1 Division of Gastroenterology and Nutrition, Centre de Recherche, Hôpital Sainte-Justine, and Departments of 2 Pediatrics and 4 Nutrition, University of Montreal, Montreal, Quebec H3T 1C5; and 3 Department of Anatomy and Cell Biology, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
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ABSTRACT |
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Structured lipids have been proposed as efficient vehicles for the supplementation of essential fatty acids (EFA) to patients with malabsorption. We investigated how a novel structured triglyceride (STG), containing purely octanoic acid in the sn-1/sn-3 and [14C]linoleic acid in the sn-2 positions, was incorporated into different lipid classes in Caco-2 cells. We also evaluated the contribution of gastric lipase in the uptake and metabolism of [14C]linoleic acid from the STG. We furthermore determined the potential of the STG to correct EFA deficiency induced in Caco-2 cells. The absorption of STG by Caco-2 cells was significantly greater compared with that of triolein. The addition of human gastric lipase significantly enhanced cellular uptake of the labeled substrate, reflecting the stereoselectivity of gastric lipase to hydrolyze medium chain FA. Analysis of the intracellular lipids synthesized revealed a predominance of phospholipids-monoglycerides. Most of the radioactivity in the lipoproteins isolated from Caco-2 cells was recovered in TG-rich lipoproteins (45%) and to a lesser extent in the high-density lipoprotein (36%) and low-density lipoprotein (17%) fractions. The administration of STG to Caco-2 cells rendered EFA deficient produced a marked increase of the cellular level of linoleic and arachidonic acids. This resulted in a lower ratio of 20:3(n-9) to 20:4(n-6), reflecting the correction of EFA deficiency in Caco-2 cells. Our data demonstrate that STG, in the presence of gastric lipase, have beneficial effects on lipid incorporation, lipoprotein production, and EFA status, utilizing Caco-2 cells as a model of EFA deficiency.
medium-chain fatty acid; intestinal malabsorption; gastric lipase
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INTRODUCTION |
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TRIGLYCERIDES (TG), the major source of dietary lipids, account for 40% of the energy provided by the typical Western diet (24). The digestion and absorption of dietary TG require a series of highly complicated processes. Lipolysis is initiated in the stomach by gastric lipase. It is active in the prepyloric region at an acidic pH, then continues the lipolytic process in the small intestine (27). Gastric lipase hydrolyzes medium-chain TG (MCT) more rapidly than long-chain TG (2, 27). The majority of TG hydrolysis occurs in the upper small intestine, under the concerted actions of biliary and pancreatic secretions (see Refs. 5, 41, and 42 for review). Lipolysis by pancreatic lipase and micellar dispersion of lipolytic products by bile acids assure their shuttle from the bulk water phase across the unstirred water layer to the region adjacent to the cell surface (45). In the enterocyte, long-chain fatty acids (FA) are reesterified into TG, incorporated into chylomicrons, and subsequently exported via the lymphatics to reach the circulation (24, 42).
Pathophysiological abnormalities in fat malabsorption can lead to inefficient intestinal transport of linoleic [18:2(n-6)] and linolenic [18:3(n-3)] acids (23), both of which are essential FA (EFA). EFA deficiency is very often associated with disorders of malabsorption, as exemplified by cystic fibrosis (23, 28), cholestasis (3), and prematurity (39). We and others (26) have shown that EFA deficiency further impairs both the intraluminal and intracellular phases of fat absorption.
Several strategies have been applied in an effort to correct EFA deficiency and improve energy intake in patients with malabsorption (44). Linoleic acid-enriched supplements can correct EFA deficiency and improve malnutrition. However, supplementation of long-chain FA to diets is limited by problems of compliance, palatability, and stability. Furthermore, their rapid metabolism to meet immediate energy needs limits the efficiency of this approach (33). Another approach has been to use MCT in the form of octanoic and decanoic FA, enhancing uptake of calories in the face of pancreatic insufficiency or other malabsorptive syndromes (15). However, despite their provision of energy, MCT contain no EFA. Thus EFA deficiency often persists in these disorders despite a dietary regimen that includes supplemental MCT (14).
Structured lipids, potentially efficient vehicles for the supplementation of EFA, have been proposed as an alternative approach (17, 33). Randomly esterified synthetic TG offer the advantage of providing medium-chain FA as a source of immediate energy through oxidative metabolism, while simultaneously providing EFA for repletion and maintenance of tissue stores (17). Preliminary studies suggested that such structured TG (STG), a mixture of long- and medium-chain FA incorporated on the same glycerol backbone, may offer several advantages. These include superior nitrogen retention (36), preservation of reticuloendothelial system function (37), attenuation of protein catabolism, and the hypermetabolic stress response to thermal injury (40). However, a recent study stressed that STG containing octanoic acid in the 1 and 3 positions and linoleic acid in the 2 position may not be advantageous when used as a sole source of dietary lipid (43).
The aims of this study were 1) to examine the cellular uptake and metabolism of a novel, purified STG by intestinal epithelial cells, in the absence of exogenous lipase, 2) to evaluate the contribution of gastric lipase in augmenting the incorporation of sn-2 EFA, and 3) to determine the potential beneficial effects of our STG in correcting experimentally induced EFA deficiency in vitro.
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MATERIALS AND METHODS |
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Cell cultures. Caco-2 cells (American Type Culture Collection, Rockville, MD) were cultured as previously reported (34). Briefly, they were grown at 37°C with 5% CO2 in complete medium consisting of DMEM (GIBCO BRL, Grand Island, NY) containing 1% penicillin/streptomycin, 1% DMEM nonessential amino acids (GIBCO BRL), and 10% decomplemented FCS (Flow Laboratories, McLean, VA). Caco-2 cells (between passages 30 and 40) were maintained in 175-cm2 flasks (Corning Glass Works, Corning, NY) in a 95% air-5% CO2 atmosphere at 37°C. Cell cultures were split (generally 1:6) when they reached 70-90% confluence, using 0.05% trypsin (0.5 nM) in EDTA (GIBCO BRL). For individual experiments, cells were plated (1 × 106 cells/well) on 24.5-mm polycarbonate Transwell filter inserts with 0.4-µm pores (Costar, Cambridge, MA) in complete medium (as described above), supplemented with 5% FCS. The inserts fit into six-well culture plates, allowing separate access to the apical and basolateral surfaces of the cell monolayers. The cells were used for experiments 18-24 days after plating, as previously described (34). The medium was refreshed every second day. Transepithelial resistance, an index of cell confluence and tight junction formation, was confirmed using a Millicel-ERS apparatus (Millipore, Bedford, MA). Data are expressed in ohms per square centimeter (±SE) (34).
Synthesis of STG substrate. The labeled STG, 1,3-dioctanoyl-2-[1-14C]linoleoylglycerol, was synthesized using the method described by Awl et al. (2), with minor modifications to optimize the yield of tracer quantities of material. All chemicals were purchased from Sigma Chemical (St. Louis, MO), except when otherwise indicated. Briefly, 1,3-dioctanoyl-2-one was prepared from octanoic acid and dihydroxyacetone in the presence of 1,1-dicyclohexylcarbodiimide and 4-dimethylaminopyridine. The purified product was reduced to 1,3-diacylglycerol with NaBH4, which was then reacted with carrier-free [1-14C]linoleic acid (DuPont, Mississauga, Ontario, Canada) in the presence of 1,1-dicyclohexylcarbodiimide and 4-dimethylaminopyridine. The tracer TG was isolated and purified by a combination of silicic acid chromatography and TLC. Gram quantities of cold STG were prepared by sn-1,3-specific lipase-catalyzed interesterification of food grade safflower oil, containing 70-80% esterified linoleic acid (HCN, Cleveland, OH) in the presence of excess octanoic acid. Briefly, safflower oil and octanoic acid (3.4:1, wt/wt) were dissolved in hexane containing 1M60 lipase immobilized on beads (a gift from Novo Nordisk, Mississauga, Ontario, Canada). After 24 h at 55°C, the STG was isolated and purified by Florisil column chromatography using a step gradient of diethyl ether in hexane. A purity of 96% of the synthesized STG was determined by gas chromatography.
The incubation substrate consisted of trace radiolabeled [14C]STG (40 mCi/mmol) or [14C]triolein (61 mCi/mmol; Amersham, Oakville, ON, Canada) as a 5% gum arabic stabilized emulsion. A stock solution containing either 1.23 mg STG/ml (unlabeled) and 8.6 µCi/ml [14C]STG (56 µCi/ml) or 3.4 mg triolein/ml (unlabeled) and 6.76 µCi/ml [14C]triolein was dried under a stream of nitrogen and then sonicated for 10 s/ml in 7 ml of gum arabic to obtain an emulsion. The final culture medium contained 0.5 µmol [14C]STG or [14C]triolein/ml (1.3 µCi/well) in DMEM with 1% nonessential amino acids, in a final volume of 1.5 ml/insert. When different concentrations were employed, the amount of unlabeled and radiolabeled substrate was proportionally increased up to fourfold.Gastric lipase.
Human gastric lipase was obtained from fetuses of 10- to 18-wk
gestation, after legal abortion. The lipase was isolated from the fetal
gastric corpus as described by Ménard et al. (35) and stored at
70°C until used. For individual experiments, 1.7 IU/ml of
purified human fetal gastric lipase were added to the gum
arabic-stabilized substrate emulsion at a pH of 7.4.
Measurement of Caco-2 cell uptake and secretion of radiolabeled STG vs. triolein. At 21 days postconfluence, Caco-2 cell monolayers were washed twice with DMEM (serum free), and the final substrate (STG or triolein, as described above) was added to the upper compartment. Cells were incubated for varying periods (2, 5, 6, 24, or 48 h) at different trace-radiolabeled substrate concentrations (0.5, 1.0, 1.5, or 2.0 µmol [14C]STG or triolein). At the end of each incubation period, cells were washed twice with nonsupplemented DMEM, scraped off with a rubber policeman in 1 ml PBS (pH 7.4), and sonicated for 30 s. Aliquots were removed and solubilized in a scintillation vial with Ready Safe (Beckman Instruments, Fullerton, CA). Total radioactivity was measured by scintillation counting (Beckman no. LS5000 TD, ON, Canada). Cell protein was quantified by the method of Lowry et al. (32), and results were expressed as dpm per milligram of cell protein. Other aliquots were taken for lipid extraction after the addition of unlabeled carrier phospholipids (PL), monoglycerides (MG), diglycerides, TG, free FA (FFA), and cholesterol esters (CE), using the standard method of Folch et al. (10). The various lipid classes synthesized from [14C]STG or triolein were separated by TLC, using a solvent mixture of hexane, diethyl ether, and acetic acid (80:20:3, vol/vol/vol) (29). The area corresponding to each lipid was then scratched off the TLC plates, and the silica powder was placed in a scintillation vial for counting. Lipids secreted into the basolateral compartment were analyzed and quantified as described above, after centrifugation (2,000 rpm for 30 min at 4°C) to remove cell debris. Results were expressed as dpm per milligram of cellular protein per hour.
Isolation of lipoproteins. Experiments were designed to determine the esterified FA content of synthesized and secreted lipoproteins derived from the STG substrate. Caco-2 cells were incubated with the [14C]STG substrate as mentioned above, to detect an appreciable amount of lipoproteins intracellularly as well as those exported into the basolateral medium. After the incubation period (18 h), the cells and their basolateral medium were collected and supplemented with antiproteases (phenylmethylsulfonyl fluoride, pepstatin, EDTA, aminocaproic acid, chloramphenicol, leupeptin, glutathione, benzamidine, dithiothreitol, sodium azide, and Trasylol), all at a final concentration of 1 mM. A lipid carrier was added (2:0.3, vol/vol), prepared from plasma obtained 1.5 h after intake of a fatty meal and incubated at 56°C (30 min) to inactivate enzymatic activity (29). The lipoprotein fractions were isolated by discontinuous density-gradient ultracentrifugation as previously described (26, 28), using a Beckman L5-65 preparative ultracentrifuge with a Ti-50 rotor (Montreal, PQ, Canada) (31). Briefly, chylomicrons, very low density lipoprotein (VLDL) (1.006 g/ml), and low-density lipoprotein (LDL) (1.063 g/ml) were separated by spinning (133,000 g) for 18 h at 4°C. The high-density lipoprotein (HDL) fraction was obtained by adjusting the LDL infranatant to a density of 1.21 g/ml and centrifuging for 48 h at 133,000 g. Each lipoprotein fraction was exhaustively dialyzed against 0.15 M NaCl and 0.001 M EDTA (pH 7.0 at 4°C). The resulting fractions were placed in a scintillation vial with Ready Safe, and radioactivity was measured by scintillation counting. Results were expressed as a percentage of the total activity found intracellularly or in the basolateral compartment.
Studies using EFA-deficient Caco-2 cells. To establish EFA-deficient Caco-2 cells, we passaged parental cells as previously described (34), using only delipidated FCS. The latter was prepared from the same dialyzed serum used for the control cells, using a procedure that eliminated >98% of total FFA (4). Cultures were split 1:6 when they reached 70-90% confluency. After two passages, they were plated at 1 × 106 cells/well on a 24.5-mm polycarbonate Transwell filter, as described previously (34). Complete medium containing 5% delipidated FCS was maintained and changed every second day.
Assessment of cellular FA was performed by gas-liquid chromatography analysis as previously described (23) after various periods to determine Caco-2 cell EFA status. In an attempt to correct EFA deficiency, we incubated the cells at day 35 postconfluency with either STG or triolein, at a concentration of 2.0 µmol/ml, in the presence of gastric lipase (as described above). Control wells consisted of normal Caco-2 cells in 5% FCS with gastric lipase. The medium was changed every second day. At the end of the incubation period, the cells were washed and scraped off in PBS on ice. The cells were centrifuged, and the pellets were stored atStatistical analysis. All results are expressed as means ± SE. Statistical analysis of data was performed by ANOVA and a Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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Uptake and metabolism of STG by Caco-2 cells.
Caco-2 cells were utilized at 20-25 days postconfluency. Adequate
functional differentiation was confirmed by the determination of
sucrase activity (12.3 ± 1.4 IU/g) and the measurement of
transepithelial resistance (2,270 ± 163 · cm2), an
index of confluence and tight junction formation (34). Very limited
uptake of labeled STG or triolein was observed in Caco-2 cells,
indicating the presence of low endogenous lipolytic activity (Fig.
1). The addition of gastric lipase to the
medium substantially enhanced cellular uptake of the radioactive
substrates. The uptake of
[14C]STG was
significantly greater than that of
[14C]triolein (Fig.
1), reflecting the stereoselectivity of gastric lipase to hydrolyze
medium-chain FA in the sn-1/3
positions. An increase in intracellular
[14C]STG uptake was
found with prolonged durations of incubation (Fig.
2A) and
higher substrate concentrations (Fig.
2B). Secretion of
radiolabeled lipids from STG into the basolateral compartment corresponded to ~2.5% of the cellular uptake. Analysis of the intracellular lipids synthesized revealed a predominance of the PL-MG
fraction when Caco-2 cells were incubated with STG as substrate, whereas the TG fraction predominated when the cells were incubated with
triolein (Table 1).
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Lipoprotein synthesis and secretion. The amount of radioactive lipids recovered from the uptake of [14C]STG was assessed in the different lipoprotein classes separated by ultracentrifugation. As depicted in Fig. 3, most of the radioactivity in the lipoproteins isolated from the Caco-2 cells was recovered in the TG-rich lipoproteins (chylomicrons/VLDL, 45%) and to a lesser extent in the HDL (36%) and LDL (17%) fractions. The total radioactivity measured in the lipoproteins from the basolateral compartment corresponded to only 2.5 ± 0.5% of the total counts found intracellularly. The pattern of labeling in the lipoprotein fractions secreted into the basolateral compartment also showed a predominance of CM plus VLDL (43%) as observed intracellularly. However, this was accompanied by a higher proportion of LDL (36%) and a lower percentage of HDL (21%).
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EFA-deficient Caco-2 cells. To establish EFA-deficient Caco-2 cells, we maintained the cultures in a medium containing delipidated FCS rather than complete FCS. After various periods of culture in these conditions, the cells were harvested, and FA analysis was performed by gas chromatography. The FA composition of normal and EFA-deficient Caco-2 cells is shown in Table 2. A significant decrease of all n-6 and n-3 EFA was noted. A compensatory increase in the percentage of nonessential n-7 and n-9 FA, including eicosatrienoic acid [20:3(n-9)] was seen. In control Caco-2 cells, the ratio of 20:3(n-9) to 20:4(n-6) was <0.1, whereas the cells cultured in delipidated medium had a ratio of >2.5, consistent with their EFA-deficient status (Fig. 4). Additional experiments were carried out to determine whether STG could correct the EFA deficiency observed in Caco-2 cells. After incubation for 60 h with STG, an increase in the linoleic acid content in the cells was noted (data not shown), without complete correction of the ratio of 20:3(n-9) to 20:4(n-6) remaining over 0.1 (0.49 ± 0.1). However, 10 days of culture with STG added to the apical compartment resulted in a decrease in the ratio back to the normal range, along with a marked increase of Caco-2 cell content of essential polyunsaturated FA (PUFA) (Table 2, Fig. 4). The decrease in the ratio of 20:3(n-9) to 20:4(n-6) was due to a significant increase in arachidonic acid [20:4(n-6), from 0.35 to 2.21%], accompanied by a decrease in 20:3(n-9) (from 0.84 to 0.12%). This reversed the process of compensatory increase of non-EFA in EFA-deficient cells. Because the ratio of n7 to 18:2(n-6) families has proven to be a sensitive discriminant for EFA deficiency, it was calculated and confirmed the pattern of the ratio of 20:3(n-9) to 20:4(n-6). When Caco-2 cells were incubated with triolein for the same time period, no correction of the EFA-deficient status was achieved, and the ratio of 20:3(n-9) to 20:4(n-6) remained persistently high (Table 2, Fig. 4).
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DISCUSSION |
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Recent studies (14, 15, 17, 33, 36, 37, 40) have examined the metabolic benefits of STG in animal models of trauma, burn injury, and endotoxic shock. Randomly esterified TG of medium- and long-chain FA have shown some utility when administered via parenteral or oral routes (17, 19, 33). In the present study, we examined the effect of a purified STG on the bioavailability and incorporation of its constitutive sn-2 EFA into intestinal epithelial cells. Our results clearly demonstrate that incubation of Caco-2 cells with the STG in the presence of gastric lipase led to its uptake and the subsequent utilization of the lipolytic products. This was reflected by enhanced FA incorporation into the major lipid classes (TG, PL, CE), as well as into various lipoprotein fractions (CM + VLDL, LDL, HDL). Furthermore, data generated in this study show that this STG was able to correct EFA deficiency experimentally induced in Caco-2 cells.
We employed Caco-2 cells to study the uptake and metabolism of STG because of their capacity to undergo spontaneous differentiation in vitro, representing a useful model of intestinal epithelial cell lipoprotein metabolism (30). Our data indicated a limited lipid secretory capacity, consistent with the persistent discrepancy between lipid formation and export processes that have been described in other studies using Caco-2 cells (30). One of our research objectives was to establish EFA-deficient Caco-2 cells as a model of cellular EFA deficiency in the context of pancreatic exocrine insufficiency, as encountered in cystic fibrosis. To our knowledge, an EFA-deficient enterocyte model has not been previously reported in the literature, although other EFA-deficient lines have been described (11, 18). We have demonstrated that this in vitro model is suitable to explore metabolic studies related to EFA deficiency, including its role in intestinal lipoprotein synthesis in the presence of exogenous gastric lipase alone, simulating pancreatic insufficiency. Despite its limitation in lipid secretion, this model can be used to examine how EFA deficiency itself impairs fat absorption, in addition to the reciprocal, established relationship showing that lipid malabsorption is a leading cause of EFA deficiency.
Normally, dietary TG require hydrolysis of the FA in positions 1 and 3 before the uptake of lipolytic products (FA and MG) by the intestine. The administration of our purified STG, 2-[14C]linoleoyl-1,3-dioctanoylglycerol, resulted in limited FA uptake. This was consistent with our recent demonstration that Caco-2 cells have some endogenous lipolytic activity. A small proportion of this enzymatic activity was found in the incubation medium, probably reflecting some secretion (38).
Gastric and pancreatic lipases participate in the digestion of TG in humans (24). Under normal circumstances, pancreatic lipase is predominant, with gastric lipase contributing ~17% of TG acyl chain hydrolysis (6). The relative deficiency of pancreatic lipase in the preterm infant or neonate, due to immature pancreatic function, is compensated for in part by increased gastric lipase activity (12, 22). In patients with severe exocrine pancreatic insufficiency such as cystic fibrosis, prepyloric lipase accounts for >90% of lipolytic activity in the upper small intestine (8). The Caco-2 cell model, displaying little endogenous lipolytic activity, thus simulates the gastrointestinal tract of subjects with intestinal malabsorption due to pancreatic insufficiency. The addition of gastric lipase to the incubation medium significantly enhanced the cellular uptake of the [14C]STG. This efficient process can be explained by the specific positional selectivity of hydrolysis of medium-chain FA in sn-1,3 localization by gastric lipase (21). Our data are in agreement with the findings of Christensen et al. (7), who demonstrated that EFA were better absorbed from TG with a defined compared with a random structure of a soybean oil and MCT mixture in rats with pancreatic insufficiency. Similarly, Jandacek et al. (19) used rats with irrigated intestinal loops as a model for pancreatic insufficiency and demonstrated that linoleic acid was better absorbed from 8:0/18:2(n-6)/8:0 than from 18:1(n-9)/18:2(n-6)/18:1(n-9). However, Tso et al. (43) assessed lymphatic transport in rats and observed that STG containing octanoic acid in the sn-1 and -3 positions and linoleic acid in the sn-2 position may not be advantageous when used as the sole source of dietary lipids, unless supplemented with long-chain TG.
EFA deficiency has been reported to occur frequently in preterm animals
(39), in patients with cholestatic liver disorders, glycogen storage
disease, and cirrhosis, as well as in malabsorptive disorders, such as
abetalipoproteinemia, cystic fibrosis, celiac, and Crohn's disease (3,
23, 28, 31). Despite adequate dietary supply, pathophysiological
mechanisms, including insufficiency of bile acids or pancreatic lipase,
diminish efficient intestinal absorption. In a further attempt to
establish the physiological role of our STG in EFA deficiency, we
cultured Caco-2 cells in medium containing delipidated serum. After 10 days, Caco-2 cells were depleted of the major EFA. The deficiency of
linoleic acid [18:2(n-6)] as well as of arachidonic acid
[20:4(n-6)] and the concomitant enrichment of
eicosatrienoic acid [20:3(n-9)] led to an elevation of the
ratio of 20:3(n-9) to 20:4(n-6), a very sensitive index of EFA
deficiency (23). Culture in the presence of our purified STG produced a
marked increase of the cellular level of 18:2(n-6). This was not
achieved with triolein supplementation. Furthermore, there was an
increased proportion of -linoleic acid [18:3(n-6)] and
20:4(n-6) in Caco-2 cells incubated with STG. EFA-deficient Caco-2
cells had a significantly lower ratio of 20:3(n-9) to 20:4(n-6) when
administered STG, reflecting correction of EFA status.
The increase in the proportion of 18:3(n-6) and 20:4(n-6) in Caco-2
cells suggests elevated 6 and
5 desaturases, respectively, as
well as elongase activity. Dias et al. (8) initially described the
presence of
6 and
5 activity in well-differentiated and polarized
Caco-2 cells and subsequently demonstrated (9) that Caco-2 cell
membrane FA composition and desaturase enzyme activity are regulated by
both dietary fat intake and the degree of cell maturation. We therefore
speculate that the differentiated status of our Caco-2 cells and the
supplementation of purified STG provided sufficient substrate to
enhance desaturase activity.
PL are integral components of all cell membranes, and PUFA are critical to PL synthesis. FA chain desaturation is a necessary step in the production of PUFA for their subsequent incorporation in membrane PL (20). The increased radioactivity in the PL fraction from Caco-2 cells incubated with our purified STG substrate implies a very high demand for PL in intestinal epithelial cells, which have the fastest turnover rate of any body tissue (13). In addition, the secretion of cellular TG containing linoleic acid was not jeopardized, compared with the TG-oleic acid output, because 1) a similar TG proportion characterized this lipid fraction secreted when Caco-2 cells were incubated with either [14C]triolein or [14C]STG, and 2) more TG with EFA were exported (2.5% of a 3-fold higher uptake of STG substrate). Our data suggest that cellular membranes may constitute active pools of linoleic acid stored for export.
Previously, we demonstrated (26) that EFA deficiency adversely affects both the intraluminal and intracellular phases of fat absorption. We were also able to show that EFA deficiency impairs the intrahepatic metabolism of cholesterol and bile acids (25), which further hinders intestinal lipid absorption. Correction of EFA deficiency can improve all of these metabolic pathways. Our observations demonstrate that the administration of our purified STG has discernible beneficial effects on Caco-2 cell lipid uptake, incorporation, and lipoprotein production. These findings suggest that STG, in the presence of gastric lipase, can correct EFA deficiency despite the absence of pancreatic lipase activity in Caco-2 cells.
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ACKNOWLEDGEMENTS |
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We thank Carole Garofalo for technical assistance and Danielle St.-Cyr Huot for preparing the manuscript.
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FOOTNOTES |
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This study was supported by a research grant from the George Weston Foundation (E. G. Seidman and E. Levy), by a Research Fellowship Award from the Swiss Nutritional Research Foundation, Medical Research Council, and the Cystic Fibrosis Foundation of Canada (J. H. Spalinger), and Research Scholarship Awards from the Fonds de la Recherche en Santé du Québec (E. G. Seidman and E. Levy).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: E. Levy, Gastroenterology and Nutrition Unit, Pediatric Research Center, Hôpital Sainte-Justine, 3175 Côte Sainte-Catherine, Montreal, PQ, Canada H3T 1C5.
Received 6 May 1998; accepted in final form 18 June 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abrams, C. K.,
M. Hamosh,
V. S. Hubbard,
S. K. Dutta,
and
P. Hamosh.
Quantitation of enzyme activity in the upper small intestine of patients with exocrine pancreatic insufficiency.
J. Clin. Invest.
73:
374-382,
1984[Medline].
2.
Awl, R. A.,
E. N. Frankel,
and
D. Weisleder.
Synthesis and characterization of triacylglycerols containing linoleate and linolenate.
Lipids
24:
866-872,
1989.
3.
Cabre, E.,
and
M. A. Gassul.
Polyunsaturated fatty acid deficiency in liver diseases: pathophysiological and clinical significance.
Nutrition
12:
542-548,
1996[Medline].
4.
Capriotti, A. M.,
and
M. Laposata.
Identification of variables critical to reproducible delipidation of serum.
J. Tissue Cult. Methods
4:
219-221,
1986.
5.
Carey, M. C.,
and
O. Hernell.
Digestion and absorption of fat.
Semin. Gastrointest. Dis.
3:
189-208,
1992.
6.
Carriere, F.,
J. A. Barrowman,
R. Verger,
and
R. Laugier.
Secretion and contribution to lipolysis of gastric pancreatic lipases during a test meal in humans.
Gastroenterology
105:
876-888,
1993[Medline].
7.
Christensen, M. S.,
A. Müllertz,
and
C. E. Hoy.
Absorption of triglycerides with defined or random structure by rats with biliary and pancreatic diversion.
Lipids
30:
521-526,
1995[Medline].
8.
Dias, V. C.,
J. M. Borkowsk,
and
H. G. Parsons.
Eicosapentaenoic acid modulates the -6 and
-5 desaturase activity in the intestinal Caco-2 cell line (Abstract).
Gastroenterology
100:
A824,
1991.
9.
Dias, V. C.,
and
H. G. Parsons.
Modulation in 9,
6 and
5 fatty acid desaturase activity in the human intestinal Caco-2 cell line.
J. Lipid Res.
36:
552-563,
1995[Abstract].
10.
Folch, J. M.,
M. Lees,
and
G. H. Sloane-Stanley.
A single method for the isolation and purification of total lipids from animal tissues.
J. Biol. Chem.
266:
497-509,
1957.
11.
Furth, E. E.,
H. Sprecher,
E. A. Fisher,
H. D. Fleishman,
and
M. Laposata.
An in vitro model for essential fatty acid deficiency: HepG2 cells permanently maintained in lipid-free medium.
J. Lipid Res.
33:
1719-1726,
1992[Abstract].
12.
Hernell, O.,
and
L. Bläckberg.
Molecular aspects of fat digestion in the newborn.
Acta Paediatr.
405:
65-69,
1994.
13.
Garg, M.,
M. Keelan,
A. B. R. Thomson,
and
M. T. Clandinin.
Desaturation of linoleic acid in the small bowel is increased by short-term fasting and by dietary content of linoleic acid.
Biochim. Biophys. Acta
1126:
17-25,
1992[Medline].
14.
Hirono, H.,
H. Suzuki,
Y. Igarashi,
and
T. Konno.
Essential fatty acid deficiency induced by total parenteral nutrition and by medium chain triglyceride feeding.
Am. J. Clin. Nutr.
30:
1670-1676,
1977[Abstract].
15.
Holt, P. R.
Studies of medium chain triglycerides in patients with differing mechanisms for fat malabsorption.
In: Medium Chain Triglycerides, edited by J. R. Senior. Philadelphia, PA: Univ. of Pennsylvania, 1968, p. 97-107.
16.
Hubbard, V. S.,
G. D. Dunn,
and
P. A. di Sant'Agnese.
Abnormal fatty-acid composition of plasma-lipids in cystic fibrosis. A primary or a secondary defect?
Lancet
2:
1302-1304,
1977[Medline].
17.
Hubbard, V. S.,
and
M. C. McKenna.
Absorption of safflower oil and structured lipid preparations in patients with cystic fibrosis.
Lipids
22:
424-428,
1987[Medline].
18.
Hyman, B. T.,
L. L. Stoll,
and
A. A. Spector.
Accumulation of (n-9)-eicosatrienoic acid in confluent 3T3-L1 and 3T3 cells.
Prog. Lipid Res.
26:
87-124,
1987[Medline].
19.
Jandacek, R. J.,
L. A. Whiteside,
B. N. Holcombe,
R. A. Voljenheim,
and
J. D. Taulbee.
The rapid hydrolysis and efficient absorption of triglycerides with octanoic acid in the 1 and 3 positions and long-chain fatty acids in the 2 position.
Am. J. Clin. Nutr.
45:
940-945,
1987[Abstract].
20.
Jeffcoat, R.,
and
A. T. James.
The regulation of desaturation and elongation of fatty acids in mammals.
In: Fatty Acid Metabolism and its Regulation, edited by S. Numa. New York: Elsevier Science, 1984, p. 85-112.
21.
Jensen, R. G.,
F. deJong,
L. G. Lambert-Davis,
and
M. Hamosh.
Fatty acid and positional selectivities of gastric lipase from premature human infants: in vitro studies.
Lipids
29:
433-435,
1994[Medline].
22.
Lee, P. C.,
M. S. Borysewics,
K. Raab,
and
S. L. Werlin.
Development of lipolytic activity in gastric aspirates from premature infants.
J. Pediatr. Gastroenterol. Nutr.
17:
291-297,
1993[Medline].
23.
Lepage, G.,
E. Levy,
N. Ronco,
L. Smith,
N. Galeano,
and
C. Roy.
Direct transesterification of plasma fatty acids for the diagnosis of essential fatty acid deficiency in cystic fibrosis.
J. Lipid Res.
30:
1483-1490,
1989[Abstract].
24.
Levy, E.
Selected aspects of intraluminal and intracellular phases of intestinal fat absorption.
Can. J. Physiol. Pharmacol.
70:
413-419,
1992[Medline].
25.
Levy, E.,
C. Garofalo,
T. Rouleau,
V. Gavino,
and
M. Bendayan.
Impact of essential fatty acid deficiency on hepatic sterol metabolism in rats.
Hepatology
23:
848-857,
1996[Medline].
26.
Levy, E.,
C. Garofalo,
L. A. Thibault,
S. Dionne,
L. Daoust,
G. Lepage,
and
C. C. Roy.
Intraluminal and intracellular phases of fat absorption are impaired in essential fatty acid deficiency.
Am. J. Physiol.
262 (Gastrointest. Liver Physiol. 25):
G319-G326,
1992
27.
Levy, E.,
R. Goldstein,
S. Freier,
and
E. Shafrir.
Gastric lipase in the newborn rat.
Pediatr. Res.
16:
69-74,
1982[Abstract].
28.
Levy, E.,
G. Lepage,
M. Bendayan,
N. Ronco,
L. Thibault,
N. Galeano,
L. Smith,
and
C. C. Roy.
Relationship of decreased hepatic lipase activity and lipoprotein abnormalities to essential fatty acid deficiency in cystic fibrosis patients.
J. Lipid Res.
30:
1197-1209,
1989[Abstract].
29.
Levy, E.,
N. Loirdighi,
L. Thibault,
T. D. Nguyen,
D. Labuda,
E. Delvin,
and
D. Ménard.
Lipid processing and lipoprotein synthesis by the developing human fetal colon.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G813-G820,
1996
30.
Levy, E.,
M. Mehran,
and
E. G. Seidman.
Caco-2 cells as a model for intestinal lipoprotein synthesis and secretion.
FASEB J.
9:
626-635,
1995
31.
Levy, E.,
L. A. Thibault,
C. C. Roy,
M. Bendayan,
G. Lepage,
and
J. Letarte.
Circulating lipids and lipoproteins in glycogen storage disease type I with nocturnal intragastric feeding.
J. Lipid Res.
29:
215-226,
1988[Abstract].
32.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
33.
McKenna, M. C.,
V. S. Hubbard,
and
J. G. Bieri.
Linoleic acid absorption from lipid supplements in patients with cystic fibrosis with pancreatic insufficiency and in control subjects.
J. Pediatr. Gastroenterol. Nutr.
4:
45-51,
1985[Medline].
34.
Mehran, M.,
E. Levy,
C. Gurbindo,
M. Bendayan,
and
E. G. Seidman.
Lipid apolipoprotein and lipoprotein synthesis and secretion during cellular differentiation in Caco-2 cells.
In Vitro Cell. Dev. Biol.
33:
118-128,
1997.
35.
Ménard, D.,
S. Monfils,
and
E. Tremblay.
Ontogeny of gastric lipase and pepsin activities.
Gastroenterology
108:
1650-1656,
1995[Medline].
36.
Mok, K. T.,
A. Maiz,
K. Yamazaki,
J. Sobrado,
V. K. Babayan,
L. L. Moldawer,
B. R. Bristian,
and
G. L. Blackburn.
Structured medium-chain triglyceride emulsions are superior to physical mixtures in sparing body protein in the burned rat.
Metabolism
33:
910-915,
1984[Medline].
37.
Sobrado, J.,
L. L. Moldawer,
J. J. Pompeselli,
E. A. Mascioli,
V. K. Babayan,
V. R. Bristrian,
and
G. L. Blackburn.
Lipid emulsions and reticuloendothelial system function in healthy and burned guinea pig.
Am. J. Clin. Nutr.
42:
855-863,
1985[Abstract].
38.
Spalinger, J. H.,
E. G. Seidman,
C. Garofalo,
D. Ménard,
and
E. Levy.
Endogenous lipase activity in Caco-2 cells (Abstract).
Gastroenterology
112:
A907,
1997.
39.
Stahl, G.,
M. Mascarenhas,
J. Fayer,
Y. Shian,
and
J. Watkins.
Passive jejunal bile salt absorption alters the enterohe-patic circulation in immature rats.
Gastroenterology
104:
163-173,
1993[Medline].
40.
Teo, T. C.,
S. J. DeMichel,
K. M. Sellek,
V. K. Babayan,
G. C. Blackburn,
and
B. R. Bristrian.
Administration of structured lipid composed of MCT and fish oil reduces net protein catabolism in enterally fed burned rats.
Ann. Surg.
210:
100-107,
1989[Medline].
41.
Thomson, A. B.,
M. Keelan,
M. L. Garg,
and
M. T. Clandinin.
Intestinal aspects of lipid absorption: a review.
Can. J. Physiol. Pharmacol.
67:
179-191,
1989[Medline].
42.
Tso, P.
Intestinal lipid absorption.
In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1994, p. 1867-1907.
43.
Tso, P.,
M. D. Karlstad,
B. R. Bistrian,
and
S. J. DeMichele.
Intestinal digestion, absorption, and transport of structured triglycerides and cholesterol in rats.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G568-G577,
1995
44.
Van Egmond, A. W. A.,
M. R. Kosorok,
A. Laxova,
and
P. M. Farrel.
Effect of linoleic acid intake on growth of infants with cystic fibrosis.
Am. J. Clin. Nutr.
63:
746-752,
1996[Abstract].
45.
Westergaad, H.,
and
J. M. Dietschy.
The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell.
J. Clin. Invest.
58:
97-108,
1976[Medline].