1 Children's Foundation Research Center at Le Bonheur Children's Medical Center, University of Tennessee Health Science Center, Memphis, Tennessee 38103; and 2 College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606
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ABSTRACT |
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We (Wang H, Berschneider HM, Du J, and Black DD. Am J Physiol Gastrointest Liver Physiol 272: G935-G942, 1997; Wang H, Lu S, Du J, Yao Y, Berschneider HM, and Black DD. Am J Physiol Gastrointest Liver Physiol 280: G1137-G1144, 2001) previously showed that different fatty acids influence synthesis and secretion of triacylglycerol (TG) and phospholipid (PL) in a newborn swine enterocyte cell line (IPEC-1). The most striking effects were produced by stearic acid (SA; 18:0), which modestly affected TG and PL synthesis but reduced TG and PL secretion, and by eicosapentaenoic acid (EPA; 20:5), which reduced TG and PL synthesis and TG secretion relative to oleic acid (OA; 18:1). To define the mechanism of these effects, differentiated IPEC-1 cells were incubated for 24 h with OA, SA, or EPA and [3H]glycerol. Endoplasmic reticulum (ER) and Golgi (G) content of labeled lipids and apolipoprotein (apo) B and apoAI protein were measured. Relative to OA, SA did not impair ER TG synthesis, but reduced movement of labeled TG from ER to G. EPA impaired both ER TG synthesis and movement of labeled TG from ER to G. PL followed the same pattern, except ER synthesis of PL was relatively unaffected by EPA. Carbonate treatment demonstrated decreased partitioning of labeled lipid from ER membrane to lumen in EPA-treated cells. Organelle apoB and apoAI content demonstrated opposite patterns after SA and EPA incubation. We conclude that SA and EPA adversely influence immature enterocyte ER to G lipid trafficking, compared with OA. Furthermore, EPA inhibits ER lipid synthesis and transfer of membrane lipid to luminal particles. Regulation of apoAI ER to G trafficking is independent of that of apoB.
oleic acid; stearic acid; eicosapentaenoic acid; apolipoprotein
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INTRODUCTION |
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UPTAKE OF LUMINAL FATTY ACIDS by the enterocyte, their movement through the cytoplasm, reesterification with monoacylglycerol into triacylglycerol in the endoplasmic reticulum (ER), packaging into nascent chylomicrons with apolipoprotein B (apoB) and other apolipoproteins, subsequent movement from the ER to the Golgi apparatus, and final secretion at the basolateral membrane are all essential steps in intestinal lipid absorption. Recent studies (8-10) have elucidated some of the details of the budding of a prechylomicron transport vesicle (PCTV) from the ER membrane and its movement to the cis-Golgi for docking mediated by membrane protein interactions. However, the regulation of many of the steps of this process remains poorly understood.
We (7, 18-20) have previously demonstrated that the chain length and degree of saturation of absorbed fatty acids have significant effects on the efficiency of subsequent triacylglycerol synthesis and secretion in a newborn swine intestinal epithelial cell line, IPEC-1. This cell line was derived from the small intestine of a newborn unsuckled piglet and can be maintained in an undifferentiated state and serially passaged in serum-containing medium (7). When plated on collagen-coated filters in serum-free medium, these cells differentiate and develop enterocytic characteristics, including cellular polarity with an apical microvillus membrane (7). These cells take up fatty acids from the apical culture medium, reesterify the absorbed fatty acids into complex lipids (predominantly triacylglycerol and phospholipid), and secrete these lipids as lipoproteins into the basolateral culture medium (7). Coincident with differentiation, these cells also acquire increased apoB mRNA editing activity to produce a higher proportion of apoB-48 and abundantly express apoAI (7).
When IPEC-1 cells were incubated with a series of fatty acids of varying chain length and degrees of saturation added to the apical culture medium compartment, the fatty acid that most efficiently stimulated triacylglycerol and phospholipid synthesis and secretion was oleic acid (OA; 18:1) (18). When the long-chain saturated fatty acid stearic acid (SA; 18:0) was used, triacylglycerol synthesis was modestly less than that observed with OA, but secretion was markedly decreased (18). When IPEC-1 cells were incubated with the very long-chain polyunsaturated fatty acid eicosapentaenoic acid (EPA; 20:5), both cellular triacylglycerol synthesis and secretion were markedly impaired, compared with OA (19). Phospholipid synthesis and secretion followed a similar pattern, except that cellular phospholipid synthesis after SA incubation was actually higher than that observed with OA, and labeled phospholipid secretion was relatively unaffected by EPA incubation compared with OA (18, 19). apoB secretion generally followed the pattern of triacylglycerol secretion after incubation with these fatty acids (18, 19).
To further delineate the cellular mechanisms of this regulation of triacylglycerol and phospholipid synthesis and secretion by OA, SA, and EPA in IPEC-1 cells, the present studies were undertaken. To define the patterns of ER synthesis and ER-to-Golgi trafficking of triacylglycerol and phospholipid after cellular uptake of SA, OA, and EPA, lipid radiolabeling and subcellular fractionation techniques were used.
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MATERIALS AND METHODS |
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Materials. [1,2,3-3H]glycerol (0.2 Ci/mmol), D-[1-14C]glucose-6-phosphate (40-60 mCi/mmol), and uridine diphospho-D-[6-3H]galactose (5-20 Ci/mmol) were purchased from DuPont New England Nuclear (Boston, MA). SA (C18:0), EPA (C20:5n-3), OA (C18:1n-9), essentially fatty acid-free bovine serum albumin, Triton X-100, phenylmethylsulfonyl fluoride (PMSF), and benzamidine were purchased from Sigma (St. Louis, MO).
Cell culture.
Derivation of the IPEC-1 cell line has been described previously
(7). Cells from passages 25 to 80 were used in these
studies, and all cell culture was carried out at 37°C in an
atmosphere containing 5% CO2. Undifferentiated IPEC-1
cells were maintained in serial passage in plastic culture flasks (75 cm2; Corning Glassworks, Corning, NY) in growth medium of
DMEM/F12 medium (GIBCO-BRL, Grand Island, NY) supplemented with 5%
fetal bovine serum (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, undifferentiated cells were
harvested by trypsinization and 2 × 106 cells/well
were plated on 24.5 (for time course studies)- or 75-mm diameter (for
cell fractionation studies) collagen-coated (type 1; Sigma) filters
(3.0-µm pore size) in Transwell culture plates (Costar; Corning).
Cells were maintained in serum-containing growth medium for 48 h
and then switched to the same medium containing 107 M
dexamethasone (Sigma), but without fetal bovine serum. Medium was then
changed every 2 days. We have previously shown that after 10 days,
IPEC-1 cells exhibit enterocytic features, including polarization with
well-defined microvilli facing the apical medium (7).
Cellular membrane integrity was assessed by measurement of apical
medium lactate dehydrogenase activity (Sigma).
Incubation of cells with fatty acids.
At 10 days postplating on Transwell filters in serum-free medium, fresh
serum-free medium was added to both the apical and basolateral
compartments. The apical medium contained fatty acid complexed with
albumin (4:1 molar ratio) at a fatty acid concentration of 0.8 mM
(12). This fatty acid concentration is in the
physiological range, and above this concentration, the basolateral
secretion of triacylglycerol begins to plateau in IPEC-1 cells
(7). For time course studies, [3H]glycerol
(12 µCi/well) was added to the apical medium, concomitant with the
addition of fatty acid/albumin. Cells were incubated for 6, 12, and
24 h followed by harvest of cells. For cell fractionation studies,
[3H]glycerol (27 µCi/plate) was added to the apical
medium, concomitant with the addition of fatty acid/albumin. Cells were
incubated for 24 h followed by harvest of culture medium and
cells. Culture medium samples containing protease inhibitors (Complete;
Roche Molecular Biochemicals, Indianapolis, IN) were stored at
80°C. Cells were harvested for subcellular fractionation as
described below.
Isolation of ER and Golgi fractions.
Cellular ER and Golgi fractions were isolated as described by Kumar and
Mansbach (8). Radiolabeled differentiated cells from four
Transwell plates were pooled for each experimental condition, and
undifferentiated, untreated cells from five flasks were added to the
preparation to serve as a carrier. After harvest, cells were suspended
in buffer D consisting of (in mM): 5 EDTA, 10 HEPES, 1 PMSF, and 2 benzamidine HCl plus 0.25 M sucrose and 10 µg/ml antipain at pH 7.3. Cells were then disrupted by N2 cavitation using a Parr
bomb (Parr Instruments, Moline, IL) set at 1,500 psi for 20 min at
4°C followed by rapid venting with return to atmospheric pressure.
Cell homogenate was then transferred to a tight-fitting Dounce
homogenizer and given five strokes. The preparation was then
centrifuged for 1.4 × 104 g/min at 4°C
in a Sorvall RC5C centrifuge (Du Pont Instruments, Wilmington, DE).
Supernatant was transferred to a polyallomar tube and centrifuged for
9 × 106 g/min at 4°C in a TH-641 rotor
in a Sorvall Ultra 80 ultracentrifuge (Du Pont Instruments). The pellet
containing the ER and Golgi fractions was suspended in buffer D
supplemented with 2% bovine serum albumin. The suspension was then
centrifuged again for 3 × 106 g/min at
4°C, followed by harvest of the pellet. The pellet was resuspended in
buffer E consisting of (in mM): 30 HEPES, 2.5 magnesium acetate, 30 KCl, 1 PMSF, and 2 benzamidine HCl plus 0.25 M sucrose and 10 µg/ml
antipain at pH 7.2 in a loose-fitting Dounce homogenizer with five
strokes. The suspension was transferred to a 12-ml polyallomar tube and
brought to 3 ml with buffer E, adjusted to 1.22 M sucrose and
successively overplayed with 2.6 ml each of (in M): 1.15, 0.86, and
0.25 sucrose. The tubes were centrifuged for 14.4 × 106 g/min at 4°C in a Sorvall Ultra 80 ultracentrifuge (Du Pont Instruments). The Golgi membranes were
collected by aspiration from the 0.25/0.85 and 0.86/1.15 M sucrose
interfaces and combined into one sample. Microsomal membranes were
collected as the pellet plus membranes remaining in the 1.22 M sucrose
layer and combined into one sample. Samples not subjected to carbonate
treatment, were immediately frozen to 80°C. Aliquots of the whole
cell homogenate and ER and Golgi fractions were used for protein
determination and measurement of galactosyltransferase activity
(4) as a Golgi marker and glucose-6-phosphatase activity
(5) as an ER marker. Compared with the whole cell
homogenate, there was a sixfold enrichment in glucose-6-phosphatase
specific activity in the ER fraction and a 20-fold enrichment in
galactosyltransferase specific activity in the Golgi fraction. The ER
fraction had less than 10% of the galactosyltransferase specific
activity found in the Golgi fraction, and the Golgi fraction had less
than 5% of the glucose-6-phosphatase specific activity found in the ER
fraction. Thus there was satisfactory marker enzyme enrichment and
minimal cross- contamination of the subcellular fractions.
Isolation of ER vesicle contents and membranes.
ER membranes and contents were isolated as described by Banerjee
(1). One volume of ice-cold 200 mM
Na2CO3, pH 11.3, was added to freshly collected
ER vesicles to achieve a final concentration of 100 mM
Na2CO3 and incubated on ice for 30 min. Samples
were centrifuged for 6.4 × 106 g/min at
4°C in a Sorvall Ultra 80 ultracentrifuge (Du Pont Instruments). Supernatant (containing organelle contents) and pellet (containing organelle membranes) were frozen at 80°C for later lipid extraction.
Triacylglycerol and phospholipid radiolabeling with [3H] glycerol. Cells were incubated with [3H]glycerol and fatty acids complexed with albumin, and the cells and media were collected and processed as described above. Total lipid in the cell homogenate, ER and Golgi fractions, ER contents and membranes, and basolateral medium were extracted as previously described (18). Extracts were applied to silica gel G plates and subjected to thin-layer chromatography using petroleum ether-diethyl ether-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 phospholipid and triacylglycerol were identified by comparison to cochromatographed standards. Total cell content of labeled lipid was expressed as specific lipid disintegrations per minute (dpm) per well, subcellular organelle content of radiolabeled lipid was expressed as specific lipid dpm per milligram cell homogenate protein, and secretion of radiolabeled lipid was expressed as specific lipid dpm per well per 24 h.
apoB and AI mass quantitation by ELISA. apoB and AI protein in subcellular fractions was quantitated by competitive ELISA assays using rabbit anti-swine apoB and AI polyclonal antibodies, respectively, as previously described (2). Standard antigens consisted of swine plasma low-density lipoproteins (LDL; apoB) and high-density lipoproteins (HDL; apoAI). All samples were run in duplicate, and variability between duplicates was less than 5%. ER and Golgi apolipoprotein content was expressed as micrograms per milligram cell homogenate protein.
Protein measurement. Cell homogenate and organelle protein were determined by the Bradford method (3).
Statistical analysis. Data in experimental groups were analyzed by one-way ANOVA followed by the Fisher's least significant difference test to compare specific groups. Statistical significance was set at a two-tailed P value of <0.05.
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RESULTS |
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To define the kinetics of intracellular triacylglycerol and
phospholipid radiolabeling in IPEC-1 cells, cells were harvested after
6, 12, and 24 h of incubation with OA, SA, and EPA with [3H]glycerol, and triacylglycerol and phospholipid were
extracted and counted. Results are shown in Fig.
1. During OA incubation, intracellular
triacylglycerol labeling rose rapidly and peaked at 12 h, followed
by a decline, as the newly synthesized triacylglycerol was secreted,
and substrate for continued synthesis was depleted in the apical medium
compartment. In the case of SA incubation, intracellular
triacylglycerol labeling also peaked at 12 h but at a lower level
than that with OA incubation and began to decline. During EPA
incubation, synthesis of triacylglycerol was much less than that of
either OA or SA, and the intracellular level of labeled triacylglycerol
plateaued at 12 h with very little decline by 24 h. This
suggests that both triacylglycerol synthesis and movement through the
secretory pathways are markedly impaired after EPA incubation,
compared with OA and SA.
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As shown in Fig. 1B, the patterns for labeled phospholipid during SA and EPA incubation were similar to those observed for triacylglycerol labeling. However, in the case of OA, phospholipid synthesis was significantly less than that for SA. This differential effect of OA and SA on phospholipid synthesis was observed in our earlier published studies (18). Although there are differences in magnitude, the kinetics of triacylglyerol and phospholipid labeling during OA, SA, and EPA incubation appear to be roughly similar in the timing of initial increase, plateau, and decline. Therefore, cell fractionation studies at 24 h of fatty acid incubation and lipid radiolabeling would be appropriate, because cellular labeled triacylglycerol and phospholipid for all three fatty acids are at a similar kinetic state at 24 h.
Figure 2 shows basolateral secretion of
radiolabeled triacylglycerol (A) and phospholipid
(B) by IPEC-1 cells after apical incubation with OA, SA, or
EPA and [3H] glycerol in the present studies. These
results confirm our previous published findings of decreased secretion
of triacylglycerol and phospholipid after SA and EPA incubation,
relative to OA (18, 19). These cells were used for cell
fractionation and isolation of ER and Golgi vesicles.
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Figure 3A, shows total ER
content of radiolabeled triacylglycerol after incubation of IPEC-1
cells for 24 h with OA, SA, or EPA in the presence of
[3H]glycerol. Treatment of cells with SA and EPA resulted
in higher and lower ER content of labeled triacylglycerol,
respectively, relative to results with OA. In Golgi fractions (Fig.
3B), SA incubation resulted in lower content of labeled
triacylglycerol, and EPA incubation caused markedly lower accumulation
of labeled triacylglycerol, compared with OA. Examination of the
Golgi/ER ratios of labeled triacylglycerol (Fig. 3C)
suggests that, relative to OA, both SA and EPA interfered with
ER-to-Golgi trafficking of radiolabeled triacylglycerol with EPA having
the greatest effect. In addition, the pattern of ER content of labeled
triacylglycerol suggests that SA resulted in the accumulation of
labeled triacylglycerol in the ER in the face of impaired movement from
ER to Golgi. In contrast, it appears that EPA incubation resulted in
decreased ER synthesis of labeled triacylglycerol, as well as impaired
movement of newly synthesized triacylglycerol from ER to Golgi. Taken
as a whole, these data suggest that SA does not significantly interfere with ER triacylglycerol synthesis, but does interfere with movement of
newly synthesized triacylglycerol from ER to Golgi, resulting in the
accumulation of radiolabeled triacylglycerol in the ER. In the case of
EPA, this fatty acid appears to both interfere with ER triacylglycerol
synthesis, as well as movement of newly synthesized triacylglycerol
from ER to Golgi.
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Figure 4 shows results of the
determination of radiolabeled phospholipid content in ER and Golgi
fractions from IPEC-1 cells incubated for 24 h with OA, SA, or EPA
added to the apical medium with [3H]glycerol. Results for
the ER (A) and Golgi (B) fractions generally parallel those obtained for triacylglycerol. However, there are two
differences. Phospholipid radiolabeling in the ER from cells incubated
with EPA was not different from that observed in cells incubated with
OA (A). Also, phospholipid radiolabeling in Golgi from cells
treated with SA was not different from that of OA-treated cells
(B). However, ER-to-Golgi trafficking of labeled
phospholipid as reflected in the Golgi-to-ER ratio (C) shows
the same pattern as that for labeled triacylglycerol.
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The relative partitioning of radiolabeled triacylglycerol and
phospholipid into ER membrane and luminal contents after fatty acid
incubation was determined by the treatment of ER vesicles with
carbonate buffer, followed by centrifugation, lipid extraction, and
thin- layer chromatography. As shown in Fig.
5, after SA incubation, this partitioning
was similar to that observed for OA. However, relatively less labeled
triacylglycerol and phospholipid partitioned into the ER lumen after
EPA incubation, compared with OA and SA.
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Figure 6 shows the total content of apoB
protein in ER and Golgi fractions from the three experimental groups.
Relative to OA, SA incubation resulted in patterns for ER, Golgi, and
ER-to-Golgi ratio very similar to those for radiolabeled
triacylglycerol (Fig. 3). However, in the case of EPA, although the
apoB content of the ER and Golgi was less than that of OA or SA, the
Golgi-to-ER ratio was comparable with that for OA (Fig. 6). This
finding is suggestive of normal trafficking of apoB from ER to
Golgi after EPA incubation, despite impaired trafficking of
triacylglycerol and phospholipid.
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Figure 7 shows the total content of apoAI
protein in the ER and Golgi fractions from IPEC-1 cells incubated with
OA, SA, or EPA. The patterns of ER and Golgi apoAI content,
as well as the Golgi-to-ER ratios, in the SA- and EPA-treated cells,
were different from those observed for apoB. In particular, the
Golgi-to-ER ratios for apoAI content after SA and EPA incubation
demonstrated inverse patterns from those for apoB. These findings
suggest that apoAI trafficking may be regulated differently from that
of apoB.
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DISCUSSION |
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We have previously reported that, relative to OA, incubation of SA and EPA in the apical medium compartment of IPEC-1 cells results in decreased basolateral secretion of newly synthesized triacylglycerol and phospholipid (18, 19). We have confirmed these findings in the present study and extended our observations to explain these results as due, at least in part, to less efficient ER-to-Golgi trafficking of newly synthesized triacylglycerol and phospholipid.
Data from the present study suggest that synthesis of triacylglycerol in the ER in response to incubation with SA is relatively unaffected, since the ER triacylglycerol radioactivity was actually higher than that of the OA-treated cells. That this increase represents an accumulation of newly synthesized triacylglycerol in the ER is supported by the observation that the content of labeled triacylglycerol in the Golgi from the SA-treated cells was significantly lower than that of the cells incubated with OA. These differences resulted in a lower Golgi-to-ER triacylglycerol dpm ratio in the SA-treated cells, relative to the OA-treated cells. The same pattern, although of a lesser magnitude, was also observed for radiolabeled phospholipid after SA incubation.
There are several possible mechanisms for the inefficient ER-to-Golgi trafficking of triacylglycerol in IPEC-1 cells after SA incubation. Impaired lipoprotein assembly in the ER could result in the defective lipidation of newly translated apoB with resultant accumulation of triacylglycerol in the ER. Such impairment could result from inhibition of translocation of triacylglycerol across the ER membrane, because the enzymes responsible for triacylglycerol reesterification are localized on the cytosolic side of the ER membrane. Also, relative dysfunction of microsomal triglyceride transfer protein (MTP) in the lipidation of apoB with triacylglycerol containing SA could result in defective particle assembly. Changes in ER membrane fluidity accompanying enrichment with SA-containing complex lipids might contribute to such defects in particle lipidation. However, after SA incubation, the percentages of labeled triacylglycerol and phospholipid in ER membranes and luminal contents were the same as those of the OA-treated cells. This observation suggests that transfer of triacylglycerol from ER membrane to luminal particles is relatively unaffected by SA. The budding of the PCTV from the ER membrane is another site of possible interference by SA incubation (10), resulting in the accumulation of labeled triacylglycerol in the ER fraction.
Because apoB mass measurements after SA incubation demonstrated an ER/Golgi profile similar to that of labeled triacylglycerol, apoB movement from ER to Golgi also may be impaired in parallel with newly synthesized triacylglycerol, relative to OA. Because there seems to be an accumulation of apoB in the ER of SA-treated cells, this would suggest that apoB in the ER is being at least partially lipidated to protect it from degradation, allowing it to accumulate in the ER. In contrast to apoB, apoAI appears to move from ER to Golgi with a higher efficiency than that of apoB, suggesting that apoAI trafficking is not dependent on the parallel movement of apoB-containing triacylglycerol-rich lipoproteins.
The relative defect in the trafficking and secretion of labeled lipid
in IPEC-1 cells incubated with EPA seems to be more complex than that
observed for SA. There appear to be two defects induced by EPA
incubation, relative to OA. First, levels of labeled triacylglycerol
are very low in ER, Golgi, and basolateral medium. Thus ER
triacylglycerol synthesis with EPA treatment appears to be impaired,
relative to that seen with OA incubation. These results are similar to
those obtained in our previous whole cell studies (19) of
the effect of EPA on lipid synthesis and secretion in IPEC-1 cells. The
suppression of triacylglycerol synthesis by EPA may potentially be
explained by several mechanisms. Previous studies (14, 21)
in rat hepatocytes suggested that n-3 fatty acids are poor substrates
for triacylglycerol synthesis, compared with OA. Other studies
(11, 16) in hepatocytes have suggested that n-3 fatty
acids inhibit the activities of diacylglycerol acyltransferase and
phosphatidate phosphohydrolase, two enzymes in the triacylglycerol
synthetic pathway. Murthy et al. (13) demonstrated that in
Caco-2 cells, phospholipids of microsomes from cells incubated with EPA
were enriched in this fatty acid. Furthermore, this microsomal lipid
compositional change was associated with a decrease in microsomal
triacylglycerol synthesis and diacylglycerol acyltransferase
activities. However, data from hepatocytes and Caco-2 cells may not
have relevance to normal enterocytes. Hepatocytes use the
-glycerophosphate pathway for triacylglycerol synthesis, whereas
normal enterocytes utilize the monoglyceride pathway. Caco-2 cells,
similar to hepatocytes, also produce triacylglycerol predominantly
through the
-glycerophosphate pathway (17). Although we
have no data on the predominant pathway of triacylglycerol synthesis in
either newborn swine small intestine or IPEC-1 cells, the EPA effect on
triacylglycerol synthesis reported here and previously
(19) may not be significant in normal newborn swine enterocytes if the effect is dependent on the presence of the
-glycerophosphate pathway for triacylglycerol synthesis.
Increased fatty acid oxidation is another potential mechanism to
explain decreased availability of fatty acids for incorporation into
intestinal lipoprotein triacylglycerol after incubation of cells with
EPA. In rat liver, EPA acts as a mitochondrial proliferator and
enhances mitochondrial -oxidation (6). In rat
experiments, EPA was noted to be hypotriglyceridemic. However, it has
been shown (13) that both OA and EPA are minimally
oxidized to CO2 in Caco-2 cells. Thus it appears that in
contrast to liver, intestinal epithelial cells shunt very limited
amounts of fatty acids into oxidative pathways. Also, decreased apical
uptake of EPA relative to OA could explain the present findings.
However, we have found similar amounts of radiolabeled OA, SA, and EPA
remaining in the apical medium compartment after incubation with IPEC-1
cells (unpublished observation). Also, EPA has been shown to be taken
up by Caco-2 cells at a somewhat higher rate than OA (15).
The second defect contributing to the decreased secretion of radiolabeled triacylglycerol by IPEC-1 cells after EPA incubation, relative to OA, appears to be inefficient movement of labeled triacylglycerol from ER to Golgi. The Golgi-to-ER triacylglycerol dpm ratio was much lower than that of either OA or SA, suggesting that even the limited amount of triacylglycerol leaving the ER was not efficiently trafficked to the Golgi compartment. The same mechanisms potentially contributing to the trafficking defect with SA incubation might also hold true for the cells incubated with EPA. However, the pattern of apoB mass content in the ER and Golgi fractions suggests some differences. The amount of ER apoB was lower than that for either OA or SA, suggesting possible degradation because of decreased apoB lipidation. In contrast, the Golgi-to-ER apoB mass ratio after EPA incubation was the same as that observed for OA. This observation might suggest that whatever apoB is available for transport in the ER is being efficiently moved to the Golgi and may represent apoB-containing particles that are poorly lipidated with a low triacylglycerol-to-apoB ratio. However, the apoB ELISA assay used in the present study may detect epitopes from apoB fragments, rather than intact apoB, and thereby overestimate the content of intact apoB mass.
The decreased partitioning of labeled triacylglycerol and phospholipid from the ER membrane into the luminal compartment observed after EPA incubation, relative to OA, suggests a defect in luminal lipoprotein particle lipidation, possibly due to altered MTP function. We have found that MTP large- subunit mRNA and protein levels are reduced in IPEC-1 cells after EPA incubation, relative to OA (unpublished observation).
As noted after SA incubation, the pattern of apoAI mass content in ER and Golgi fractions after EPA treatment is the opposite of that observed for apoB, suggesting that apoAI trafficking from ER to Golgi may be independent of that of apoB. This is consistent with our previous observation in IPEC-1 cells that the apoAI secreted into the basolateral medium in response to EPA treatment is associated with the high-density lipoprotein density range, not the apoB-containing chylomicron and very low-density lipoprotein density range (19). These findings are also consistent with the observation that the PCTV isolated from rat enterocytes contains very little apoAI, despite abundant apoAI in ER and Golgi fractions (9). These data in the rat studies were interpreted as providing evidence that apoAI is carried to the Golgi by a route different from that of apoB and triacylglycerol.
In summary, the present studies suggest that SA and EPA adversely influence immature enterocyte ER- to-Golgi trafficking, compared with OA. Furthermore, EPA appears to inhibit ER lipid synthesis and transfer of ER membrane lipid to luminal particles. Our data support the concept that apoAI ER-to-Golgi trafficking may be independent of the movement of apoB through the enterocyte secretory pathway.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Child Health and Human Development Grant HD-22551 (to D. D. Black), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42100 (to H. M. Berschneider), and funding from the Children's Foundation Research Center of Memphis at Le Bonheur Children's Medical Center (to D. D. Black).
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. D. Black, Children's Foundation Research Center of Memphis, Le Bonheur Children's Hospital, Rm. 401, W. Patient Tower, 50 N. Dunlap, Memphis, TN 38103 (E-mail: dblack{at}utmem.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 23, 2002;10.1152/ajpgi.00397.2001
Received 10 September 2001; accepted in final form 15 January 2002.
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