Biliary lipids, composed of bile acids,
cholesterol, and phosphatidylcholine, are a major source of luminal
lipid in the small intestine. In the present study in a newborn swine
intestinal epithelial cell line (IPEC-1), taurocholate and
phosphatidylcholine were found to have no effect on apolipoprotein B
(apo B) secretion but did significantly increase the basolateral
secretion of apo A-I. This regulation of apo A-I secretion occurred at
the pretranslational level for taurocholate and at the
posttranslational level for phosphatidylcholine. The regulation of apo
A-I secretion by phosphatidylcholine did not involve changes in apo A-I
degradation and may involve mobilization of a preformed pool of apo
A-I. Cholesterol, whether solubilized with taurocholate or
phosphatidylcholine, had no effect on the secretion of either apo B or
apo A-I. However, when taurocholate, phosphatidylcholine, and
cholesterol were combined, apo B secretion was decreased, and the
increase in apo A-I secretion noted with taurocholate and
phosphatidylcholine alone was ablated. Another primary bile acid,
taurochenodeoxycholate, was found to decrease apo B secretion but had
no effect on apo A-I secretion. However, the significance of this
effect is uncertain, since this bile acid caused significant cellular
membrane injury, as evidenced by increased apical medium lactate
dehydrogenase activity. Phosphatidylcholine, but not taurocholate,
dramatically increased the basolateral secretion of radiolabeled
phospholipid with a modest increase in cellular triglyceride
radiolabeling. Furthermore, this effect of phosphatidylcholine on lipid
synthesis did not require significant hydrolysis or uptake of the
phosphatidylcholine molecule. Studies using radiolabeled taurocholate
did not demonstrate active transport of taurocholate by these cells.
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INTRODUCTION |
THE DIGESTION, UPTAKE, processing, and packaging of
lipid with apolipoproteins to form nascent lipoprotein particles by
enterocytes for basolateral secretion are crucial events in the
absorption and assimilation of luminal lipid in the small intestine.
Although dietary lipid is a major nutrient in the neonatal mammal, the small intestine is also the site of absorption of endogenous lipids (26). One important source of endogenous luminal lipids is bile. Biliary lipids consist of bile acids, cholesterol, and phospholipid (predominantly phosphatidylcholine) and normally undergo a
quantitatively important enterohepatic circulation (24). Biliary
phosphatidylcholine, in particular, appears to play an important role
in the mucosal phase of lipid absorption by providing the surface coat
for the nascent chylomicron particle (21, 26). Studies in the adult rat
have demonstrated that biliary phosphatidylcholine is preferentially used for this purpose over dietary phosphatidylcholine (18, 22), and
the presence of additional luminal phosphatidylcholine enhances
triglyceride absorption in the rat (2). The effects of biliary lipids
on intestinal apolipoprotein synthesis and secretion have been studied
in vivo in the intact adult rat (10-13) and in vitro in the human
colon carcinoma cell line Caco-2 (14, 19). However, studies in a
neonatal model system are lacking.
We have previously described (3-7, 25, 29) the regulation of
intestinal apolipoprotein expression in the newborn swine, a model very
similar to the human infant with regard to intestinal development and
lipoprotein metabolism. In addition to these in vivo studies, we have
also characterized (15, 27) the specific effects of the uptake of
various fatty acids on apolipoprotein secretion and lipid synthesis in
vitro in a cell line (IPEC-1) derived from a newborn unsuckled piglet.
These cells differentiate when plated in serum-free medium on
collagen-coated filters in Transwell culture plates. The cells become
polarized with an apical microvillus membrane and take up fatty acids
from the apical culture medium. The fatty acids are reesterified,
packaged into lipoprotein particles, and secreted into the basolateral
medium (15). Apical uptake of fatty acids is also associated with
increased basolateral secretion of apolipoprotein B (apo B) and apo A-I
by IPEC-1 cells (15, 27). These cells also switch from exclusive
production of apo B-100 in the undifferentiated state to production of
apo B-48 with differentiation, accompanied by the appearance of apo B
mRNA editing (15). Overall, these cells may be a more physiological model for the immature enterocyte compared with Caco-2 cells, which
were derived from a human colon carcinoma (17).
Modulation of apolipoprotein secretion and lipid synthesis by the
enterocyte in response to biliary lipid is a potentially important
regulatory mechanism in the neonatal mammal. However, at present such
regulation is poorly understood in the newborn. The aim of the present
study was to determine the effects of incubation of major bile
constituents (taurocholate, cholesterol, and phosphatidylcholine) on
the secretion of apo B and apo A-I, as well as the synthesis and
secretion of triglyceride and phospholipid, by IPEC-1 cells.
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MATERIALS AND METHODS |
Materials.
[1,2,3-3H]glycerol
(200 mCi/mmol),
trans-[35S]methionine
(600 mCi/mmol),
[dioleoyl-1-14C]phosphatidylcholine
(120 mCi/mmol), and
[24-14C]taurocholic
acid (46.3 mCi/mmol) were purchased from DuPont NEN (Boston, MA).
Cholesterol, taurocholate, taurochenodeoxycholate, egg yolk
phosphatidylcholine, essentially fatty acid-free BSA, Triton X-100,
phenylmethylsulfonyl fluoride (PMSF), and benzamidine were purchased
from Sigma Chemical (St. Louis, MO). IgG-SORB was obtained from the
Enzyme Center (Malden, MA), and protein A bound to Sepharose was
purchased from Pharmacia Biotechnology (Piscataway, NJ).
Cell culture.
The derivation of the IPEC-1 cell line has been described previously
(15). Cells from passages
37 to
119 were used in these studies.
Undifferentiated IPEC-1 cells were maintained in serial passage in
plastic culture flasks (75 cm2;
Corning Glassworks, Corning, NY) in growth medium (GM) composed of
DMEM/Ham's F-12 medium (GIBCO BRL, Grand Island, NY) supplemented with
5% fetal bovine serum (FBS) (GIBCO BRL), insulin (5 µg/ml), transferrin (5 µg/ml), selenium (5 ng/ml) (ITS Premix, Collaborative Research, Bedford, MA), epidermal growth factor (5 µg/l)
(Collaborative Research), penicillin (50 µg/ml), and streptomycin (4 µg/ml) (GIBCO BRL). To induce differentiation, we
harvested undifferentiated cells by trypsinization and plated 2 × 106 cells/well on 24.5-mm-diameter
collagen-coated filters (3.0-µm pore size) in Transwell-COL six-well
culture plates (Costar, Cambridge, MA). Cells were maintained in
serum-containing GM for 48 h, then switched to the same medium
containing 10
7 M
dexamethasone (Sigma Chemical) but without FBS. Medium was then changed
every 2 days. We have previously shown (15) that after 10 days IPEC-1
cells exhibit enterocytic features, including polarization with
well-defined microvilli facing the apical medium. Cellular membrane
integrity was assessed by measurement of apical medium lactate
dehydrogenase (LDH) activity (Sigma Chemical).
Incubation of cells with lipids.
At 10 days postplating on Transwell filters in serum-free medium, fresh
serum-free medium was added to both the apical and basolateral
compartments. Cells were incubated with various combinations of
taurocholate, taurochenodeoxycholate, cholesterol, and
phosphatidylcholine in the apical medium for 24 h. Cholesterol and
phosphatidylcholine were dispersed in the culture medium by first
evaporating the chloroform in which these lipids were dissolved under
an N2 stream in a sterile glass
tube. Culture medium containing 1 mM taurocholate (for cholesterol
dispersion) or medium alone (for phosphatidylcholine) was added,
followed by sonication on ice for 1 min at the maximum setting with the
microprobe of an ultrasonic dismembranator (Fisher, Pittsburgh, PA).
Cells were incubated for 24 h followed by harvest of culture medium and
cells. After experimental incubations, cells were rinsed and disrupted
in ice-cold PBS containing 1% Triton X-100, 1 mM PMSF, and 1 mM
benzamidine, using an ultrasonic dismembranator (Fisher). Cell
homogenates were stored at
80°C. Culture medium samples
containing the same concentrations of PMSF and benzamidine were also
stored at
80°C.
Apo B and A-I quantitation by ELISA.
Apo B and A-I protein in cell homogenates and culture medium was
quantitated by competitive ELISA assays using rabbit anti-swine apo B
and A-I polyclonal antibodies, respectively, as previously described
(5). Standard antigens consisted of swine plasma low-density
lipoprotein (apo B) and high-density lipoprotein (apo A-I). All samples
were run in duplicate, and variability between duplicates was <5%.
Secretion of apolipoprotein mass was expressed as nanograms per
microgram of cell protein per 24 h, and cell apolipoprotein content was
expressed as nanograms per microgram of cell protein.
Measurement of apo A-I synthesis and degradation.
Radiolabeling of apo A-I with
[35S]methionine in
IPEC-1 cells was carried out in two separate experiments. To assess the
effects of taurocholate and phosphatidylcholine on apo A-I synthesis, we added fresh medium to differentiated cells at 10 days postplating with 2.0 mM taurocholate, 1.0 mM phosphatidylcholine, or no additive (control) in the apical medium. Cells were incubated for 23 h, followed
by the addition of fresh methionine-free medium containing the same
additives. One hour later,
[35S]methionine (0.5 mCi/well) was added to the apical medium. Cells and basolateral medium
were harvested after a 15-min incubation for apo A-I
immunoprecipitation as described below. Synthesis was expressed as apo
A-I dpm in cell homogenate per microgram of cell protein after the
15-min incubation. During this short radiolabeling period, there was
negligible appearance of labeled apo A-I in the basolateral culture medium.
To assess the effects of phosphatidylcholine treatment on apo A-I
residence time as an index of degradation, we carried out a pulse-chase
experiment in differentiated cells 10 days postplating incubated with
and without 1.0 mM phosphatidylcholine added to the apical culture
medium. After incubation for 20.5 h, fresh methionine-free medium was
added to both the apical and basolateral compartments maintaining the
same additives in the apical medium. One hour later,
[35S]methionine (1.0 mCi/well) was added to the apical medium. After a 20-min pulse, the
apical medium was removed, and the filter inserts were transferred to
new plates with fresh serum-free medium containing 10 mM
nonradiolabeled methionine with the same additives present during the
pulse period. At 0, 0.5, and 2 h into the chase period, cells and
basolateral medium were harvested.
After radiolabeling experiments were completed as described above,
cells were rinsed and disrupted in ice-cold PBS containing 1% Triton
X-100, 1 mM PMSF, and 1 mM benzamidine using an ultrasonic dismembranator (Fisher). Cell homogenates were stored at
80°C. Culture medium samples containing the same
concentrations of PMSF and benzamidine were also stored at
80°C. Apo A-I immunoprecipitation was carried out as
described below.
Apolipoprotein immunoprecipitation.
After [35S]methionine
radiolabeling, cell homogenates and culture medium were subjected to
immunoprecipitation, using a technique adapted from Murthy et al. (20)
with rabbit anti-swine apo A-I polyclonal antibodies. The basolateral
medium and cell homogenate collected from each well were precleared by
incubation with 0.2 ml of IgG-SORB (10% solution wt/vol) for 1 h at
4°C with constant agitation. The samples were then centrifuged, and
the supernatant was collected. Rabbit polyclonal antibodies to swine
apo A-I were purified by ammonium sulfate precipitation from serum and
added to the supernatant. The amount of antibody added was determined to be in excess by reimmunoprecipitation of samples. Samples were incubated for 18 h at 4°C with gentle agitation. The
antigen-antibody complexes were harvested by the addition of 50 µl of
protein A-Sepharose (10%, wt/vol) and incubation for 2 h followed by
centrifugation to harvest the pellet. The pellet was washed six times
with immunoprecipitation buffer (10 mM
NaH2PO4,
5 mM Na2EDTA, 100 mM NaCl, 0.02%
sodium azide, 0.1% SDS, 1% Triton X-100, 1 mM PMSF, and 1 mM
benzamidine) followed by suspension of the pellet in 50 µl of Laemmli
reducing buffer. Samples were heated at 95°C for 5 min and
centrifuged, and the supernatant was subjected to SDS-PAGE using a 15%
acrylamide gel under reducing conditions, according to the method of
Laemmli (16). After electrophoresis, gels were dried at 80°C for 4 h. Autoradiography was performed by exposing the gels to Kodak X-Omat AR film for 3-5 days. Apolipoprotein bands were identified by comparison to coelectrophoresed molecular weight standards (GIBCO BRL).
After autoradiography, gel bands containing immunoprecipitated apolipoproteins were sliced out, solubilized, and subjected to liquid
scintillation counting.
Determination of apo A-I mRNA levels by slot blot hybridization.
Total RNA was extracted from IPEC-1 cells by the method of Chomczynski
and Sacchi (9). Intactness of each RNA preparation was verified by
agarose gel electrophoresis and visualization of ribosomal RNA
subunits. Ten micrograms of RNA were applied to nitrocellulose filters
using a slot blot apparatus (Hoefer, San Francisco, CA). Filters were
serially hybridized with a radiolabeled swine apo A-I cDNA (25) and an
18S ribosomal subunit cDNA (generously provided by Dr. Nicholas
Davidson, University of Chicago, Chicago, IL). Hybridization signals
were quantitated using a Bio-Rad model GS-525 molecular imager system
and associated software (Hercules, CA). Apo A-I mRNA abundance was
expressed as apolipoprotein mRNA-to-18S ribosomal subunit RNA signal
intensity ratio.
Triglyceride and phospholipid radiolabeling with
[3H]glycerol and determination
of fate of
[14C]phosphatidylcholine added
to apical medium.
To determine the effects of taurocholate and phosphatidylcholine on
triglyceride and phospholipid synthesis, we incubated cells for 24 h
with [3H]glycerol (12 µCi/well) and taurocholate (2.0 mM) and/or
phosphatidylcholine (1.0 mM). To determine the fate of
phosphatidylcholine added to the apical medium,
[14C]phosphatidylcholine
(0.5 µCi/well) and unlabeled phosphatidylcholine (1.0 mM) were added
to the apical medium and incubated for 24 h. After experimental
incubations, cells and medium were collected, processed, and stored as
described above. Total lipid in the cells and medium was extracted as
previously described (28). Extracts were applied to silica gel G plates
and subjected to TLC using a mixture of petroleum ether, diethyl ether,
and acetic acid (80:20:1, vol/vol/vol). Lipid bands were identified by
exposure to iodine vapor and scraped off the plate for liquid
scintillation counting. Bands corresponding to phospholipids,
diglycerides, monoglycerides, cholesterol, free fatty acids,
triglycerides, and cholesteryl esters were identified by comparison to
cochromatographed standards. Cellular content of radiolabeled lipid was
expressed as specific lipid dpm per well, and secretion of radiolabeled
lipid was expressed as specific lipid dpm per well per 24 h.
Determination of fate of
[14C]taurocholate added to
apical medium.
The ability of IPEC-1 cells to transport taurocholate was tested by
measuring both apical to basolateral and basolateral to apical movement
of radiolabeled taurocholate against a concentration gradient
(0.5-2.0 mM).
[14C]taurocholate was
added to either apical or basolateral medium containing the lower cold
taurocholate concentration to achieve a final concentration of 0.17 µCi/ml. Cells were incubated for 24 h, followed by harvest of apical
and basolateral media and cells for scintillation counting.
Phospholipid and protein measurement.
Total lipid was extracted from cell homogenates and basolateral medium
as previously described (28), followed by phospholipid measurement by
the method of Bartlett (1). Phospholipid mass was expressed as
micrograms per well for cells and basolateral medium. Cell homogenate
protein was determined by the Bradford method (8).
Statistical analysis.
Data from multiple experimental groups were analyzed by one-way ANOVA,
followed by the Fisher's least significant differences test to compare
specific groups. Data from two experimental groups were compared by
Student's t-test. Statistical
significance was set at two-tailed P < 0.05.
 |
RESULTS |
Effect of bile acids, cholesterol, and phosphatidylcholine on apo B
and A-I cellular mass and basolateral secretion in IPEC-1 cells.
Figure
1A shows
IPEC-1 cell apo B content after incubation with no added lipid
(control), taurocholate alone, cholesterol solubilized with
taurocholate, phosphatidylcholine alone, phosphatidylcholine plus
taurocholate, and cholesterol solubilized with phosphatidylcholine. There were no significant differences among the six experimental groups
of cells after statistical analysis by ANOVA. Figure
1B shows basolateral secretion of apo
B for the six groups. Again, there were no significant differences.
Figure 2A
shows cell apo A-I content. As observed for apo B, there was no
significant difference among the experimental groups. Figure
2B shows apo A-I basolateral secretion. Secretion of apo A-I was significantly higher in all groups
compared with the control group. Whether solubilized with taurocholate
or phosphatidylcholine, cholesterol did not further enhance apo A-I
secretion. To test the effects of a complete bile mixture (bile acid,
phosphatidylcholine, and cholesterol) as well as another bile acid
species on apolipoprotein cellular mass and secretion, we performed the
experiments depicted in Figs. 3 and 4. Figure
3A shows cell apo B content after
incubation with no added lipid (control), taurocholate alone,
taurocholate plus phosphatidylcholine and cholesterol, and
taurochenodeoxycholate. Although cell apo B content appeared to be
lower than control after incubation with all three lipids and
taurochenodeoxycholate, these differences did not reach statistical
significance. Figure 3B shows
basolateral secretion of apo B for the four groups. As shown, apo B
secretion was significantly reduced after incubation of the combination of taurocholate, phosphatidylcholine, and cholesterol and
taurochenodeoxycholate, relative to the control group. Figure
4A shows cellular apo A-I mass. No
significant differences were noted among the four groups. Figure
4B shows apo A-I basolateral
secretion. The increase in apo A-I secretion induced by taurocholate
was attenuated when this bile acid was combined with both
phosphatidylcholine and cholesterol. Taurochenodeoxycholate had no
effect on apo A-I secretion. None of the experimental groups
demonstrated a significant increase in LDH activity in apical medium
compared with control, except in the case of taurochenodeoxycholate.
Incubation with this bile acid resulted in a six-fold increase in LDH
activity (data not shown), indicating significant cellular membrane
injury. Figure 5 shows a dose-response
curve for basolateral apo A-I secretion with increasing concentrations
of phosphatidylcholine and taurocholate added to the apical medium. As
shown, apo A-I secretion peaks at a phosphatidylcholine concentration
of 1.0 mM and begins to decline at a concentration of 2.0 mM. Secretion
peaks at a taurocholate concentration of 2.0 mM and begins to decline
at a concentration of 4.0 mM. In subsequent experiments,
phosphatidylcholine and taurocholate were added to the apical culture
medium at concentrations of 1.0 and 2.0 mM, respectively.

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Fig. 1.
Effect of biliary lipids added to the apical medium on IPEC-1 cellular
apolipoprotein B (apo B) content (A)
and basolateral secretion (B).
Incubations were carried out for 24 h. Control, no additives; TC,
taurocholate at 1.0 mM; TC + CH, taurocholate + cholesterol at 1.0 and
0.5 mM, respectively; PC, phosphatidylcholine at 0.5 mM; TC + PC, taurocholate + phosphatidylcholine at 1.0 and 0.5 mM, respectively;
PC + CH, phosphatidylcholine + cholesterol at 0.5 mM each. Bars
represent means ± SE; n = 5-8
per group. Group means were analyzed by ANOVA.
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Fig. 2.
Effect of biliary lipids added to the apical medium on IPEC-1 cellular
apo A-I content (A) and basolateral
secretion (B). Incubations were
carried out for 24 h. See Fig. 1 legend for description of groups. Bars
represent means ± SE; n =5-8
per group. Group means were analyzed by ANOVA. Bars with different
superscripts are significantly different at
P < 0.05.
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Fig. 3.
Effect of biliary lipids added to the apical medium on IPEC-1 cellular
apo B content (A) and basolateral
secretion (B). Incubations were
carried out for 24 h. Control, no additives; TC, taurocholate at 1.0 mM. TC + PC + CH, taurocholate + phosphatidylcholine + cholesterol at
1.0, 0.5, and 0.5 mM, respectively; TCDC, taurochenodeoxycholate at 1.0 mM. Bars represent means ± SE; n = 3-7 per group. Group means were analyzed by ANOVA. Bars with
different superscripts are significantly different at
P < 0.05.
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Fig. 4.
Effect of biliary lipids added to the apical medium on IPEC-1 cellular
apo A-I content (A) and basolateral
secretion (B). Incubations were
carried out for 24 h. See Fig. 3 legend for description of groups. Bars
represent means ± SE; n = 3-7
per group. Group means were analyzed by ANOVA. Bars with different
superscripts are significantly different at
P < 0.05.
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Fig. 5.
Apo A-I secretion after incubation of IPEC-1 cells with varying
concentrations of taurocholate and phosphatidylcholine in the apical
culture medium.
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Effect of taurocholate and phosphatidylcholine on apo A-I synthesis
and mRNA levels in IPEC-1 cells.
Figure 6 shows apo A-I synthesis by IPEC-1
cells after incubation with taurocholate and phosphatidylcholine.
Compared with the control group, taurocholate incubation resulted in an
increase in apo A-I synthesis, whereas phosphatidylcholine did not
change apo A-I synthesis. To determine whether the increased apo A-I mass secretion after incubation with phosphatidylcholine might be due
to changes in apo A-I degradation, we performed a pulse-chase radiolabeling experiment with
[35S]methionine as
shown in Fig. 7. Plots of the decline in
specific apo A-I counts in cells and medium over the chase period show almost identical slopes, indicating no significant difference in apo
A-I degradation with or without phosphatidylcholine. Therefore, the
mechanism of the induction of apo A-I secretion by IPEC-1 cells with
phosphatidylcholine incubation appears to be posttranslational and does
not involve changes in apo A-I degradation. This appears to be similar
to the mechanism of the stimulation of apo A-I secretion by oleic acid
by these cells (15), possibly involving mobilization of a preformed
intracellular pool of apo A-I with a slow turnover.

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Fig. 6.
Effect of incubation of IPEC-1 cells with 2.0 mM taurocholate (TC) and
1.0 mM phosphatidylcholine (PC) added to the apical medium on apo A-I
synthesis as determined by
[35S]methionine
radiolabeling. Bars represent means ± SE;
n = 3-4 per group. Group means
were analyzed by ANOVA. Bars with different superscripts are
significantly different at P < 0.05.
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Fig. 7.
Effect of incubation of IPEC-1 cells with 1.0 mM phosphatidylcholine
(PC) added to the apical medium on apo A-I degradation as assessed by
[35S]methionine
pulse-chase radiolabeling. Cells were pulsed with
[35S]methionine for 20 min. Total dpm in both cells and basolateral medium as a % of the
total at time
0 of the chase are depicted on the
y-axis and chase time (in min) is
depicted on the x-axis.
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We next wished to determine whether the observed increases in apo A-I
secretion and synthesis induced by incubation with taurocholate were
mediated by changes in apo A-I mRNA levels. Figure
8 shows apo A-I mRNA levels in IPEC-1 cells
after incubation with either taurocholate or phosphatidylcholine,
compared with control incubations. As expected, phosphatidylcholine
incubation did not result in a change in apo A-I mRNA levels. However,
taurocholate incubation resulted in a significant increase in apo A-I
mRNA levels, paralleling changes in apo A-I secretion and synthesis.
Therefore, taurocholate upregulates apo A-I synthesis and secretion at
the pretranslational level in IPEC-1 cells.

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Fig. 8.
Effect of incubation of IPEC-1 cells with 2.0 mM taurocholate (TC) and
1.0 mM phosphatidylcholine (PC) added to the apical medium on apo A-I
mRNA levels as measured by slot blot hybridization with a radiolabeled
swine apo A-I cDNA. Hybridization with a radiolabeled 18S ribosomal
subunit cDNA was used to control for RNA loading. Apo A-I mRNA
abundance is expressed as apolipoprotein mRNA-to-18S ribosomal subunit
RNA signal intensity ratio. Bars represent means ± SE;
n = 4 per group. Group means were
analyzed by ANOVA. Bars with different superscripts are significantly
different at P < 0.05.
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Effect of taurocholate and phosphatidylcholine on triglyceride and
phospholipid synthesis and secretion by IPEC-1 cells.
Figure 9 shows the incorporation of
[3H]glycerol into
triglyceride and phospholipid in cell homogenate and basolateral medium after incubation with the combination of taurocholate and
phosphatidylcholine for 24 h. As shown, secretion of radiolabeled
phospholipid into basolateral medium was increased 13-fold after
incubation with the combination of taurocholate and
phosphatidylcholine. In the cell homogenate, there was a modest
increase in triglyceride labeling with no change in phospholipid
labeling. Figure 10 shows the
incorporation of
[3H]glycerol into
triglyceride and phospholipid in cell homogenate and basolateral medium
after separate incubations with either taurocholate or
phosphatidylcholine for 24 h. As shown in Fig. 10, phosphatidylcholine
incubation was associated with a 12-fold increase in basolateral
secretion of radiolabeled phospholipid and a modest increase in
cellular triglyceride labeling. Taurocholate had no effect. To
determine whether the striking increase in the basolateral secretion of
radiolabeled phospholipid after phosphatidylcholine incubation was
associated with a similar increase in the secretion of phospholipid
mass, we measured phospholipid mass in the cell homogenate and
basolateral medium (Fig. 11). As shown,
there was an approximate six-fold increase in basolateral secretion of
phospholipid after phosphatidylcholine incubation without change in
cellular phospholipid mass.

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Fig. 9.
Effect of incubation of IPEC-1 cells with 2.0 mM taurocholate (TC) and
1.0 mM phosphatidylcholine (PC) added together to the apical medium on
incorporation of
[3H]glycerol into
triglyceride (TG) and phospholipid (PL) in cell homogenate and
basolateral medium. Bars represent means ± SE;
n = 4 per group.
a P < 0.000001, b P < 0.05 compared with control (Con).
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Fig. 10.
Effect of incubation of IPEC-1 cells with 2.0 mM taurocholate (TC) and
1.0 mM phosphatidylcholine (PC) added to the apical medium separately
on the incorporation of
[3H]glycerol into
triglyceride (TG) and phospholipid (PL) in cell homogenate and
basolateral medium. Bars represent means ± SE;
n = 4 per group. Con, control. Group
means were analyzed by ANOVA, followed by Fisher's least significant
squares test. Bars with different superscripts are significantly
different at P < 0.05.
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Fig. 11.
Effect of incubation of IPEC-1 cells with 2.0 mM taurocholate (TC) and
1.0 mM phosphatidylcholine (PC) added to the apical medium separately
on phospholipid mass in the cell homogenate and secreted into the
basolateral medium. Bars represent means ± SE;
n = 4 per group. Group means
were analyzed by ANOVA, followed by Fisher's least significant squares
test. Bars with different superscripts are significantly different at
P < 0.05.
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Uptake of radiolabeled phosphatidylcholine by IPEC-1 cells.
To determine whether the observed effects on apo A-I secretion and
phospholipid synthesis and secretion after phosphatidylcholine incubation resulted from cellular uptake of the phosphatidylcholine, IPEC-1 cells were incubated with
[14C]phosphatidylcholine
added to the apical medium for 24 h, followed by harvest of the apical
and basolateral media and cell homogenate. Total lipid was extracted,
and various lipid classes were separated by TLC and subjected to liquid
scintillation counting. Of the total counts recovered, 96% remained in
the apical medium, 1% was present in the cell homogenate, and 3% was
recovered from the basolateral medium. As shown in Table
1, the majority of these counts were
present in intact phospholipid in all three locations.
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Table 1.
Distribution of 14C in various lipid classes in apical
and basolateral media and cell homogenate after 24-h incubation with
[14C]phosphatidylcholine
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Fate of radiolabeled taurocholate after incubation with IPEC-1
cells.
Figure 12 shows the results of an
experiment designed to test whether IPEC-1 cells actively transport
taurocholate from apical to basolateral medium against a concentration
gradient. First, radiolabeled taurocholate was added to the apical
medium at a concentration of 0.5 mM, and cold taurocholate was added to
the basolateral medium at a concentration of 2.0 mM. After a 24-h incubation, 0.160 µmol of radiolabeled taurocholate appeared in the
basolateral medium (Fig. 12). Next, labeled taurocholate was added to
the basolateral medium at a concentration of 0.5 mM, and unlabeled
taurocholate was added to the apical medium at a concentration of 2.0 mM. After incubation, 0.123 µmol of labeled taurocholate appeared in
the apical medium (Fig. 12). These results were not statistically
different, demonstrating no net transfer of taurocholate from apical to
basolateral medium against a concentration gradient, except for a small
amount apparently due to diffusion, possibly by a paracellular route.
Less than 1% of radiolabel was recovered in the cell homogenate under
both conditions, indicating negligible accumulation of taurocholate
within the cells.

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Fig. 12.
Translocation of radiolabeled taurocholate across IPEC-1 cell monolayer
against a concentration gradient. First,
[14C]taurocholate (0.5 mM) was added to the apical medium, and cold taurocholate (2.0 mM) was
added to the basolateral medium and incubated for 24 h. Radiolabeled
taurocholate in the basolateral medium was measured (AP to BL). Next,
labeled taurocholate (0.5 mM) was added to the basolateral medium, and
unlabeled taurocholate (2.0 mM) was added to the apical medium and
incubated for 24 h. Radiolabeled taurocholate in the apical medium was
measured (BL to AP). Bars represent means ± SE;
n = 4 per group. Group means were
analyzed by Student's t-test. No
significant difference was found between the 2 groups.
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DISCUSSION |
Biliary lipids, composed of bile acids, cholesterol, and
phosphatidylcholine, are a major source of luminal lipid in the small intestine. In the present study in a newborn swine intestinal epithelial cell line (IPEC-1), taurocholate and phosphatidylcholine were found to have no effect on apo B secretion but did significantly increase the basolateral secretion of apo A-I. This regulation of apo
A-I secretion occurred at the pretranslational level for taurocholate
and at the posttranslational level for phosphatidylcholine. The
regulation of apo A-I secretion by phosphatidylcholine did not involve
changes in apo A-I degradation. Cholesterol, whether solubilized with
taurocholate or phosphatidylcholine, had no effect on the secretion of
either apo B or apo A-I. However, when taurocholate, phosphatidylcholine, and cholesterol were combined, apo B secretion was
decreased, and the increase in apo A-I secretion noted with taurocholate and phosphatidylcholine alone was ablated. Another primary
bile acid, taurochenodeoxycholate, was found to decrease apo B
secretion, but had no effect on apo A-I secretion. However, we are
uncertain of the significance of this effect, since this bile acid
caused significant cellular membrane injury, as evidenced by markedly
increased apical medium LDH activity. We also found that
phosphatidylcholine, but not taurocholate, dramatically increased the
basolateral secretion of radiolabeled phospholipid with a modest
increase in cellular triglyceride radiolabeling. Furthermore, this
effect of phosphatidylcholine on lipid synthesis did not require
significant hydrolysis or uptake of the phosphatidylcholine molecule.
Studies using radiolabeled taurocholate did not support the active
transport of taurocholate by these cells.
The results of our studies in the IPEC-1 cell line differ in some
respects from those of previous studies in the adult rat and the Caco-2
intestinal epithelial cell line. In vivo studies in the adult rat have
demonstrated that jejunal apo B synthesis falls after bile diversion,
and this fall may be completely prevented by continuous replacement
with taurocholate (12). In these same studies (12), ileal apo B
synthesis was downregulated to an even greater degree by biliary
diversion but was only partially restored by bile acid replacement.
Further studies (10) in a chronic bile-diverted rat model demonstrated
that the reinfusion of either taurocholate or taurocholate plus
lysophosphatidylcholine caused a reexpression of apo B synthesis.
However, since apo B expression may also be regulated at the
posttranslational level (23), these changes in synthesis may not fully
reflect the complete spectrum of regulation under these experimental
conditions. Other studies by this same group have demonstrated
downregulation of apo A-I synthesis in ileum, but not jejunum, by
biliary diversion in the adult rat. However, normal apo A-I synthesis
levels could not be restored by bile acid infusion, suggesting the
importance of other bile constituents, such as phospholipid (11). In
vitro studies in the human colon carcinoma cell line, Caco-2, have
demonstrated regulation of apo B synthesis and secretion by bile acids
and phosphatidylcholine without any effect on apo A-I synthesis or secretion (14, 19).
In adult rat studies, it was shown that cholesterol absorption was
negatively correlated with jejunal apo B synthesis (10). Although in
vivo data from the rat suggest that changes in intestinal cholesterol
absorption may regulate apo B expression (13), it is very difficult to
sort out cholesterol as an independent variable, since this sterol
requires solubilization with molecules such as bile acids, which may
exert an independent effect on apo B. Caco-2 cells incubated with
cholesterol or 25-hydroxycholesterol did not demonstrate a change in
apo B secretion compared with controls (14). Intestinal apo A-I
synthesis was not influenced by either acute or chronic perturbations
in intestinal cholesterol flux in the rat (13). Studies in Caco-2 cells
have demonstrated no regulation of apo A-I synthesis or secretion by
cholesterol (14).
The variance of our results with the findings of others in the adult
rat or in Caco-2 cells may be due to several factors. The regulatory
patterns described in the present study may be peculiar to the newborn
piglet with species and/or developmental specificity. We have
previously found major differences between the adult rat and newborn
swine in in vivo studies of the effects of dietary lipid on intestinal
apolipoprotein expression (3-5, 29). Biliary diversion in the
newborn piglet results in a decrease in jejunal apo A-I mass and
synthesis without effect on apo B expression (5). Although Caco-2 cells
are of human origin, they are derived from a colon carcinoma and may
not respond in a completely physiological manner (17). Although caution
should also be applied to the interpretation of results obtained from the IPEC-1 cell line, these cells were originally derived from the
small intestine of a normal newborn unsuckled piglet, and data derived
from our in vivo studies to date in the newborn piglet have generally
agreed with in vitro IPEC-1 cell data.
An unexpected finding in this study was the marked increase in
secretion of radiolabeled phospholipid into the basolateral medium
after incubation of IPEC-1 cells with phosphatidylcholine. This
increase in secretion of radiolabeled phosphatidylcholine was
accompanied by a parallel increase in the basolateral secretion of
phospholipid mass. However, experiments with radiolabeled
phosphatidylcholine added to the apical medium demonstrated very
limited uptake (4%) of the radiolabel with the majority of radiolabel
recovered from the cell homogenate and both apical and basolateral
media remaining as intact and unhydrolyzed phospholipid. The mechanism
and significance of the uptake of this limited amount of mainly intact
phosphatidylcholine are not known. It is possible that the small amount
recovered in the cell homogenate may have been nonspecifically bound to the cell membrane, despite extensive washing, and the limited quantity
recovered in the basolateral medium may have entered this compartment
via the paracellular route. An alternative, but unproven, explanation
would involve the hydrolysis of phosphatidylcholine at the apical
membrane by a membrane-bound phospholipase, uptake of the hydrolytic
products, reesterification within the cell, and basolateral secretion
of intact phosphatidylcholine. However, if uptake of the
phosphatidylcholine does occur, it is a quantitatively minor process.
Overall, these findings suggest that the intact phosphatidylcholine
molecule may interact with the apical cell membrane in some fashion to
transduce an intracellular signal to upregulate phospholipid synthesis
in the smooth endoplasmic reticulum. Mathur et al. (19) demonstrated an
increase in basolateral secretion of triacylglycerol accompanied by a
modest increase in triacylglycerol synthesis after incubation of Caco-2
cells with phosphatidylcholine. However, in the study by Mathur et al. (19) phospholipid synthesis and secretion were not affected. Determination of the cellular mechanism of this signaling in IPEC-1 cells will be the focus of future studies. We were unable to test the
effects of lysophosphatidylcholine in our IPEC-1 cell culture system,
since even low concentrations (100 µM) caused excessive cellular
injury as reflected by high culture medium LDH activity (data not
shown). Regulation of intestinal apolipoprotein expression and lipid
synthesis by intact undigested phosphatidylcholine in the intestinal
lumen may be important in the neonate with developmental deficiency of
pancreatic phospholipase A2, which
would lead to significant amounts of intact phosphatidylcholine in the
small intestinal lumen.
The cellular mechanisms of the regulation of apo A-I secretion by
taurocholate and phosphatidylcholine in IPEC-1 cells are not known at
present. With regard to the regulation by taurocholate, our studies
using radiolabeled taurocholate demonstrated limited bidirectional
diffusion of the bile acid but did not provide evidence for active
transport of taurocholate from apical to basolateral medium against a
concentration gradient. Less than 1% of the radiolabeled taurocholate
was recovered from cell homogenates. Although the cells were washed
thoroughly, it is impossible to accurately determine how much of the
associated radiolabel was intracellular or bound to the cell surface.
Therefore, it is not known whether taurocholate exerted its effect by
interacting with the microvillus membrane or acting intracellularly.
However, it is likely that taurocholate induced a cellular signal
transduction pathway to the cell nucleus to increase apo A-I mRNA
transcript stability or increase transcription of the apo A-I gene to
result in increased mRNA levels. Field et al. (14) found that
taurocholate inhibited apo B secretion in Caco-2 cells by increasing
its rate of degradation. Because treatment with a calcium ionophore
produced the same effect, these investigators (14) speculated that
taurocholate might produce its effect on apo B secretion through the
same mechanism by causing the release of intracellular calcium.
Although this may represent a potential mechanism for the regulation of
apo A-I secretion in IPEC-1 cells, in the studies in Caco-2 cells
taurocholate had no effect on apo A-I secretion. Field et al. (14) also
found that apo B secretion was increased by phosphatidylcholine,
independent of its hydrolysis, through increasing apo B synthesis. In
the present study, phosphatidylcholine, also independent of hydrolysis, increased apo A-I secretion but had no effect on apo B secretion. This
effect of phosphatidylcholine was not accompanied by an increase in apo
A-I synthesis or mRNA levels or a decrease in apo A-I degradation. We
have previously reported a similar posttranslational mechanism responsible for the increase in both apo B and A-I secretion after incubation of IPEC-1 cells with oleic acid, possibly involving the
mobilization of a preformed intracellular pool of apolipoprotein with a
slow turnover to account for the lack of observable differences in
radiolabeling (15). In the present study, it is possible that the
increased apo A-I secretion by IPEC-1 cells after phosphatidylcholine incubation may be related to the accompanying increase in secretion of
newly synthesized phospholipid and the two secretory processes may be
somehow coupled.
In summary, taurocholate and phosphatidylcholine in physiological
concentrations, both alone and in combination, induced basolateral secretion of apo A-I, but not apo B, by IPEC-1 newborn swine intestinal epithelial cells. The mechanism of this upregulation of apo A-I secretion appeared to be different for taurocholate (pretranslational) and phosphatidylcholine (posttranslational). Cholesterol, whether dispersed with taurocholate or phosphatidylcholine, had no effect on
either apo B or A-I secretion. However, the combination of taurocholate, phosphatidylcholine, and cholesterol decreased apo B
secretion but had no effect on apo A-I secretion. Phosphatidylcholine, but not taurocholate, stimulated the synthesis and basolateral secretion of phospholipid. Significant hydrolysis and/or uptake of phosphatidylcholine was not necessary for these effects on apo A-I
and phospholipid secretion. The effects of taurocholate on apo A-I
expression were not associated with demonstrable active taurocholate
transport in IPEC-1 cells. We speculate that these effects of
phosphatidylcholine and taurocholate, both major components of bile,
may be important in the regulation of apo A-I and phospholipid synthesis and secretion in the newborn mammalian small intestine.
This study was supported by National Institutes of Health Grants
HD-22551 (D. D. Black) and DK-42100 (H. M. Berschneider).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: D. D. Black, Crippled Children's
Foundation Research Center, Le Bonheur Children's Hospital, 50 N. Dunlop, Memphis, TN 38103.