Department of Nutritional Sciences, Rutgers University, New Brunswick, New Jersey 08901-8525
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ABSTRACT |
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Free fatty acids (FFA) and sn-2-monoacylglycerol (sn-2-MG), the two hydrolysis products of dietary triacylglycerol, are absorbed from the lumen into polarized enterocytes that line the small intestine. Intensive studies regarding FFA transport across the brush-border membrane of the enterocyte are available; however, little is known about sn-2-MG transport. We therefore studied the kinetics of sn-2-MG transport, compared with those of long-chain FFA (LCFA), by human intestinal Caco-2 cells. To mimic postprandial luminal and plasma environments, we examined the uptake of taurocholate-mixed lipids and albumin-bound lipids at the apical (AP) and basolateral (BL) surfaces of Caco-2 cells, respectively. The results demonstrate that the uptake of sn-2-monoolein at both the AP and BL membranes appears to be a saturable function of the monomer concentration of sn-2-monoolein. Furthermore, trypsin preincubation inhibits sn-2-monoolein uptake at both AP and BL poles of cells. These results suggest that sn-2-monoolein uptake may be a protein-mediated process. Competition studies also support a protein-mediated mechanism and indicate that LCFA and LCMG may compete through the same membrane protein(s) at the AP surface of Caco-2 cells. The plasma membrane fatty acid-binding protein (FABPpm) is known to be expressed in Caco-2, and here we demonstrate that fatty acid transport protein (FATP) is also expressed. These putative plasma membrane LCFA transporters may be involved in the uptake of sn-2-monoolein into Caco-2 cells.
albumin; taurocholate; fatty acid transport protein
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INTRODUCTION |
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SMALL INTESTINAL EPITHELIUM contains morphologically and functionally polarized enterocytes that play a central role in lipid absorption. The average Western diet is composed of ~100 g of lipids per day, and >90% of dietary fat is triacylglycerol (TG) (54). Free fatty acids (FFA) and sn-2-monoacylglycerol (sn-2-MG) are the major products of pancreatic lipase hydrolysis of dietary TG (4). After digestion, these lipolytic products are dispersed as vesicles and bile salt-mixed micelles (17), and it is thought that absorption across the apical brush-border membrane (BBM) of the enterocytes takes place in large part from the micellar phase (48). Although long-chain FFA (LCFA) uptake has been examined a great deal (1, 22, 33, 38, 50, 51), surprisingly little is known about intestinal MG absorption. Not only does sn-2-MG represent one-third of the products of luminal dietary TG hydrolysis, but the food industry has used MG (presumably a mixture of sn-1 and sn-2 species) as an emulsifying agent and food additive since the 1950s (5). Thus we sought to examine the basic mechanism by which sn-2-MG is transported across the BBM of the enterocyte.
MG also may be transported across the basolateral (BL) membrane of the enterocyte. In the bloodstream, TG-rich lipoprotein particles are hydrolyzed by lipoprotein lipase (LPL) that extends into the vascular space from the outer endothelial cell membrane. Several studies have shown that circulating plasma lipoproteins accumulate MG after LPL hydrolysis (11, 12). Thus if MG is produced by LPL activity, it would be, like FFA, either taken up directly into peripheral tissues or bound to serum albumin and circulated in the plasma. In fact, it has been shown that MG can bind to serum albumin, with dissociation constants (Kd) in the micromolar range (49). Because FFA from the plasma are taken up into the enterocyte via the BL membrane (15), it is likely that plasma albumin-bound MG, or perhaps lipoprotein-derived MG, also can be taken up into the enterocyte across the BL membrane. This study was designed to examine the uptake of sn-2-monoolein (sn-2-18:1) at both the apical (AP) and BL membranes of Caco-2 cells, compared with two major dietary FFAs, palmitate (16:0) and oleate (18:1).
Caco-2 is a human colon adenocarcinoma cell line that was established in 1974 by Fogh et al. (14). When grown in culture, Caco-2 cells spontaneously develop many functions characteristic of mature villus epithelial cells of the small intestinal epithelium. The cells form a polarized monolayer of columnar epithelium with intercellular tight junctions (16) and an AP membrane with a brush border of organized microvilli. When grown on permeable filters, both AP and BL compartments can be separately accessed. Caco-2 secrete chylomicrons (24) and very low-density lipoproteins (25) from the BL membrane. Furthermore, Caco-2 cells express both liver (L-FABP) and intestinal fatty acid-binding proteins (I-FABP) that may play important roles in trafficking LCFA and MG intracellularly (10, 22, 52). However, Caco-2 cells have a number of traits that do not resemble those of intestinal absorptive cells: Caco-2 cells synthesize primarily apolipoprotein B (apoB)-100, while normal human intestine produces apoB-48 (20); Caco-2 cells accumulate glycogen; Caco-2 cells possess some characteristics of fetal cells and colonic crypt cells; Caco-2 cells express lower levels of monoacylglycerol acyltransferase (MGAT) activity than do jejunal cells (53); and Caco-2 cells express only 10% of the I-FABP protein level of rat enterocytes (10). Nevertheless, Caco-2 cells remain the most well-characterized human intestinal cell line with respect to lipid metabolism and, on balance, perform most of the functions associated with intestinal lipid absorption, transport, and metabolism (23). Because Caco-2 cells express low MGAT activity (53), sn-2-MGs may not be rapidly metabolized immediately after entering the cells, assuming there are no alternative pathways. Therefore, Caco-2 cells should be a good model in which to study the transmembrane transport of sn-2-MG, where uptake and metabolism are more readily distinguished.
There are two mechanisms that have been proposed for LCFA transport across the AP membrane of the enterocyte: passive diffusion through the lipid bilayer (21, 32) and carrier-mediated transport (1, 2, 38, 43, 46). Arguments for facilitated LCFA transport include the observed saturable kinetics as a function of unbound LCFA concentration in different tissues and cells (2, 43, 46) and the cloning and characterization of several putative transport proteins (1). In contrast, little is currently known about how MG transport across cellular membranes occurs. In the present studies, we examined the mechanisms of 3H-labeled sn-2-18:1 uptake into Caco-2 enterocytes. To mimic the physiological situation, we examined the kinetics of LCFA and long-chain MG (LCMG) uptake using taurocholate (TC)-mixed micelles at the AP surface to model the luminal situation and using albumin-bound MG to model the plasma situation. A direct comparison of TC-bound and bovine serum albumin (BSA)-bound lipid uptake at the AP surface also was made, to distinguish the effects of membrane surface from that of substrate presentation.
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MATERIALS AND METHODS |
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Materials. Polycarbonate Transwell filter inserts (6.5-mm diameter, 0.4-µm pore) were purchased from Costar (Cambridge, MA). Tritium-labeled triolein, oleate, and palmitate (9,10-3H) were from NEN (Boston, MA). Unlabeled fatty acids and monoacylglycerols were obtained from Nu-Chek Prep (Elysian, MN) and Serdary Research Lab (Englewood Cliffs, NJ), respectively. BSA (essentially FFA free), trypsin, pronase, Triton X-100, and pancreatic lipase (type VI) were obtained from Sigma (St. Louis, MO). Sodium taurocholate (TC) was purchased from Calbiochem (La Jolla, CA). Polyvinylidene difluoride (PVDF) Immobilon-P blotting membranes were from Millipore (Bedford, MA). The YM2 ultrafiltration membranes were purchased from Amicon (Beverly, MA). Dulbecco's modified Eagle's medium (DMEM), nonessential amino acids, fetal bovine serum (FBS), penicillin, streptomycin, trypsin-EDTA, and oligo(dT)18 were from Life Technologies (Grand Island, NY). The sodium borate (3%)-coated preparative thin-layer chromatography (TLC) plates were obtained from Analtech (Newark, DE). Human monoclonal CD36 antibody was purchased from Alexis (San Diego, CA). The simian virus (SV) total RNA isolation kit and avian myeloblastosis virus (AMV) reverse transcriptase were obtained from Promega (Madison, WI). Primers for RT-PCR were made by Genosys (Woodlands, TX).
Cell culture.
Caco-2 cell cultures were obtained from American Type Culture
Collection and were grown in DMEM with 4.5 g/l glucose, 4 mM glutamine,
100 U/ml penicillin, 100 mg/ml streptomycin, 1% nonessential amino
acids, and 20% FBS in a 95% air-5% CO2 atmosphere at
37°C, as described previously (23). The medium was
changed every other day. Cells were plated at a density
104 cells/cm2 in 75-cm2 flasks
and split with 0.25% trypsin-1 mM EDTA when they reached 70-90%
confluence, as previously described (51, 52). For
experiments, cells were plated at a density of 3 × 105 cells/cm2 onto 6.5-mm polycarbonate
Transwell filter inserts with 0.4-µm pores (Costar) or at
104 cells/cm2 onto 12-mm-diameter glass
coverslips, which were placed in 24-well tissue culture plates. Cells
were grown to 14-18 days postconfluence for the experiments.
Transepithelial resistance (TER) measurements were made to ensure tight
junction formation, and only monolayers with TER
250
· cm2 were used for experiments
(52). Most experiments were done with cells grown on the
filters; however, a few AP uptake experiments were done with cells
grown on glass coverslips; the AP uptake results from these two
conditions were similar.
Preparation of radiolabeled sn-2-MG.
Because there is no commercially available radiolabeled
sn-2-MG, and because the isomerization of monoacylglycerides
between sn-1 and sn-2 positions is likely to
occur by acid, alkali, or heat (28),
[3H]sn-2-monoolein was freshly prepared before
each experiment. [3H]triolein (specific activity: 28 Ci/mmol) that has 3H labeled on the double bond of all acyl
chains was used as substrate. [3H]sn-2-18:1 was prepared by digestion
of radiolabeled triolein with pancreatic lipase (type VI) (Sigma) at
37°C for 2 h, followed by 3% sodium borate-coated preparative
TLC separation (27). The
[3H]sn-2-monoolein was prepared freshly before
each uptake experiment to minimize the possibility of acyl group
isomerization between sn-1 and sn-2 positions and
was always used within 2 wk. We found that <10% of the
[3H]sn-2-monoolein migrated as
[3H]sn-1-monoolein after 1 mo of storage at
20°C.
Determination of critical micellar concentrations of sn-2-monoolein. The critical micellar concentration (CMC) of [3H]sn-2-monoolein in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM NaHPO4, pH 7.4) was determined at room temperature by ultrafiltration with the use of a stirred ultrafiltration cell (Amicon) with Amicon YM2 filters [exclusion limit: relative molecular weight (Mr) of 1,000] (39, 50). Briefly, solutions and dispersions of 3H-labeled lipids in PBS were filtered through the Amicon ultrafiltration cell, and the radioactivities of the filtrate and of the dispersion retained on the filter were determined. It is worth noting that the 1,000 Mr cut-off filter would retain dimolecular as well as polymolecular monoolein aggregates; thus this method determines an apparent CMC, which indicates the concentration at which monomolecular monoolein is markedly diminished.
Preparation of uptake media: TC-bound lipids. Radiolabeled oleic acid (18:1), palmitic acid (16:0), and sn-2-monoolein (sn-2-18:1) were dried under N2. The dried lipids were then dissolved in ethanol (0.5% volume in final volume) and subsequently dispersed in 10 mM TC (typical luminal bile salt level) (9) in PBS to a concentration of 9 mM LCFA/MG and incubated for 1 h at 37°C with 90-rpm shaking to obtain an optically clear solution. Serial dilutions of TC-mixed lipids were then made by adding desired volumes of 10 mM TC in PBS to obtain total LCFA/MG concentrations of 25-8,000 µM. The specific activities of the uptake solutions were 0.5 µCi/nmol for each ligand. The monomer concentrations of LCFA in TC mixtures were determined using the fluorescent probe ADIFAB (Molecular Probes, Eugene, OR) (37) and the ultrafiltration method described by Tranchant et al. (50) and Schulthess et al. (39). The monomer concentrations of sn-2-18:1 were determined only by the ultrafiltration method because ADIFAB does not bind MG.
Preparation of uptake media: BSA-bound lipids. Radiolabeled 18:1, 16:0, and sn-2-18:1 were dried under N2. The dried lipids were dissolved in 0.5% (vol/vol) ethanol relative to final volume before being dispersed in 100 µM BSA (typical plasma level) (3) in PBS, pH 7.4. Media were incubated for 1 h at 37°C with 90-rpm shaking to obtain an optically clear solution. Serial dilutions of BSA-bound lipids were made by adding desired volumes of 100 µM BSA in PBS to obtain total LCFA(MG) concentrations of 25-1,800 µM. The specific activities of the uptake solutions were 0.5 µCi/nmol for each ligand. The monomer concentrations of LCFA bound to BSA were calculated from the equilibrium binding constants as reported by Spector et al. (44) as well as those reported by Richieri and Kleinfeld (35), and the monomer concentration of sn-2-18:1 bound to BSA was calculated from the binding constant determined by Thumser et al. (49).
LCFA and MG uptake assay. Initial rates of uptake of TC-mixed lipids at the AP membrane as well as the initial rates of uptake of BSA-bound lipids at the BL membrane were determined as previously described (51). Briefly, the cells were washed with PBS twice, and then the uptake medium for either side was added to the cells. After designated times of incubation, the uptake medium was rapidly aspirated off, and the filter insert was immediately dipped into an ice-cold 0.5% BSA solution ("stop" solution) to stop cellular uptake and remove surface-bound lipid. The cells were then washed with ice-cold stop solution two more times, followed by washing with ice-cold PBS three times, and were then scraped into a 0.05% Triton X-100 solution (51). Protein content (6) and radioactivity were determined after sonication of cells for 15 s with a Branson sonifier equipped with a microtip (Danbury, CT). Initial rates of uptake were determined over a range of ligand concentrations to obtain the apparent Michaelis-Menten constant (Km) and maximum velocity (Vmax) of uptake, as described previously (51). Because the levels of "donor" lipid are far exceeded by levels of "acceptor" (i.e., cell phospholipid), it was assumed that unidirectional uptake was being monitored, rather than exchange.
Competition studies. For competition studies, excess unlabeled lipids were added to radiolabeled BSA-bound lipids, and the uptake assays were performed as described above. The monomer concentration for each concentration of lipid used in these studies was calculated as described above and was always below the corresponding CMC value (36).
Protease studies. The effect of trypsin on sn-2-18:1, 18:1, and 16:0 uptake in Caco-2 cells was determined. As previously found, the AP surface remained intact at higher protease concentrations than did the BL surface (51); therefore, the protease studies were performed with 15-min preincubation with trypsin at 0.5 mg/ml at the AP surface and 0.05 mg/ml at the BL surface. The cells were then washed, the TER was measured to ensure the integrity of the tight junctions, and the uptake experiments were performed within 5 min.
mRNA determination.
RT-PCR was used to evaluate the presence of mRNA transcripts for the
membrane transporters fatty acid translocase (FAT/CD36) and fatty acid
transport protein (FATP) in Caco-2. All determinations were done in
triplicate, at minimum. RNA from rat adipose tissue was used as a
positive control for both transcripts. RNA was prepared from Caco-2
cells grown in 75-cm2 flasks at designated times
postconfluence, as indicated. Total RNA was extracted from cells by
using the SV total RNA isolation kit from Promega. The first-strand DNA
synthesis was done using the oligo(dT)18 primer from Life
Technologies and AMV transcriptase from Promega. cDNA first-strand
synthesis was performed at 42°C for 1 h, followed by 94°C for
10 min for enzyme inactivation. PCR was then carried out by using
primers designed on the basis of a human intestinal form of FATP,
hsFATP4 (13) , as follows: 5' GTGCT GTTCT
CCAAG CTGGT GCTGA AACTG 3' (nt 31-60) and 5' AGATA GAACA GCGGG
TCTTC ACAAT AGCCG 3' (nt 1849 to
1820). The primers for FAT/CD36
were designed using the cDNA of human CD36 (55) as
follows: 5' CCAGG AGTTT GCAAG AAACA GGTGC TTAAC 3' (nt 81-110) and
5' AGCAA CAAAC ATCAC CACAC CAACA CTGAG 3' (nt
1510 to
1481). A
10-µl amount of the first-strand mix was added to 100 µl of PCR
buffer containing 10 µl of 10× buffer for Vent polymerase [2 µl
of 10 mM dNTP, 1 µl of 100 µM hsFATP4 or CD36 primers,
0.5 µl of 100 mM MgSO4, and 2 µl of Vent polymerase
(New England Biolabs, Beverly, MA)]. After the initiation incubation
of 2 min at 94°C, 30 PCR cycles consisted of 1 min at 94°C, 1 min
at 71°C, and 2 min at 72°C. PCR was completed with a final 10 min
at 72°C. Amplified DNA was separated through a 1% agarose gel and
visualized by ethidium bromide staining. The size of the amplified
fragments (1,800 bp for hsFATP4 and 1,400 bp for human
CD36) corresponded to an amplification of cDNA rather than genomic DNA.
PCR was also performed using
-actin primers obtained from Promega.
Western blot to detect FAT/CD36. Caco-2 cells were harvested and lysed by sonication. The lysed cells were centrifuged at 12,000 g for 10 min at 4°C. Total protein in the lysate was measured according to Bradford (6). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% gels. The proteins were electrophoretically transferred to PVDF membranes according to method of Matsudaira (26). The blotting membrane was blocked with 5% nonfat dry milk and then incubated with the monoclonal mouse anti-human CD36 antibody at a 1:10,000 dilution. Antigen-antibody complexes were detected by incubating with anti-mouse IgG-horseradish peroxidase conjugate at a 1:10,000 dilution. The blots were developed by enhanced chemiluminescence (ECL; Amersham).
Statistical methods and calculations. Within each experiment, triplicate samples were analyzed. Each separate experiment was performed at least three times, and results are expressed as means ± SD. A two-way repeated-measures analysis of variance was used to determine whether differences existed among Vmax or Km values for each lipid by various combinations of cell surface (i.e., AP or BL) and lipid carrier (i.e., TC or BSA). This analysis was carried out with the SAS program. Results were considered statistically significant at P < 0.05.
Kinetics of lipid uptake were analyzed by constructing Woolf plots, as described previously (29, 51). Theoretical competition curves were generated by assuming Km = Ki and using the equation
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RESULTS |
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Determination of CMC values for sn-2-monoolein.
The CMC of [3H]sn-2-monoolein was determined
using ultrafiltration (39, 50). When the
sn-2-monoolein concentration was below its CMC, the
radioactivity ratio of the filtrate and the solution in the filtration
cell was nearly 1:1. After the concentration of 3H-labeled
lipid in PBS reached its CMC, the radioactivity ratio was dramatically
decreased. The midpoint of this linear break was estimated as its CMC,
as shown in Fig. 1. The average CMC value
obtained for sn-2-monoolein was 4.2 ± 0.5 µM. The
CMC values for oleic acid (6.0 µM) and palmitic acid (4.0 µM) were
obtained from Richieri et al. (35).
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Determination of initial rates of lipid uptake by Caco-2.
Initial rates of uptake of AP TC-bound lipids as well as the initial
rates of BL BSA-bound lipid uptake were determined as described in
MATERIALS AND METHODS. The AP uptake of TC-mixed lipids as
well as BL uptake of BSA-bound lipids was found to be a linear function
of time within 20 s (Fig. 2,
A and B). Therefore, the kinetic parameters of
uptake were determined using the 10-s point for various concentrations
of lipids, to ensure initial rate conditions.
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Kinetic studies of TC-mixed lipid uptake at the AP surface. To mimic the postprandial intestine, we used bile salt micellar solutions for AP uptake studies. The monomer concentrations of 18:1, 16:0, and sn-2-18:1 in TC-mixed micelles were obtained by using the ADIFAB probe method and/or the filtration method, as described in MATERIALS AND METHODS. Because ADIFAB is very sensitive to even trace amounts of ethanol (53), the unbound concentrations of LCFA in TC micelles were measured in an ethanol-free environment, as reported (53). The unbound concentrations of LCFA obtained from ADIFAB and from the filtration method were consistently similar (data not shown).
AP uptakes of TC-mixed 18:1, 16:0, and sn-2-18:1 AP uptake were plotted as a function of unbound (monomer) concentration of lipid (Fig. 3A). The results show that AP uptake of TC-mixed sn-2-18:1, 18:1, and 16:0 appears to be a saturable function of monomer lipid concentration. This finding suggests that uptake of both sn-2-18:1 and LCFA at the AP surface of the Caco-2 occurs by a facilitated transport process. Woolf plots, which display the lipid monomer concentration [S] on the x-axis and [S]/V (uptake velocity) on the y-axis, were constructed from the data to determine the apparent Km and Vmax values, as described previously (51). The results in Table 1 demonstrate that for AP uptake of TC-mixed lipids, 18:1 has the highest Vmax (18,276 ± 3,764 pmol · mg
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Kinetic studies of BSA-bound lipid uptake at BL and AP surfaces.
To model plasma LCFA and MG uptake by enterocytes, we used BSA-bound
lipids for BL surface uptake studies. Uptake of BSA-bound lipids across
the BL surface was determined, and the results were plotted as a
function of the monomer concentrations of the lipids using
Kd values reported by Spector et al.
(44) and Thumser et al. (49) (Fig.
3B). The results show apparent saturable kinetics of
BSA-bound lipids, suggesting that facilitated uptake is occurring at
the BL side of Caco-2 cells. The Km values for
all three ligands were within the range of 0.2-0.3 µM, which was
slightly higher than the Km values for AP uptake
of TC-bound lipids (0.03-0.2 µM) (Table 1). In contrast to AP
uptake of TC-mixed lipids, 16:0 had the highest
Vmax (85.6 ± 7.4 pmol · mg1 · 10 s
1),
followed by 18:1 (29.3 ± 9.2 pmol · mg
1 · 10 s
1) and
then sn-2-18:1 (21.8 ± 5.8 pmol · mg
1 · 10 s
1) (Table
1). These absolute values for Vmax were two
orders of magnitude lower than those obtained for AP uptake of TC-bound lipids, suggesting that TC-mixed lipids are more efficiently taken up
by Caco-2 than are BSA-bound lipids.
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Protease studies.
To determine whether protease-sensitive membrane proteins were involved
in the saturable kinetics of uptake of LCFA and MG, we preincubated
Caco-2 cells with trypsin and performed uptake studies within 5 min
after incubation. The results show that preincubation with trypsin at
the AP surface and at the BL surface inhibits the uptake of TC-mixed
18:1, 16:0, and sn-2-18:1 uptake by 20-40% compared with control cells (Table 2 ).
Trypsin preincubation also inhibited BSA-bound
sn-2-18:1 uptake at both AP and BL membranes by 11%
and 30%, respectively. These results support the hypothesis that
membrane proteins may be involved in the uptake of LCFA and sn-2-18:1 across the AP and BL surfaces of the Caco-2
cell.
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Competitive inhibition studies.
To further explore the saturable nature of LCFA and MG transport in
Caco-2 cells, we conducted studies to determine whether competitive
inhibition of uptake occurred. The AP uptake of radioactive 18:1 or
sn-2-18:1 (monomer concentrations of 0.01 and 0.02 µM, respectively) was measured in the presence of increasing
concentrations of unlabeled 18:1 and MG, with total concentrations
always below their CMC. Figure
5A shows that the uptake of
radiolabeled 18:1 was competitively inhibited by excess unlabeled 18:1,
as well as by LCMG [sn-2-18:1 and
sn-2-monopalmitin (sn-2-16:0)] but not by
medium-chain MG [sn-2-caprin (sn-2-10:0)],
in a dose-dependent manner. Similarly, Fig. 5B shows that
the uptake of [3H]sn-2-monoolein was
competitively inhibited by the addition of excess unlabeled LCFA (18:1)
and MG (sn-1-18:1, sn-2-18:1, and sn-2-16:0) but not by medium-chain FFA [capric acid
(10:0)] or medium-chain MG [sn-2-caprin
(sn-2-10:0)]. These competition studies further
suggest that LCFA and LCMG uptake is, at least in part, protein
mediated. Furthermore, they imply that LCFA and LCMG may compete for
the same protein(s) on the AP membrane of Caco-2. Figure
6 shows the experimental (solid line) and
theoretical (dashed line) competitive inhibition curves for
sn-2-18:1. To obtain a measure of relative inhibition
of uptake, we assumed that the Km of BSA-bound
[3H]sn-2-18:1 was unchanged upon addition
of competitive ligands. The theoretical values, which were calculated
on the basis of competitive kinetics, assuming the apparent
Km equals the Ki
(40), are very similar to those obtained experimentally.
In this case, the calculated Ki values for
sn-1-18:1, sn-2-16:0, and 18:1 were similar, in the range of 0.2 µM. In addition, the calculated
Ki values for sn-2-18:1 and
sn-2-16:0 inhibition of [3H]18:1 uptake
were 0.16 ± 0.04 and 0.17 ± 0.02 µM, respectively (Table
3). The Ki values
obtained for these ligands suggest similar competitive kinetics for
LCFA and sn-2-18:1.
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Transcript and protein expression of candidate membrane transport
proteins.
We previously reported that Caco-2 cells express the 43-kDa plasma
membrane fatty acid binding protein (FABPpm), though preincubation with
anti-FABPpm antibodies did not inhibit LCFA uptake (51). At that time, FABPpm was the only putative transmembrane transporter known to be present in the intestine. It is now thought that several membrane transporters, which also may participate in LCFA uptake, may
coexist in Caco-2 cells. Therefore, blocking only one membrane transporter may not substantially inhibit LCFA uptake. FATP and FAT/CD36 are putative LCFA transporters that are expressed in various
tissues, including intestine (13, 31). In this study, we
determined the mRNA expression of FATP and FAT/CD36 in Caco-2 cells
using RT-PCR. The results show that Caco-2 cells do express the human
intestinal FATP (hsFATP4) (Fig.
7); however, no detectable FAT/CD36 was
found at either the mRNA or protein level (data not shown).
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DISCUSSION |
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The enterocytes of the small intestine are responsible for uptake of the hydrolysis products of dietary TG, FFA, and sn-2-MG. In the present studies, the kinetics of sn-2-MG as well as LCFA at the both AP and BL surfaces were evaluated using human intestinal Caco-2 cells as an enterocyte model. To ensure that all uptake studies were performed using monomeric lipid, we measured the CMC of sn-2-18:1, since it has not been previously reported (Fig. 1). The result obtained for sn-2-18:1 (4.2 ± 0.5 µM) is in the same range as that reported for 1-O-hexadecylglycerol (2.8 ± 0.3 µM) by Schulthess et al. (39) and those reported for 18:1 (6 µM) and 16:0 (4 µM) by Richieri et al. (36). The maximum unbound concentrations of lipids used in the uptake experiments were 1.53, 1.26, and 1.32 µM for sn-2-18:1, 18:1, and 16:0, respectively, all below their CMCs.
Uptake kinetics were evaluated, and the results demonstrate that uptake of MG and LCFA across both AP and BL plasma membranes of Caco-2 cells appears to be a saturable function of monomer lipid concentrations (Fig. 3). The apparent Km for uptake of BSA-bound sn-2-18:1 was ~0.3 µM at both surfaces and was of a range similar to the apparent Km values for LCFA uptake by Caco-2 cells (Table 1), as well as Km values reported for FFA uptake using other intestinal cell preparations (46, 51).
The lower Km for sn-2-18:1 than 18:1 at the AP surfaces, observed for both TC-mixed and BSA-bound lipids, suggests that the AP membrane has a slightly higher affinity for sn-2-18:1 compared with 18:1. On the other hand, the higher Vmax for 18:1 compared with sn-2-18:1 may reflect, at least in part, a lower capacity of Caco-2 cells for sn-2-18:1 uptake relative to 18:1. Caco-2 cells express high concentrations of L-FABP (52), which has a higher affinity for oleic acid relative to monoolein (18); thus it is possible that the intracellular L-FABP could play a role in this differential uptake.
It was demonstrated that the rate of LCFA uptake as a function of the unbound (monomer) concentration of LCFA displays saturable kinetics by using the historical Kd values for fatty acids bound to BSA reported by Spector et al. (44), suggesting a carrier-mediated mechanism for transmembrane movement (Fig. 3C). When the data were evaluated using the unbound LCFA concentrations calculated from the newer Kd values reported by Richieri et al. (34), a saturable component was observed only at unbound LCFA concentrations below 15 nM, and a diffusive component was seen when unbound LCFA concentrations were higher (Fig. 4). It is worth noting that normal levels of unbound LCFA in the bloodstream are ~7 nM (49). Levels of unbound FA in the complex and dynamic milieu of the intestinal lumen are not known, although they are likely to be widely variable based on food intake. These observations suggest that both carrier-mediated and passive diffusion mechanisms for LCFA uptake by Caco-2 cells coexist. Thus saturable uptake of LCFA and MG is supported by both treatments of the data, although the absolute values for kinetic parameters and the contribution of a diffusive component to net uptake are considerably different between the two. Stump et al. (47) have also recently documented dual pathways for fatty acid uptake in isolated rat adipocytes, and Chow and Hollander (8) came to a similar conclusion regarding linoleic uptake by everted sacs of rat jejunum. It appears, then, that coexistence of facilitated and diffusive transport mechanisms may be the norm. The protein-mediated pathways may serve to direct fatty acids and MG to specific intracellular targets when lipid levels are low, while the diffusive uptake may represent a largely unregulated "overflow" pathway that could nevertheless effect uptake of large quantities of lipid.
In agreement with others (22, 33, 51), TC-mixed LCFA uptake had dramatically increased Vmax values compared with those of BSA-bound LCFA. This difference was also evident for MG. We ensured that the differential AP vs. BL uptake capacities were not simply a result of micellar disruption of the AP plasma membrane during the TC incubations. Preincubation of Caco-2 monolayers with identical micellar solutions did not have any effect on subsequent uptake (data not shown), indicating that damage to the membrane during the micelle incubation did not occur. Hofmann (19) estimated that the product of the concentration gradient and the aqueous diffusion coefficient was 150 times higher for micellar aggregates compared with a monomer solution. Therefore, it may be reasonable to get similarly higher Vmax values for micellar TC-mixed LCFA uptake than for BSA-bound LCFA. At present, however, the precise role of TC in the uptake kinetics requires further exploration.
When the AP and BL uptake of LCFA from BSA solutions was compared, it was found that AP uptake had a greater Vmax than did BL uptake (Table 1). This could be related to differences in the plasma membrane composition between the AP and BL domains of the enterocytes (7). It has been shown that the AP membrane of the enterocyte has more intramembrane particles, higher protein/lipid and cholesterol/phospholipid ratios, and lower fluidity than the BL membrane. In addition, we previously showed that increased AP uptake of LCFA is not due to greater AP plasma membrane surface area in Caco-2 cells but, rather, is likely to reflect enterocyte-specific FFA transport and metabolism (52). In contrast, the AP and BL uptake of BSA-bound LCMG shows similar Vmax and Km values (Table 1). However, as we previously determined the ratio of the surface area of AP and BL membranes of Caco-2 to be 1:3 (52), this implies more effective uptake of MG at the AP surface, similar to LCFA.
The observation that AP uptake of TC-mixed 18:1 has a higher Vmax than TC-mixed 16:0, whereas BSA-bound 18:1 has a lower Vmax compared with BSA-bound 16:0, was very consistent over more than seven separate experiments (Table 1). Similarly, in 1968, Simmonds et al. (41) showed that 18:1 in TC solutions has a higher absorption rate than 16:0, using the intestinal perfusion technique in rats. In addition, Ockner et al. (30) showed that 16:0 in TC micelles has a lower rate of absorption compared with linoleic acid. This finding was ascribed to the slower esterification rate of 16:0 in the rat intestine (30). The present studies were performed at 10-s time periods, and we found that >90% of radioactive LCFA remained unesterified in Caco-2 cells at this time interval (Ref. 51 and data not shown). Therefore, the lower Vmax value obtained for TC-mixed 16:0 was not likely related to a slower esterification rate. It is possible that a somewhat lower Kd of L-FABP for 18:1 than that for 16:0 (37) may result in binding or transferring more 18:1 relative to 16:0 away from the plasma membranes by L-FABP.
As proposed by Sorrentino et al. (43), the monomer concentration of LCFA in BSA-bound LCFA solution is the major factor that determines the uptake velocity. In such a case, the solubility of lipid in the aqueous phase may play an important role. On the basis of the free energy of transfer from hydrocarbon to water, sn-2-18:1 has the highest aqueous solubility, followed by 18:1 and then 16:0 (42). Therefore, the adsorption of lipids to the plasma membranes is presumably highest for 16:0, followed by 18:1 and then sn-2-18:1. Indeed, studies have shown that rabbit BBM bound more 16:0 than 18:1 (32), suggesting a higher membrane solubility for 16:0 relative to 18:1. Thus the higher Vmax values obtained for BSA-bound 16:0 relative to 18:1, at both the AP and BL surfaces of the Caco-2 cells, may reflect the intrinsic binding of monomeric lipids by the membrane.
In contrast to BSA-bound lipids, it has been proposed that TC-mixed lipids may be taken up through a collisional transfer mechanism (48). In such a mechanism, the rate and number of effective collisions between micelles and the plasma membrane, as well as the rate of ligand dissociation from the micelle into the membranes, must be taken into consideration. If the size of the micelle is smaller, the micelle numbers would be greater at a fixed concentration of LCFA and TC. This would result in more effective collisions between micelles and the plasma membrane. Therefore, the Vmax would, presumably, be higher. In addition, the Vmax could also be higher if the rate of ligand dissociation from the micelle to the membrane were greater. Studies of fluorescent FFA transfer in micelles indicate that unsaturated FFA dissociation occurs more rapidly than saturated FFA dissociation and that collisional transfer of lipid occurs at micelle concentrations similar to those used in the present studies (45). Thus a higher dissociation rate for 18:1 may contribute to the observed higher Vmax in TC uptake studies, compared with that of 16:0. In addition, a collision-based transfer mechanism could account, in part, for the higher uptake rates observed with TC micelles compared with BSA-bound lipids and, perhaps, for the different apparent Km values for TC-mixed lipids relative to BSA-bound lipids.
There are at least three membrane proteins that have been proposed to participate in LCFA transport in the small intestine: FABPpm (46), FAT/CD36 (1), and FATP (38). Previously, we demonstrated the expression of FABPpm in Caco-2 cells (51); here we studied additional intestinal membrane protein(s) that may also contribute to total LCFA uptake. We report, for the first time, that Caco-2 cells do express the transcript of FATP (Fig. 7). In contrast, mRNA as well as protein expression of FAT/CD36 was not detectable in Caco-2 cells (data not shown). Thus both FABPpm and FATP in Caco-2 cells may contribute to the saturable component of MG and FFA uptake observed in this study. Trypsin preincubation inhibited the uptake of sn-2-18:1 at both the AP and BL membranes by up to 40% (Table 2), further supporting the hypothesis that membrane protein(s) may be involved in facilitating MG and FFA uptake in Caco-2. In addition, the competition studies suggest that sn-2-18:1 and 18:1 could compete through the same putative carrier(s) in Caco-2 cells. Indeed, Stremmel (46) has shown that both FFA and MG interact with FABPpm. No such data are presently available for FATP.
In the present studies, we examined the kinetics and mechanism of sn-2-monoolein uptake at both the AP and BL plasma membrane of Caco-2 cells, using substrate incubations that resemble normal physiological conditions. The kinetic data obtained are consistent with the presence of saturable uptake mechanisms at both the AP and BL plasma membranes. The observation of competitive sn-2-monoolein uptake by LCFA and by LCMG suggests that the same membrane protein(s) may be involved in the absorption of these ligands.
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ACKNOWLEDGEMENTS |
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We thank Dr. Alfred Thumser for helpful discussions.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38389 (J. Storch) and by state funds.
Present address of S.-Y. Ho: Dept. of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson Univ., Philadelphia, PA 19107.
Address for reprint requests and other correspondence: J. Storch, Dept. of Nutritional Sciences, Rutgers Univ., New Brunswick, NJ 08901-8525 (E-mail: storch{at}aesop.rutgers.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 10 January 2001; accepted in final form 15 May 2001.
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REFERENCES |
---|
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---|
1.
Abumrad, NA,
el-Maghrabi MR,
Amri EZ,
Lopez E,
and
Grimaldi PA.
Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation. Homology with CD36.
J Biol Chem
268:
17665-17668,
1993
2.
Abumrad, NA,
Harmon CM,
and
Ibrahimi A.
Membrane transport of long-chain fatty acids: evidence for a facilitated process.
J Lipid Res
39:
2309-2318,
1998
3.
Berk, PD,
and
Stump DD.
Mechanisms of cellular uptake of long chain free fatty acids.
Mol Cell Biochem
192:
17-31,
1999[ISI][Medline].
4.
Borgström, B.
Influence of bile salt, pH, and time on the action of pancreatic lipase; physiological implications.
J Lipid Res
5:
522-531,
1964
5.
Boyle, E,
and
German JB.
Monoglycerides in membrane systems.
Crit Rev Food Sci Nutr
36:
785-805,
1996[ISI][Medline].
6.
Bradford, M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
7.
Brasitus, TA,
and
Schachter D.
Lipid dynamics and lipid-protein interactions in rat enterocyte basolateral and microvillus membranes.
Biochemistry
19:
2763-2769,
1980[ISI][Medline].
8.
Chow, SL,
and
Hollander D.
A dual, concentration-dependent absorption of linoleic acid by rat jejunum in vitro.
J Lipid Res
20:
349-356,
1979[Abstract].
9.
Dakka, T,
Dumoulin V,
Chayvialle JA,
and
Cuber JC.
Luminal bile salts and neurotensin release in the isolated vascularly perfused rat jejuno-ileum.
Endocrinology
134:
603-607,
1994[Abstract].
10.
Darimont, C,
Gradoux N,
Cumin F,
Baum HP,
and
De Pover A.
Differential regulation of intestinal and liver fatty acid-binding proteins in human intestinal cell line (Caco-2): role of collagen.
Exp Cell Res
244:
441-447,
1998[ISI][Medline].
11.
El-Maghrabi, MR,
Waite M,
and
Rudel LL.
Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.
Biochem Biophys Res Commun
81:
82-88,
1978[ISI][Medline].
12.
Fielding, BA,
Humphreys SM,
Allman RF,
and
Frayn KN.
Mono-, di- and triacylglycerol concentrations in human plasma: effects of heparin injection and of a high-fat meal.
Clin Chim Acta
216:
167-173,
1993[ISI][Medline].
13.
Fitscher, BA,
Riedel HD,
Young KC,
and
Stremmel W.
Tissue distribution and cDNA cloning of a human fatty acid transport protein (hsFATP4).
Biochim Biophys Acta
1443:
381-385,
1998[ISI][Medline].
14.
Fogh, J,
Fogh JM,
and
Orfeo T.
One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.
J Natl Cancer Inst
59:
221-226,
1977[ISI][Medline].
15.
Gangl, A,
and
Ockner RK.
Intestinal metabolism of plasma free fatty acids. Intracellular compartmentation and mechanisms of control.
J Clin Invest
55:
803-813,
1975[ISI][Medline].
16.
Grasset, E,
Pinto M,
Dussaulx E,
Zweibaum A,
and
Desjeux JF.
Epithelial properties of human colonic carcinoma cell line Caco-2: electrical parameters.
Am J Physiol Cell Physiol
247:
C260-C267,
1984[Abstract].
17.
Hernell, O,
Staggers JE,
and
Carey MC.
Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 2. Phase analysis and aggregation states of luminal lipids during duodenal fat digestion in healthy adult human beings.
Biochemistry
29:
2041-2056,
1990[ISI][Medline].
18.
Ho, SY,
and
Storch J.
Monoacylglycerol transfer from rat liver fatty acid-binding protein to phospholipid vesicles.
FASEB J
8:
A731,
1994[ISI].
19.
Hofmann, AF.
Fat digestion: the interaction of lipid digestion products with micellar bile salt solutions.
In: Lipid Absorption: Biochemical and Clinical Aspects, edited by Rommel K,
and Goebell H.. Lancaster, UK: MTP, 1976, p. 3-21.
20.
Hughes, TE,
Ordovas JM,
and
Schaefer EJ.
Regulation of intestinal apolipoprotein B synthesis and secretion by Caco-2 cells. Lack of fatty acid effects and control by intracellular calcium ion.
J Biol Chem
263:
3425-3431,
1988
21.
Kamp, F,
Zakim D,
Zhang F,
Noy N,
and
Hamilton JA.
Fatty acid flip-flop in phospholipid bilayers is extremely fast.
Biochemistry
34:
11928-11937,
1995[ISI][Medline].
22.
Levin, MS,
Talkad VD,
Gordon JI,
and
Stenson WF.
Trafficking of exogenous fatty acids within Caco-2 cells.
J Lipid Res
33:
9-19,
1992[Abstract].
23.
Levy, E,
Mehran M,
and
Seidman E.
Caco-2 cells as a model for intestinal lipoprotein synthesis and secretion.
FASEB J
9:
626-635,
1995
24.
Luchoomun, J,
and
Hussain MM.
Assembly and secretion of chylomicrons by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly.
J Biol Chem
274:
19565-19572,
1999
25.
Luchoomun, J,
Zhou Z,
Bakillah A,
Jamil H,
and
Hussain MM.
Assembly and secretion of VLDL in nondifferentiated Caco-2 cells stably transfected with human recombinant ApoB48 cDNA.
Arterioscler Thromb Vasc Biol
17:
2955-2963,
1997
26.
Matsudaira, P.
Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes.
J Biol Chem
262:
10035-10038,
1987
27.
Mattson, FH,
and
Volpenheim RA.
The use of pancreatic lipase for determining the distribution of fatty acids in partial and complete glycerides.
J Lipid Res
2:
58-62,
1961
28.
Mattson, FH,
and
Volpenheim RA.
Synthesis and properties of glycerides.
J Lipid Res
3:
281-296,
1962
29.
Neame, KD,
and
Richards TG.
Elementary Kinetics of Membrane Carrier Transport. Oxford, UK: Blackwell Scientific, 1972.
30.
Ockner, RK,
Pittman JP,
and
Yager JL.
Differences in the intestinal absorption of saturated and unsaturated long chain fatty acids.
Gastroenterology
62:
981-992,
1972[ISI][Medline].
31.
Poirier, H,
Degrace P,
Niot I,
Bernard A,
and
Besnard P.
Localization and regulation of the putative membrane fatty-acid transporter (FAT) in the small intestine. Comparison with fatty acid- binding proteins (FABP).
Eur J Biochem
238:
368-373,
1996[Abstract].
32.
Proulx, P,
Aubry H,
Brglez I,
and
Williamson DG.
Studies on the uptake of fatty acids by brush border membranes of the rabbit intestine.
Can J Biochem Cell Biol
63:
249-256,
1985[ISI][Medline].
33.
Ranheim, T,
Gedde-Dahl A,
Rustan AC,
and
Drevon CA.
Fatty acid uptake and metabolism in Caco-2 cells: eicosapentaenoic acid (20:5(n-3)) and oleic acid (18:1(n-9)) presented in association with micelles or albumin.
Biochim Biophys Acta
1212:
295-304,
1994[ISI][Medline].
34.
Richieri, GV,
Anel A,
and
Kleinfeld AM.
Interactions of long-chain fatty acids and albumin: determination of free fatty acid levels using the fluorescent probe ADIFAB.
Biochemistry
32:
7574-7580,
1993[ISI][Medline].
35.
Richieri, GV,
and
Kleinfeld AM.
Unbound free fatty acid levels in human serum.
J Lipid Res
36:
229-240,
1995[Abstract].
36.
Richieri, GV,
Ogata RT,
and
Kleinfeld AM.
A fluorescently labeled intestinal fatty acid binding protein. Interactions with fatty acids and its use in monitoring free fatty acids.
J Biol Chem
267:
23495-23501,
1992
37.
Richieri, GV,
Ogata RT,
and
Kleinfeld AM.
Equilibrium constants for the binding of fatty acids with fatty acid binding proteins from adipocyte, intestine, heart, and liver measured with the fluorescent probe ADIFAB.
J Biol Chem
269:
23918-23930,
1994
38.
Schaffer, JE,
and
Lodish HF.
Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein.
Cell
79:
427-436,
1994[ISI][Medline].
39.
Schulthess, G,
Lipka G,
Compassi S,
Boffelli D,
Weber FE,
Paltauf F,
and
Hauser H.
Absorption of monoacylglycerols by small intestinal brush border membrane.
Biochemistry
33:
4500-4508,
1994[ISI][Medline].
40.
Segal, IH.
Simple inhibition systems.
In: Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. New York: Wiley, 1975, p. 100-125.
41.
Simmonds, WJ,
Redgrave TG,
and
Willix RL.
Absorption of oleic and palmitic acids from emulsions and micellar solutions.
J Clin Invest
47:
1015-1025,
1968[ISI][Medline].
42.
Small, DM.
Substituted aliphatic hydrocarbons: alcohols and acids.
In: Handbook of Lipid Research. The Physical Chemistry of Lipids: From Alkanes to Phospholipids. New York: Plenum, 1986, vol. 4, p. 233-284.
43.
Sorrentino, D,
Robinson RB,
Kiang CL,
and
Berk PD.
At physiologic albumin/oleate concentrations oleate uptake by isolated hepatocytes, cardiac myocytes, and adipocytes is a saturable function of the unbound oleate concentration. Uptake kinetics are consistent with the conventional theory.
J Clin Invest
84:
1325-1333,
1989[ISI][Medline].
44.
Spector, AA,
Fletcher JE,
and
Ashbrook JD.
Analysis of long chain free fatty acid binding to bovine serum albumin by determination of stepwise equilibrium constants.
Biochemistry
10:
3229-3232,
1971[ISI][Medline].
45.
Storch, J,
and
Kleinfeld AM.
Transfer of long-chain fluorescent free fatty acids between unilamellar vesicles.
Biochemistry
25:
1717-1726,
1986[ISI][Medline].
46.
Stremmel, W.
Uptake of fatty acids by jejunal mucosal cells is mediated by a fatty acid binding membrane protein.
J Clin Invest
82:
2001-2010,
1988[ISI][Medline].
47.
Stump, DD,
Fan X,
and
Berk PD.
Oleic acid uptake and binding by rat adipocytes define dual pathways for cellular fatty acid uptake.
J Lipid Res
42:
509-520,
2001
48.
Thomson, ABR,
Schoeller C,
Keelan M,
Smith LC,
and
Clandinin MT.
Lipid absorption: passing through the unstirred layers, brush- border membrane, and beyond.
Can J Physiol Pharmacol
71:
531-555,
1993[ISI][Medline].
49.
Thumser, AEA,
Buckland AG,
and
Wilton DC.
Monoacylglycerol binding to human serum albumin: evidence that monooleoylglycerol binds at the dansylsarcosine site.
J Lipid Res
39:
1033-1038,
1998
50.
Tranchant, T,
Besson P,
Honard C,
Delane J,
Antoine JM,
Couet C,
and
Gore J.
Mechanisms and kinetics of alpha linolenic acid uptake in Caco-2 clone TC7.
Biochim Biophys Acta
1345:
151-161,
1997[ISI][Medline].
51.
Trotter, PJ,
Ho SY,
and
Storch J.
Fatty acid uptake by Caco-2 human intestinal cells.
J Lipid Res
37:
336-346,
1996[Abstract].
52.
Trotter, PJ,
and
Storch J.
Fatty acid uptake and metabolism in a human intestinal cell line (Caco-2): comparison of apical and basolateral incubation.
J Lipid Res
32:
293-304,
1991[Abstract].
53.
Trotter, PJ,
and
Storch J.
Fatty acid esterification during differentiation of the human intestinal cell line Caco-2.
J Biol Chem
268:
10017-10023,
1993
54.
Tso, P,
and
Crissinger K.
Digestion and absorption of lipids.
In: Biochemical and Physiological Aspects of Human Nutrition, edited by Stipanuk MH.. Philadelphia, PA: Saunders, 2000, p. 125-141.
55.
Wyler, B,
Daviet L,
Bortkiewicz H,
Bordet JC,
and
McGregor JL.
Cloning of the cDNA encoding human platelet CD36: comparison to PCR amplified fragments of monocyte, endothelial and HEL cells.
Thromb Haemost
70:
500-505,
1993[ISI][Medline].