2 Crippled Children's Foundation Research Center, Le Bonheur Children's Medical Center, University of Tennessee Health Science Center, Memphis, Tennessee 38103; 1 Arkansas Children's Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202; and 3 College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606
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ABSTRACT |
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Long-chain polyunsaturated fatty acids (LC-PUFA) are important in the development of the immature nervous system, and adding these fatty acids to infant formula has been proposed. To determine the effect of n-3 LC-PUFA on apolipoprotein secretion and lipid synthesis in newborn swine enterocytes, differentiated IPEC-1 cells were incubated for 24 h with docosahexaenoic acid (DHA; 22:6) or eicosapentaenoic acid (EPA; 20:5) complexed with albumin at a fatty acid concentration of 0.8 mM or albumin alone (control) added to the apical medium. Oleic acid (OA; 18:1) was used a control for lipid-labeling studies. Both DHA and EPA reduced apolipoprotein (apo) B secretion by one-half, whereas EPA increased apo A-I secretion. The increased apo A-I secretion occurred primarily in the high-density lipoprotein fraction. These changes in apoprotein secretion were not accompanied by significant changes in synthesis. Modest decreases in apo B mRNA levels were observed for DHA and EPA, whereas there were no changes in apo A-I mRNA abundance. EPA reduced cellular triacylglycerol labeling by one-half, and DHA and EPA decreased cellular phospholipid labeling compared with OA. Labeled triacylglycerol secretion was decreased 75% by EPA, and DHA doubled labeled phospholipid secretion. If present in vivo, these effects should be considered before supplementing infant formula with these fatty acids.
docosahexaenoic acid; eicosapentaenoic acid; messenger ribonucleic acid; phospholipid; triacylglycerol
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INTRODUCTION |
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THE LONG-CHAIN
POLYUNSATURATED fatty acids (LC-PUFA), arachidonic acid [AA;
20:4(n-6)], and docosahexaenoic acid [DHA; 22:6(n-3)] are important
for mammalian central nervous system and retinal development (2,
3, 6-8, 15, 16). Humans do not synthesize n-3 or n-6
long-chain fatty acids. Therefore, AA and DHA may be synthesized from
linoleic acid [18:2(n-6)] and linolenic acid [18:3(n-3)],
respectively, which must be derived from the diet and are considered
essential nutrients. However, human infants, especially preterm
infants, may have limited ability to elongate and desaturate linoleic
and linolenic acids to AA and DHA, thereby predisposing to a deficiency
state, even with adequate stores of the precursor fatty acids
(14). As a result, there is interest in supplementing
infant formula with LC-PUFA. However, the effects of these fatty acids
on immature enterocyte lipid and apolipoprotein synthesis and secretion
are not known. There is particular concern regarding the n-3 LC-PUFA,
DHA, and eicosapentaenoic acid [EPA; 20:5(n-3)], because these fatty
acids have been shown (18, 20, 23, 29, 30, 34, 36, 37) to
inhibit lipoprotein secretion from cultured enterocytes (Caco-2 cells)
and hepatocytes. A study (13) in the lymph-fistula rat
demonstrated impaired transport of DHA and EPA into mesenteric lymph
relative to oleic acid (OA). However, direct evidence for impairment of
triacylglycerol secretion from enterocytes in vivo is lacking, because
enterocytes in vivo synthesize triacylglycerol via the monoglyceride
pathway, whereas Caco-2 cells produce triacylglycerol through the
-glycerophosphate pathway (19). These fatty acids have
also been shown to reduce hepatic very low density lipoprotein (VLDL)
secretion in humans (25, 26).
In the present study, we focused on two n-3 LC-PUFA: DHA and EPA. EPA is a precursor of DHA, and both are found in marine oils. DHA and EPA have both been found to lower serum triacylglycerol levels in adult human studies (12, 28). The aim of our study was to determine the effect of DHA and EPA on apolipoprotein and lipid synthesis and secretion in a newborn swine intestinal epithelial cell line (IPEC-1). This cell line was derived from a newborn unsuckled piglet, and we (11, 32, 33) have previously characterized the apical uptake, cellular processing, and basolateral secretion of several fatty acids and biliary lipids, as well as associated changes in apolipoprotein secretion. Although these cells do differentiate in serum-free medium to resemble enterocytes functionally and morphologically, several features suggest that the cells do not mature to ultimately resemble adult enterocytes but rather remain in a relatively immature state, more closely resembling late fetal or early neonatal enterocytes (11). For example, in undifferentiated IPEC-1 cells, there is no expression of apolipoprotein B (apo B) mRNA editing activity, and only apo B-100 is synthesized and secreted, as is the case in the early human and swine fetus (11). In differentiated cells cultured in serum-free medium on collagen-coated filters for 10-12 days, ~50% of the apo B mRNA is edited, and these cells synthesize and secrete both apo B-100 and B-48. In neonatal and adult swine, >90% of the apo B mRNA is edited in small intestine, and their enterocytes produce predominantly apo B-48 (11). In addition, even when maximally differentiated, these cells tend to be cuboidal and have glycogen inclusions, a feature of immature enterocytes (22). In the present study, lipid and apolipoprotein synthesis and secretion were characterized after incubation of IPEC-1 cells with DHA and EPA in the apical culture medium compartment.
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MATERIALS AND METHODS |
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Materials. [1,2,3-3H]glycerol (0.2 Ci/mmol) was purchased from Dupont New England Nuclear (Boston, MA). DHA [C22:6(n-3)], EPA [C20:5(n-3)], OA [C18:1(n-9)], essentially fatty acid-free BSA, Triton X-100, phenylmethylsulfonyl fluoride (PMSF), and benzamidine were purchased from Sigma Chemical (St. Louis, MO).
Cell culture.
The derivation of the IPEC-1 cell line has been described previously
(11). 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 (GM): DMEM/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,
undifferentiated cells were harvested by trypsinization, and 2 × 106 cells/well were plated on 24.5-mm diameter
collagen-coated filters (3-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 107 M dexamethasone (Sigma) but without FBS.
Medium was then changed every two 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
(11). Cellular membrane integrity was assessed by
measurement of apical medium lactate dehydrogenase (LDH) activity
(Sigma Chemical).
Incubation of cells with fatty acids.
At 10 days postplating on Transwell filters in serum-free medium, we
added fresh serum-free medium to both the apical and basolateral
compartments. The apical medium contained fatty acid complexed with
albumin (4:1 molar ratio) at a concentration of 0.8 mM
(23). 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
(11). Cells were incubated for 24 h followed by
harvest of culture medium and cells. In lipid radiolabeling
experiments, [3H]glycerol (12 µCi/well) was also added
to the apical medium, concomitant with the addition of fatty acid and
albumin. 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, Pittsburgh,
PA). Cell homogenates were stored at 80°C. Culture medium samples
containing the same concentrations of PMSF and benzamidine were also
stored at
80°C.
Triacylglycerol and phospholipid radiolabeling with [3H]glycerol. Cells were incubated for 24 h with [3H]glycerol and fatty acids complexed with albumin, and the cells and medium were collected and processed as described above. Total lipid in the cells and medium was extracted as previously described (32). Extracts were applied to silica gel G plates and subjected to TLC 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 phospholipids and triacylglycerols 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.
Isolation of basolateral medium lipoprotein fractions and
lipoprotein-free fraction.
After incubation of cells with fatty acids, basolateral culture medium
was subjected to sequential density ultracentrifugation using a Beckman
SW 41 Ti rotor (Palo Alto, CA) at 17°C (32). The density
classes separated were chylomicron (CM) plus VLDL [density (d) 1.006 g/ml], low-density lipoprotein (LDL; 1.006 g/ml
d
1.063 g/ml), and high-density lipoprotein (HDL; 1.063 g/ml
d
1.21 g/ml). The d > 1.21 g/ml lipoprotein-free fraction was also collected. Lipoprotein fractions were subjected to apo A-I
quantitation by ELISA, as well as lipid extraction followed by
phospholipid measurement.
Apo B and A-I mass quantitation by ELISA. Apo B and A-I protein in cell homogenates and culture medium was quantitated by competitive ELISA using rabbit anti-swine apo B and A-I polyclonal antibodies, respectively, as previously described (4). Standard antigens consisted of swine plasma LDL (apo B) and HDL (apo A-I). All samples were run in duplicate, and variability between duplicates was <5%. Secretion of apolipoprotein mass was expressed as micrograms per milligram cell protein per 24 h, and cell apolipoprotein content was expressed as micrograms per milligram cell protein.
Measurement of apo B and A-I synthesis. Radiolabeling of apo B and A-I with [35S]methionine in IPEC-1 cells was carried out to assess the effect of DHA and EPA on apolipoprotein synthesis. Fresh medium was added to differentiated cells at 10 days postplating with 0.8 mM DHA or EPA complexed with albumin or albumin only (control), as described in previous experiments (11, 32), 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 B and A-I immunoprecipitation as described below. Synthesis was expressed as apolipoprotein dpm in cell homogenate per well after the 15-min incubation. We (11) have demonstrated previously the negligible appearance of labeled apo B or A-I in the basolateral culture medium during this short radiolabeling period.
After radiolabeling experiments were completed, cells were rinsed and disrupted in ice-cold PBS containing 1% Triton X-100, 1 mM PMSF, and 1 mM benzamidine by using an ultrasonic dismembranator (Fisher). Cell homogenates were stored atApolipoprotein immunoprecipitation. After [35S]methionine radiolabeling, cell homogenates and culture medium were subjected to immunoprecipitation using a technique adapted from Murthy et al. (23) with rabbit anti-swine apo B and A-I polyclonal antibodies. The cell homogenates collected from each well were precleared by incubation with 0.2 ml of IgGsorb (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 B and 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 (Na2)EDTA, 100 mM NaCl, 0.02% Na 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 (17). Samples were heated at 95°C for 5 min and centrifuged, and the supernatant was subjected to SDS-PAGE using a 3% to 20% acrylamide gradient gel under reducing conditions according to the method of Laemmli (17). 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 apo B-48, B-100, and A-I were sliced out, solubilized, and subjected to liquid scintillation counting.
Apo B and A-I mRNA quantitation by RT-PCR.
Total RNA was extracted from cells after experimental incubations
(9). Aliquots (2-10 µg) were treated with 0.5 U of
DNase RQ1 (Promega, Madison, WI) at 37°C for 60 min in 50 µl 40 mM
Tris · HCl, pH 7.5, 6 mM MgCl2, 10 mM NaCl, 10 mM
dithiothreitol, and 20 U RNase inhibitor (RNasin, Promega). The RNA was
then sequentially extracted with phenol-chloroform and chloroform,
precipitated with ethanol, washed once (with 70% ethanol), and
resuspended in 20-40 µl H2O. For reverse
transcription, 5 µg total RNA were used. Reverse transcription was
performed at 42°C for 15 min in a final volume of 20 µl in buffer
containing 10 mM Tris · HCl, pH 8.3, 90 mM KCl, 1 mM
MnCl2, 200 µM of each dNTP, 0.5 µg
oligo(dT)15 as primer, and 15 U apical membrane vesicle RT
(Promega). After reverse transcription, the single-strand cDNA was
amplified using the Qiagen Taq PCR core kit (Santa Clarita,
CA) with 1 µl cDNA and 100 pmol of each specific primer (B100F and
B100R for apo B, AIF and AIR for apo A-I, and B2MGF and B2MGR for
2-microglobulin) in a total volume of 50 µl. After
incubation for 3 min at 95°C, PCR was performed for 18 cycles in a
thermal cycler (Perkin-Elmer Cetus) as follows: 45 s at 94°C,
45 s at 55°C, and 45 s at 72°C. For each RNA sample, a
negative control was run to check for DNA contamination by using
AmpliTaq (Perkin-Elmer Cetus), leaving the sample on ice during reverse
transcription. Additionally, each reaction contained a tube with all of
the above buffers and enzymes but without RNA to exclude PCR product
contamination. After RT-PCR, two-fifths of the reaction products were
subjected to 1.5% agarose electrophoresis. Expected product sizes were
confirmed as follows:
2-microglobulin, 172 bp; apo B,
290 bp; and apo A-I, 371 bp. RT-PCR products were transferred to
nitrocellulose filters (Trans-Blot, Bio-Rad, Hercules, CA) and
hybridized with sequence-confirmed relevant PCR fragments. Quantitation
was carried out using a GS-700 scanning densitometer (Bio-Rad). Results
were expressed as a ratio of apolipoprotein to
2-microglobulin arbitrary densitometric units.
Oligonucleotides. The following primers were used for PCR: B2MGF, 5'-GAA GAT GAA GGC GGA GCA GT-3' (5' at 192 nt); B2MGR, 5'-TGC CGG TTA GTG GTC TCG AT-3' (5' at 363 nt); B100F, 5'-GCA GCT CCA CCA TTC AGT TC-3' (5' at 5912 nt); B100R, 5'-CTG TTC TAA GGC CAC AGT GC-3' (5' at 6201 nt); AIF, 5'-GAA CAA GTG GCA GGA GGA GA-3' (5' at 384); and AIR, 5'-GGA TGC TGA CCT TGA GGT TC-3' (5' at 751 nt).
Protein and phospholipid measurement. Cell homogenate protein was determined by the Bradford method (5). Phospholipid mass content in cell homogenates and culture medium lipoprotein fractions was determined by the Bartlett method (1) after lipid extraction.
Statistical analysis. Data in experimental groups were analyzed by one-way ANOVA followed by Fisher's least significant difference test to compare specific groups. Statistical significance was set at two-tailed P < 0.05.
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RESULTS |
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Effect of DHA and EPA on apolipoprotein secretion.
Figure 1, top, shows IPEC-1
cellular apo B and A-I content in control cells and in cells incubated
for 24 h with either DHA or EPA. There were no differences among
experimental groups for either apo B or A-I. Basolateral secretion of
apo B and A-I, as depicted in Fig. 1, bottom, was regulated
by LC-PUFA. For apo B, both DHA and EPA reduced apo B mass secretion by
approximately one-half. In the case of apo A-I, EPA treatment increased
apo A-I secretion relative to the control group. Figure
2 shows that the additional apo A-I
secreted in response to EPA treatment was secreted in the HDL range,
making it extremely likely that this apo A-I is associated with lipid
particles. Note that there is a significant amount of free apo A-I in
the lipoprotein-free d > 1.21 density fraction, but this does not
change with EPA treatment. DHA treatment did not result in a
significant increase in apo A-I secretion relative to controls (Fig.
1). None of the fatty acids caused significant cellular injury as
assessed by measurement of culture medium LDH activity (data not
shown).
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Effect of DHA and EPA on apolipoprotein mRNA levels and synthesis.
Figure 3, top, shows apo B and
A-I synthesis as assessed by [35S]methionine
radiolabeling, followed by immunoprecipitation and SDS-PAGE. The use of
SDS-PAGE allowed the resolution of both apo B-48 and apo B-100
immunoprecipitates. There were no statistically significant differences
among the three experimental groups, although apo A-I synthesis tended
to parallel apo A-I secretion for the three experimental groups.
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Effect of DHA and EPA on triacylglycerol and phospholipid synthesis
and secretion.
Figure 4, top, shows the
incorporation of [3H]glycerol into cellular
triacylglycerol and phospholipid after incubation with OA (control),
DHA, and EPA. Cellular triacylglycerol labeling was significantly
reduced by EPA to 55% of the control value, with DHA having no
significant effect. Cellular phospholipid labeling was reduced 42% by
DHA and 61% by EPA relative to the control group. In basolateral
medium (Fig. 4, bottom), radiolabeled triacylglycerol secretion paralleled cellular radiolabeling with EPA, reducing secretion of labeled triacylglycerol by 75% relative to the OA control
group. Incubation with DHA increased the secretion of labeled
phospholipid approximately twofold, and EPA had no effect relative to
the OA control group.
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DISCUSSION |
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This study is the first to demonstrate that the n-3 LC-PUFA, EPA and DHA, regulate apolipoprotein secretion and lipid synthesis in newborn swine intestinal epithelial cells. Apo B secretion was reduced by ~50% by both DHA and EPA. In contrast, apo A-I secretion, predominantly in the basolateral medium HDL fraction, was significantly increased by EPA. Cellular triacylglycerol labeling was reduced by EPA, and cellular phospholipid labeling was reduced by both DHA and EPA. Secretion of radiolabeled triacylglycerol was reduced by EPA, whereas secretion of labeled phospholipid was doubled by DHA.
Inhibition of apo B secretion by EPA has previously been described (23, 29, 30) in the human colon carcinoma cell line Caco-2. Murthy et al. (23) demonstrated that apo B synthesis and secretion were reduced in Caco-2 cells after EPA treatment for 48 h, relative to OA. Apo B mRNA levels were also reduced, suggesting pretranslational regulation by EPA. Ranheim et al. (29, 30) also observed reduced apo B secretion from Caco-2 cells with EPA incubation relative to OA after relatively shorter incubation times. No such studies have been performed in Caco-2 cells by using DHA. In addition to these observations in Caco-2 cells, n-3 fatty acids have been shown to reduce apo B secretion by cultured rat hepatocytes (18, 34), primary human hepatocytes (20), Hep G2 cells (36), and perfused rat liver (37). Furthermore, n-3 fatty acids appear to inhibit apo B secretion from hepatocytes by stimulating apo B degradation (34). In the present study in IPEC-1 cells, we found reduced secretion of apo B by both EPA and DHA relative to the fatty acid-free control. We also found modest, but significant, accompanying decreases in apo B mRNA levels, suggesting at least partial regulation at the pretranslational level. However, there were no significant differences in apo B synthesis after incubation with either DHA or EPA compared with controls. It is possible that we may have observed greater reduction in apo B mRNA levels and/or synthesis with longer incubation times.
With regard to the cellular mechanism of posttranslational downregulation of apo B secretion by DHA and EPA in IPEC-1 cells, there are four possibilities. First, although not specifically addressed in the present study, apo B degradation may be enhanced by DHA and EPA. Second, mobilization of a preformed, intracellular pool of apo B with slow turnover, as we (11) have previously described as a mechanism for the increase in apo B secretion after OA incubation in IPEC-1 cells, may be inhibited by DHA and EPA. Third, n-3 LC-PUFA may impair the assembly of nascent triglyceride-rich lipoproteins in the endoplasmic reticulum, perhaps by interfering with the normal function of microsomal triglyceride transfer protein in apo B lipidation. Finally, because we (32) have found previously that apo B secretion in IPEC-1 cells is positively correlated with triacylglycerol secretion for several types of fatty acids of varying chain lengths and degrees of saturation, the decreased apo B secretion associated with DHA and EPA treatment may be related to reduced availability of newly synthesized triacylglycerol for incorporation into lipoproteins. This may be the case especially for EPA, which caused significant decreases in both cellular triacylglycerol radiolabeling and basolateral secretion of labeled triacylglycerol, in addition to reduction of apo B secretion.
The enhanced secretion of apo A-I in the HDL fraction of the basolateral medium induced by EPA in IPEC-1 cells is a novel finding and not previously reported in any other model system. We (32) have previously demonstrated a progressive increase in apo A-I secretion with increasing desaturation of 18-carbon fatty acids in IPEC-1 cells. Therefore, a similar effect of EPA on apo A-I secretion was not unexpected. The mechanism of this increase appears to be posttranslational, because significant changes in apo A-I synthesis and mRNA levels were not present. We (11) have previously described a posttranslational mechanism for the increase in apo A-I secretion induced by OA, which seems to involve the mobilization of a preformed intracellular pool of apo A-I with a relatively slow turnover. This mechanism may also mediate the increased basolateral secretion of apo A-I induced by EPA.
The suppression of triacylglycerol synthesis and secretion by EPA may
potentially be explained by at least three mechanisms. First, EPA may
inhibit triacylglycerol synthesis. Previous studies (27,
35) in rat hepatocytes suggested that n-3 fatty acids are poor
substrates for triacylglycerol synthesis compared with OA. Other
studies (21, 31) 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. (24) 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. Increased fatty acid oxidation is another potential
mechanism to explain decreased availability of fatty acids for
incorporation into intestinal lipoprotein triacylglycerol. In rat
liver, EPA acts as a mitochondrial proliferator and enhances
mitochondrial -oxidation, whereas prolonged DHA feeding increases
peroxisomal
-oxidation (10). In these rat experiments
(10), EPA was noted to be hypotriglyceridemic and DHA was
not. However, it has been shown that both OA and EPA are minimally
oxidized to CO2 in Caco-2 cells (24). Thus it
appears that in contrast to liver, intestinal epithelial cells shunt
very limited amounts of fatty acids into oxidative pathways. Finally,
decreased apical uptake of EPA relative to OA could explain the present
findings. However, this is doubtful, because EPA has been shown to be
taken up by Caco-2 cells at a somewhat higher rate than OA
(30).
Both DHA and EPA reduced cellular phospholipid radiolabeling relative to OA. However, relative to OA, DHA doubled the basolateral secretion of labeled phospholipid and EPA had no effect. The mechanism of these effects of DHA and EPA on phospholipid synthesis and secretion in IPEC-1 cells is not clear. However, these observations may represent a depletion of the intracellular pool of phospholipid to maintain an equivalent secretion of labeled phospholipid, as in the case of EPA, or an enhanced secretion, as in the case of DHA, relative to OA. Compared with control cells, both OA and DHA induced increased phopholipid mass in cell homogenate and the CM plus VLDL and HDL fractions of the basolateral medium. Contrary to our findings with radiolabeled phospholipid, there was no difference in cell homogenate total phospholipid content between OA- and DHA-treated cells. There was a modest increase in basolateral medium phospholipid mass in the lipoprotein-free fraction from DHA-treated cells compared with OA-treated cells. This change paralleled that observed for total basolateral medium labeled phospholipid in the phospholipid-labeling experiments, although not to the same magnitude. Together, these findings suggest that phospholipid mass changes lag behind the changes observed for phospholipid radiolabeling. Furthermore, both radiolabeled phospholipid and phospholipid mass data suggest that DHA stimulates production of new phospholipid, which is promptly secreted into the lipoprotein-free basolateral medium fraction compared with OA-treated cells and does not accumulate in the cell. Detailed studies of the intracellular trafficking of phospholipid, as well as triacylglycerol, are needed to further elucidate these mechanisms.
In summary, the present studies demonstrate that apo B and A-I secretion, as well as triacylglycerol and phospholipid synthesis and secretion, are regulated by DHA and EPA in newborn swine intestinal epithelial cells. Although DHA decreases apo B secretion, it has only a modest effect on triglyceride synthesis and secretion and actually increases phospholipid secretion. A novel finding not previously reported in either cultured Caco-2 cells or hepatocytes is the induction of basolateral apo A-I secretion in the medium HDL fraction by EPA in IPEC-1 cells. Whether this might be beneficial to the neonate by increasing serum apo A-I and HDL levels and cholesterol transport is not known. Although EPA increases apo A-I secretion, it decreases both apo B secretion and triglyceride synthesis and secretion, which may be potentially deleterious. These findings may have important clinical implications for infants fed formulas supplemented with these fatty acids. Further studies may be needed before infant formulas are routinely supplemented with LC-PUFA, particularly from marine oil sources.
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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 (D. D. Black), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42100 (H. M. Berschneider), and the Crippled Children's Foundation Research Center at Le Bonheur Children's Medical Center (D. D. Black).
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. D. Black, Crippled Children's Foundation Research Center, 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.
Received 27 June 2000; accepted in final form 24 January 2001.
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