied by pulse-chase analysis (Fig 2). The rate of intracellular
disappearance of apoB48 in BSA- and OA-treated cells was similar (Fig 2A). The amounts of apoB48 recovered from cells and media after a
2-hour chase were 35% and 6% in BSA-treated cells and 37% and 9% in
OA-treated cells, respectively. Thus, recovery of apoB48 was 41% and
46% in BSA- and OA-treated cells, indicating that >50% of newly
synthesized apoB48 was degraded. The rate of intracellular
disappearance of apoB100 was slower in cells treated with OA (Fig 2B).
After 2 hours, apoB100 distribution in cells and media was 24% and 4%
in BSA-treated cells and 33% and 9% in OA-treated cells. The recovery
of apoB100 increased from 28% in BSA-treated cells to 43% in
OA-treated cells, suggesting that OA protected some (14%) of the
apoB100 from degradation. The intracellular disappearance and secretion
of apoA-I were not affected by OA treatment (Fig 2C). The recovery of
apoA-I in BSA-treated and OA-treated cells was 88% and 84%,
respectively. These studies indicated that the majority of apoB48 and
apoB100 was degraded in both BSA- and OA-treated cells. Incubation of
these cells with OA resulted in increased secretion of apoB100 but had
no effect on the secretion of apoB48 and apoA-I.
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10% from original data) from two independent
experiments are plotted as line graphs.
Secretion of Recombinant Human ApoB48 as Lipoprotein Particles in
Transfected, Nondifferentiated Caco-2 Cells
The flotation properties of secreted lipoproteins were
analyzed by density gradient
ultracentrifugation and Western blot analysis
(Fig 3). Apo B48 was observed by day 3 at
a density (1.17 to 1.20 g/mL) corresponding to that of HDL
particles in the conditioned medium of transfected cells. In contrast,
apoB48 was not observed in the conditioned medium of nontransfected
cells (data not shown). After differentiation (day 15), transfected and
nontransfected cells secreted apoB48-containing particles of
predominantly HDL density. Some particles had a flotation density
similar to that of human LDL. ApoB100 was first detectable on day 12.
At this time very little apoB48 was observed, suggesting that induction
of apoB mRNA editing activity may occur at a later stage. By day 15,
apoB100 was the predominant protein in both transfected and
nontransfected cells and was associated with lipoproteins of
d=1.05 to 1.10 g/mL. These studies indicate that both
recombinant and endogenous apoBs are secreted as
lipoprotein particles.
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Transfected Cells Secrete Lipoproteins of Different Flotation
Densities Under Different Culture Conditions
We studied the effect of different culture conditions on the
assembly and secretion of apoB-containing lipoproteins by
ultracentrifugation and ELISA (Fig 4). Cells cultured in serum-free medium
secreted lipoproteins that had a flotation density corresponding to
human plasma HDL (Fig 4A). When cells were cultured in serum-containing
medium, the secreted particles had a flotation density similar to that
of plasma LDL (Fig 4B). In addition, these particles exhibited a size
similar to that of human plasma LDL (data not shown), as indicated by
the elution pattern during gel filtration (Superose-6 HR 10/30 column).
Similar particles were not observed when the conditioned medium from
nontransfected Caco-2 cells was analyzed by gel filtration
chromatography.
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Next, we studied the effects of OA supplementation on the flotation
properties of secreted lipoproteins. Addition of OA to the
serum-containing medium increased apoB secretion twofold to threefold.
These cells predominantly secreted VLDL- and IDL-density particles (Fig 4C
). As a control, cells were cultured in serum for 48 hours, and the
medium was removed and incubated with OA for an additional 48 hours in
the absence of cells. VLDL/IDL–density particles were not observed in
this conditioned medium, indicating that the change in density was not
due to extracellular acquisition of lipids. In a separate study,
conditioned medium from cells cultured in serum and OA was
analyzed further by sequential density
ultracentrifugation. The majority of apoB was
associated with VLDL/IDL (68%). The LDL/HDL fraction contained 32%.
No significant amounts of apoB48 were present in a density
corresponding to chylomicrons/chylomicron remnants
(Sf<200).
The secretion of different populations of lipoproteins under different conditions was also observed in two other (JL-4 and JL-9) clones (data not shown). Supplementation with OA-BSA complexes in the presence of serum for 3, 6, 9, and 12 hours resulted in the secretion of VLDL, indicating that incubation of cells with OA for 3 hours was sufficient for the induction of VLDL assembly. Not only fetal bovine serum but also horse serum and OA supported the secretion of VLDL. As little as 2.5% fetal bovine serum was sufficient for the secretion of VLDL in the presence of OA. Incubation of cells for 6 hours with OA and serum resulted in the secretion of VLDL-like particles, but subsequent incubation in serum-free medium for 48 hours resulted in the secretion of LDL-like particles. An additional 48-hour incubation in serum-free medium resulted in the secretion of HDL-like particles. These studies indicated that the induction of secretion of VLDL-like particles by OA-supplemented serum in different clones was rapid and reversible.
Secretion of VLDL by Transfected Cells Requires TG
Synthesis
The major reason for the lower density of larger lipoprotein
particles is the incorporation of neutral lipids, TGs, and cholesteryl
esters into the core of nascent lipoproteins. Thus, we evaluated the
need for the synthesis of these lipids in the assembly of VLDL by
transfected cells. Triacsin C, a competitive inhibitor of
long-chain fatty acyl-CoA synthase,34 35 36 inhibited TG and
phospholipid synthesis by 90% and 63%, respectively, in cells
cultured in serum-containing medium (Table 2). In a separate experiment under
identical conditions, triacsin C completely inhibited the secretion of
apoB (data not shown). Supplementation with OA resulted in a threefold
increase in the synthesis of TGs and a 30% decrease in phospholipid
synthesis. The reason for the decrease in phospholipid synthesis is not
clear at this time. In cells cultured in serum and OA, triacsin C
inhibited the synthesis of TGs by 66% but had no significant effect on
phospholipid synthesis (Table 2). Inhibition of TG synthesis had a
profound inhibitory effect on the secretion and flotation
properties of secreted lipoproteins (Fig 5). Cells incubated with serum and OA
secreted mainly VLDL and IDL particles. In the presence of triacsin C,
secretion of these larger lipoproteins was significantly inhibited
(>80%). Note also that the total amount of apoB secreted was
significantly (>80%) reduced. Similar results (ie, >80% inhibition
of VLDL secretion) were also observed in a second independent
experiment. Subsequent removal of triacsin C from the medium resulted
in the secretion of VLDL particles, indicating that the effect of
triacsin C was reversible (data not shown). Triacsin C had no effect on
protein synthesis. Furthermore, the amount of apoA-I secreted and the
flotation properties of apoA-I–containing lipoproteins were not
affected by the presence of triacsin C (Fig 5). Most apoA-I was
secreted as HDL. These studies indicated that triacsin C specifically
inhibited secretion of apoB-containing lipoproteins.
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A possible requirement for the active synthesis of cholesteryl esters
for the secretion of VLDL was also evaluated by using the ACAT
inhibitor CD113,818 (Table 3). CD113,818 inhibited cholesteryl ester
synthesis by 72% but did not significantly affect the incorporation of
oleate into TGs or phospholipids. This degree of inhibition of
cholesteryl ester synthesis affected neither the amounts of apoB
secreted nor the flotation properties of the secreted particles (data
not shown).
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MTP Activity in Nondifferentiated Caco-2 Cells
Studies in nonhepatic and nonintestinal cells have demonstrated
that efficient lipoprotein assembly requires expression of apoB and
MTP. In mouse mammary fibroblasts,37 however, transfection
of apoB has been shown to result in the secretion of lipoproteins in
the absence of MTP. Thus, we determined whether MTP activity was
required for the assembly and secretion of lipoproteins in
nondifferentiated Caco-2 cells. Direct measurement of MTP activity
revealed that microsomal fractions from both transfected and
nontransfected nondifferentiated Caco-2 cells transferred 0.2% of TGs
per hour per microgram of protein (Table 4). The MTP activity in these cells was
30% to 60% of that observed in HepG2 and McA-RH7777 cells. Next, we
determined whether this activity was necessary for lipoprotein assembly
and secretion (Fig 6). For this purpose,
we used an MTP inhibitor (CP-10,447) that has been shown to
inhibit MTP activity in vitro and apoB secretion in HepG2 and Caco-2
cells but has no effect on TG synthesis.38 Addition of
CP-10,447 to the culture medium abolished the secretion of
apoB-containing lipoproteins but had no effect on apoA-I secretion (Fig 6). These studies indicated that nondifferentiated Caco-2 cells express
MTP and that it is required for lipoprotein assembly and secretion.
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Discussion |
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Properties of the Transfected Cell Lines
We have developed novel intestine-derived cell lines that express human recombinant apoB48 cDNA. These cells, in contrast to differentiated Caco-2 cells, could be studied within 2 to 4 days of plating. Furthermore, these cells are unique with respect to their ability to secrete lipoproteins whose flotation densities can be modulated by culture conditions. They secreted HDL, LDL, or VLDL, depending on the fatty acid supply and the presence or absence of serum. The flotation properties of lipoproteins secreted by transfected nonhepatic and nonintestinal cells are not modulated by nutritional factors such as lipids, carbohydrates, etc.37 39 40 41 Thus, the cells described in this study may be useful for study of the regulation of the biosynthesis of various lipoproteins by such factors. Furthermore, the availability of these cell lines may help in developing different models expressing different candidate genes in the study of intestinal lipid metabolism and lipoprotein assembly.
Lipid requirements for the assembly of lipoproteins in these cells are
similar to those observed in other cells. In general, TG synthesis has
been shown to be necessary for the assembly and secretion of hepatic
apoB-containing lipoproteins.36 42 Our data in
intestine-derived cells (Fig 5) are in agreement with these studies.
The requirements for cholesteryl esters in lipoprotein assembly,
however, are not yet resolved.36 43 44 45 Our data are
consistent with the studies of Wu et al,36 in that
the ACAT inhibitor does not inhibit apoB secretion in HepG2
cells. Many but not all cells46 47 respond to OA
supplementation and secrete more apoB.13 42 48 49 50 The
nondifferentiated, transfected Caco-2 cells described in this study
responded to OA and secreted larger lipoproteins (Fig 4).
Several reports have shown that nondifferentiated Caco-2 cells do not synthesize and secrete apoB.13 15 17 51 52 We detected small amounts of apoB100 in these cells due to the very sensitive methods of detection used, but these amounts were too low to study lipoprotein assembly. After transfection, however, these cells secreted mainly apoB48 and could be used to study lipoprotein assembly. ApoB48 secretion was not affected by OA supplementation and is in agreement with studies demonstrating no effect on apoB48 secretion.20 53 In contrast, OA increased the secretion of apoB100 by decreasing the rates of intracellular degradation. OA has been shown to increase apoB100 secretion in Hep G2 and McA-RH7777 cells.20 54
Implications for Intestinal Lipoprotein Assembly
Since apoB is always found associated with TG-rich lipoproteins
and is a nonexchangeable apolipoprotein, it has been assumed that apoB
is obligatory for the assembly of these lipoproteins. Deletion of the
apoB gene in mice has been shown to be lethal to the
fetus,55 so the absolute requirement for apoB in the
assembly of TG-rich lipoproteins could not be addressed. In the
present study, we have provided evidence that transfection of
apoB48 cDNA leads to lipoprotein assembly in nondifferentiated Caco-2
cells, indicating that the amount of apoB synthesized is a limiting
factor for lipoprotein assembly in these cells.
Another limiting factor could have been MTP. Most nonintestinal and
nonhepatic cells do not assemble lipoproteins even when transfected
with apoB, most likely because they do not express
MTP.39 40 41 56 57 In mouse mammary-derived carcinoma C127
cells, however, transfection with apoB results in lipoprotein secretion
even in the absence of MTP.37 In contrast, the assembly of
lipoproteins in transfected Caco-2 cells and most other cell types
requires MTP activity (Fig 6). Intestinal cells appear to express MTP
at all times in sufficient amounts to support the assembly of larger
lipoproteins. Lipoprotein assembly in these cells is limited by the
availability of apoB. In liver-derived cells, however, apoB and MTP
expression is constitutive and these cells assemble apoB-containing
lipoproteins at all times. Thus, intestinal cells appear to express all
the factors required for the assembly of lipoproteins, and lipoprotein
secretion is thus determined by the availability of apoB.
The apoB48-transfected, nondifferentiated Caco-2 cells described in
this study appear to be very efficient in the assembly and secretion of
larger lipoproteins (Figs 4 through 6). The majority of cell culture
models developed to study lipoprotein assembly are incapable of
assembling larger lipoproteins.37 39 40 41 42 48 49 56 58 The
only cell lines that have been shown to secrete larger lipoproteins are
McA-RH777750 59 and differentiated
Caco-213 14 15 16 cells. In McA-RH7777 cells, different
proportions of apoB48 are secreted as VLDL- or HDL-size particles after
OA supplementation.50 60 Similar to McA-RH7777 cells,
differentiated Caco-2 cells also secrete apoB in lipoproteins of
different densities after OA supplementation.13 14 15 16 Both of
these cell lines secrete significantly higher amounts of apoB100 than
apoB48.
The different populations of secreted lipoproteins probably represent intermediates in the assembly of intestinal VLDL. For example, the apoB-containing HDL/LDL particles may represent primordial lipoproteins (the first step). Such particles may be secreted or degraded intracellularly. Induction of TG synthesis may result in expansion of the core of these primordial lipoproteins, resulting in the biosynthesis and secretion of VLDL (the second step). A similar mechanism involving two lipidation steps has been proposed for the secretion of hepatic VLDL.28 50 Thus, VLDL assembly may be similar in the liver and intestine except for the use of the apoB species (apoB100 in the liver and apoB48 in the intestine). However, more studies are required to understand the molecular mechanisms involved in lipoprotein assembly in these two tissues.
In summary, the present studies have demonstrated that apoB and not MTP is the limiting factor for the assembly of lipoproteins by nondifferentiated Caco-2 cells. It is clear from these studies that nondifferentiated Caco-2 cells have all the "machinery" required for TG-rich, buoyant lipoproteins. Using these cell models, we have demonstrated that in addition to apoB, active TG synthesis and MTP activity are required for the assembly of larger lipoproteins. The transfected Caco-2 cells are very efficient in the assembly of larger lipoproteins compared with presently available cell models used to study lipoprotein assembly. Thus, studies with these cells may lead to the definition of specific factors that cooperate with apoB and MTP in the assembly of larger intestinal VLDL.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Financial support from the National Institutes of Health (DK-46900 and HL-22633), the American Heart Association (National Center and southeastern Pennsylvania Affiliate), and the W.W. Smith Charitable Trust (to M.M.H.); the technical assistance of Shuyun Zhang; and helpful discussions with Drs Edward Fisher, Julian Marsh, Michael Phillips, and Zemin Yao are gratefully acknowledged.
Received February 26, 1997; accepted June 24, 1997.
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