Role of insulin and nutritional factors in intestinal glycoprotein fucosylation during postnatal development

Marie-Claire Biol1, Didier Lenoir1, Sandrine Greco1, Dany Galvain2, Irene Hugueny1, and Pierre Louisot1

1 Institut National de la Santé et de la Recherche Médicale Unité 189-Structure d'intervention, Centre National de la Recherche Scientifique, Faculté de Médecine Lyon-Sud, 69600 Oullins; and 2 Laboratoire de Radioanalyse, Centre Hospitalier Lyon-Sud, 69310 Pierre-Bénite, France

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

This study deals with the role of insulin in the regulation of the intestinal glycoprotein fucosylation process during postnatal development in the rat. Circulating insulin level was found to increase at weaning time in parallel with alpha -1,2-fucosyltransferase activity and with the appearance of alpha -1,2-fucoproteins in brush-border membranes. Insulin treatment of young suckling rats induced a precocious increase in fucosyltransferase activity and in the biosynthesis of its substrate (GDP-fucose), but the sensitivity to insulin disappeared after weaning. The insulin level was lower in 22-day-old rats that received prolonged nursing (on a high-fat diet) compared with age-matched normally weaned rats (on a high-carbohydrate diet), whereas the appearance of alpha -1,2-fucoproteins and the increase in activity of alpha -1,2-fucosyltransferase were delayed, as was the decrease in the degradation of GDP-fucose. In 22-day-old animals that received prolonged nursing and insulin treatment, the alpha -1,2-fucosyltransferase activity reached a level close to that observed in age-matched weaned rats, and several alpha -1,2-fucoproteins appeared in brush-border membranes with a molecular mass similar to that found in weaned rats. These results suggest that changes in insulin levels at weaning time (as caused, in the present case, by dietary modifications) may be responsible for the regulation of the glycoprotein fucosylation process, essentially by increasing fucosyltransferase activity.

fucosyltransferase; fucoproteins; weaning

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

DURING THE THIRD WEEK OF postnatal life in the rat, important ontogenic changes occur in the small intestine. These include changes in the activity of the digestive glycoproteinic enzymes (e.g., glycosidases, aminopeptidase) that enable the animal to cope with the solid high-carbohydrate diet of the adult (15). The role of the glycanic chains of these glycoproteins on their activity is not yet clear. However, the glycosylation processes are also drastically modified during postnatal development, in that, between postnatal life and adulthood, there is a shift from sialylation to fucosylation in the apical and basolateral plasma membranes of the epithelial cells, as demonstrated by lectin cytochemistry (28), and, after cellular fractionation, in the purified brush-border membrane glycoproteins (7, 18, 29) and in the mucins (26). This shift is accompanied by parallel variations in the activity of the glycosyltransferases involved in the sialylation and/or the fucosylation processes (3, 12). The involvement of hormones in the regulation of intestinal maturation is well known; the operative hormonal signal is probably provided by corticoids, thyroid hormones, and/or insulin (10), whose serum levels increase markedly in the rat toward the end of the third week of life (6). Although insulin may influence cell differentiation and be a possible candidate for glycosylation regulation during postnatal development, its role in the intestinal glycosylation process has not so far been properly studied. The glycoprotein fucosylation process involves both different enzymic systems for the synthesis and degradation of the GDP-fucose substrate and also a regulatory protein (fuctinin) that acts on intestinal fucosyltransferase, and which, it has been suggested, may play a physiological role in the developmental changes of the fucosylation pathway (20, 23). The present study examines the effect of insulin on the different enzymic and regulatory systems involved in the fucosylation process in the rat small intestine to find out if the normal increase in hormone levels at weaning time might explain the large associated increase in glycoprotein fucosylation that also occurs at this time.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals and insulin treatment. A developmental study was carried out on litters of 10 male suckling pups of the Sprague-Dawley strain (IFFA CREDO), which were maintained at a controlled temperature (23°C) on a 12:12-h light-dark cycle in special cages and were given no solid food before weaning time. Some male rats were weaned on a solid high-carbohydrate diet (Souriffarat, UAR) at 19 days of age (after the removal of the dams) and maintained with this food until adulthood. For experiments performed on 14-day-old rats, in each litter of ten 10-day-old suckling rats, the first group of five pups (the insulin group) was given, via intraperitoneal injection for 4 days, 16 mU of porcine insulin (Sigma Chemical, St. Louis, MO) in 0.9% NaCl per gram of body weight, twice a day (at 8 and 18 h). The second group of rats (the control group) received only 0.9% NaCl but under the same conditions. Experiments on these rats were carried out at 14 days of age. A second set of experiments performed on 22-day-old animals involved either prolonged nursing (high-fat diet) without solid food up to the 22nd day under conditions previously described (4) or an abrupt weaning change to a solid high-carbohydrate diet (Souriffarat) at 19 days of age (weaned rats). Some animals in each diet group received insulin under the conditions described above between the 18th and the 22nd day, while the others received 0.9% NaCl. For postweaning treatment, rats were weaned on the 19th day, treated with insulin or 0.9% NaCl under the same conditions between the 24th and the 28th day, and killed at 28 days of age.

Cell fractionations. At the end of the treatment, the animals were killed, and their small intestines were removed, flushed with cold 0.9% NaCl, and opened. The mucosae were harvested with a glass slide and homogenized in 10 mM Tris · HCl, 10 mM KCl, 10 mM MgCl2, and 250 mM sucrose, pH 7.4. Depending on the age, cytosols and microsomal pellets were prepared from the small intestine of between one (adult) and three (young) rats for each control and assay group, as previously described (4). The processing of the brush-border membranes was carried out by the CaCl2 precipitation technique of Kessler et al. (17).

Enzymic and regulatory activity. Glycoprotein fucosyltransferase activity was measured using asialofetuin as an exogenous acceptor in the cytosol or the microsomal fraction (4). This made it possible to determine the alpha -1,2-linkage of fucose to the galactose residue and the alpha -1,3-linkage of fucose to the N-acetylglucosamine residue of the galactose(beta 1-4)-N-acetylglucosamine terminals of the oligosaccharidic chains. The reaction mixture (250 µl total) contained between 150 and 250 µg protein, 20 µM asialofetuin, 5 mM MnCl2, 10 mM AMP, 0.25% Triton X-100, and 6.5 µM GDP-[14C]fucose (sp act 10.1 GBq/mmol; NEN). The incubations were carried out at 23°C for 30 min, and the reactions were stopped with 20% TCA. The radioactive products were collected on GF/B fiberglass filters (Whatman), and the radioactivity was measured using a Toluene Scintillator (Packard). The alpha -1,2-fucosyltransferase activity (galactoside 2-alpha -L-fucosyltransferase, E.C. 2.4.1.69) was more specifically determined in the conditions described above, but with phenyl beta -D-galactoside (50 mM) as an acceptor instead of asialofetuin. The reaction was stopped by the addition of 750 µl water at 4°C; the [14C]fucosylated phenyl beta -D-galactoside was separated by chromatography, using a Sep-Pak C18 cartridge (Waters-Millipore), according to the method of Palcic et al. (22). The cartridge was washed with 20 ml H2O, the [14C]fucosylated product was eluted with 10 ml CH3OH, and the radioactivity was detected using an Emulsifier Scintillator Plus (Packard) after evaporation of the different fractions.

The synthesis and degradation of the GDP-fucose substrate were studied in cytosol, and the reaction products were separated by HPLC as previously described (24).

Fuctinin activity (fuctinin being a fucosyltransferase inhibitor) was determined, as previously described (24), in a fraction semipurified from cytosol with partially purified alpha -1,2-fucosyltransferase (23). The measurement of this activity (taken as the quantity of proteins giving 50% inhibition) was based on concentration curves obtained for the different dilutions of the inhibitor.

Fucoprotein detection. After electrophoresis of the proteins of the intestinal brush-border membrane and transfer to nitrocellulose membranes, the alpha -1,2-fucoproteins were detected by a lectin sorbent assay, using the lectin of Ulex europeus (UEA-I), as described previously (24).

Chemical determination. The DNA content of the homogenate prepared from intestinal mucosae was determined according to the method of Burton (8) and the protein content according to the method of Schaffner and Weissmann (25). Serum insulin concentrations were obtained using an insulin RIA-GNOST kit (Behring, Germany).

Statistical treatment of results. The results were expressed as means ± SE. For developmental variations, the results were submitted to a one-factor ANOVA. When the F test indicated a significant effect, we analyzed the differences between the means by the Newman-Keuls test. Differences were considered significant at P < 0.05. For the comparison of results between two groups only, the Student's t-test or the Mann-Whitney U test was used, depending on the number of values. For studies on the variations due to two factors (e.g., diet and treatment for the 22-day-old rats, or treatment and time for the 10-day-old rats), the results were submitted to a two-factor ANOVA and, where significant results were found, the means were compared with the Newman-Keuls test.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Developmental changes in circulating insulin and intestinal fucosyltransferase activity. A large and significant increase in circulating insulin was observed just after weaning (Table 1). Concomitant with the postweaning appearance of many alpha -1,2-fucoproteins in the brush-border membranes, which we have already described (24), a large and significant increase in both the membrane-bound and soluble fucosyltransferase activity (as determined using asialofetuin as an acceptor), and especially in that of alpha -1,2-fucosyltransferase (as determined using phenyl beta -D-galactoside as an acceptor) was observed (Table 1). Given the large increase in insulinemia that accompanies this increase in intestinal glycoprotein fucosylation, we considered it interesting to study the role of insulin (as a possible maturation factor) on the evolution of the fucosylation process during postnatal development.

                              
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Table 1.   Developmental changes in circulating insulin and intestinal fucosyltransferase activity

Effect of insulin treatment on young suckling rats. The fucosylation process involves a set of proteins and enzymes that can modulate the transfer of fucose onto glycoproteins by the control of fucosyltransferase activity or substrate availability (Fig. 1). We have studied the effect of exogenous insulin on the fucosylation process in the small intestine of the suckling rats, in which the base insulin level and that of fucosyltransferase activity are normally low.


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Fig. 1.   Metabolic pathway of glycoprotein fucosylation.

After each injection of insulin, the circulating insulin level remained significantly higher than the normal level for >4 h. Two hours after injection, it was 2.5-fold as high as that of the adult, returning to something similar to the adult level for 4 h after injection, at which time the circulating insulin level had fallen back to near its initial value, which is also that of untreated rats (Table 2). Insulin treatment of suckling rats between the 10th and 14th day of life induced a significant increase in the specific activity of fucosyltransferase, as well as that of alpha -1,2-fucosyltransferase (Table 3). These increases were observed both in the soluble and membrane-bound forms of the enzymes. The levels of mucosal protein and DNA were not significantly modified by insulin treatment (7.7 ± 1.2 vs. 9.0 ± 0.9 mg DNA/g intestine for insulin-treated rats vs. control rats, n = 12, not significant), which suggests that insulin does not affect intestinal cellularity significantly. In fact, when expressed in terms of total activity per gram of intestine or relative to DNA content, fucosyltransferase activity varied in an analogous way in the nontreated and treated animals than when expressed in terms of specific activity.

                              
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Table 2.   Circulating insulin level after insulin injection

                              
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Table 3.   Effect of insulin on fucosyltransferase and fuctinin activity

In contrast, the activity of fuctinin was not significantly modified by the administration of insulin (Table 3).

Because insulin increases the biosynthesis of GDP-fucose but does not affect the degradation of GDP-fucose by GDP-fucose pyrophosphatase (Table 4), overall GDP-fucose substrate availability is probably increased by insulin treatment.

                              
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Table 4.   Effect of insulin on activity of enzymes involved in substrate synthesis or degradation

Effect of insulin treatment after weaning. Insulin treatment of weaned rats between the 24th and 28th days of life had no effect on fucosyltransferase activity, on GDP-fucose biosynthesis or degradation, or on fuctinin activity (data not shown).

Effect of insulin treatment at time of weaning. The increase in intestinal fucosylation, which is normally observed at the end of the third week of life, was delayed by prolonged nursing. Indeed, the activity levels of fucosyltransferase (with asialofetuin) and alpha -1,2-fucosyltransferase (with phenyl beta -D-galactoside) were significantly lower in the intestine of 22-day-old rats that received prolonged nursing (high-fat diet) than in those of age-matched weaned rats (high-carbohydrate diet) and were in fact close to the levels observed in very young rats (Fig. 2A). Fuctinin activity in the weaned rats was lower than in the rats that received prolonged nursing, but the decrease in activity was not significant when the results were analyzed using two-factor ANOVA (Fig. 2B). The level of GDP-fucose degradation (Fig. 2D) was higher in rats that received prolonged nursing than in weaned rats, but GDP-fucose biosynthesis was not affected by the difference in diet (Fig. 2C). Moreover, prolonged nursing delayed the appearance of alpha -1,2-fucoproteins in the brush-border membranes of the 22-day-old rats that received prolonged nursing, whereas the fucoproteins were present in the membranes of the 22-day-old weaned rats (Fig. 3). These results indicate that the glycoprotein glycosylation process in the rat intestine is particularly sensitive to diet manipulations at weaning time. However, it is also to be noted that the circulating insulin level in the 22-day-old rats that received prolonged nursing was lower than that of the age-matched weaned rats and similar to that of young rats (Fig. 4).


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Fig. 2.   Effect of insulin on fucosylation regulation process in relationship to weaning. PN, 22-day-old rats that received prolonged nursing alone. PN-Ins, 22-day-old rats that received prolonged nursing accompanied by insulin treatment. W, 22-day-old rats abruptly weaned at 19 days of age. W-Ins, 22-day-old rats that were weaned and treated with insulin. A: alpha -1,2-fucosyltransferase (determined with phenyl beta -D-galactoside as acceptor) results for 8 assays. B: fuctinin activity for 7 assays. C: GDP-fucose biosynthesis for 8 assays. D: GDP-fucose degradation for 8 assays. Results for the 4 groups of animals were analyzed by 2-factor ANOVA and means ± SE were compared by Newman-Keuls test. a,b Significant diet-related differences (P < 0.05). c Significant difference resulting from insulin treatment (P < 0.05).


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Fig. 3.   Effect of insulin on appearance of alpha -1,2-fucoproteins in brush-border membranes in relationship to weaning. Fucoproteins were detected by Ulex europeus lectin (UEA-I) in brush-border membranes (7 µg protein/lane) prepared from small intestine of 22-day-old rats that received prolonged nursing (PN), 22-day-old rats that received prolonged nursing and insulin treatment (PN-Ins), 22-day-old weaned rats (W), and 22-day-old weaned rats treated with insulin (W-Ins). MMC, molecular mass control.


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Fig. 4.   Circulating insulin level in 22-day-old rats that either received prolonged nursing (PN) or were weaned at day 19 (W). Values are means ± SE for 5 rats. Comparisons were made using Mann-Whitney U test. * P < 0.05.

When rats that received prolonged nursing were treated with insulin, the activity levels of fucosyltransferase (with asialofetuin) and alpha -1,2-fucosyltransferase (Fig. 2A) were significantly higher than for untreated rats that received prolonged nursing and were at a level between that of the untreated rats that received prolonged nursing and that of both groups of weaned rats. Although the fuctinin activity of the insulin-treated rats that received prolonged nursing tended to decrease and to present a level between that of the control rats that received prolonged nursing and both groups of weaned rats, the differences between the four groups were not found to be significant by two-factor ANOVA (Fig. 2B), due perhaps to a rather high degree of individual dispersion of the results. The level of GDP-fucose biosynthesis was not modified by either dietary change or insulin treatment (Fig. 2C), and GDP-fucose degradation was higher in the two groups of rats that received prolonged nursing than in the two groups of weaned rats, although insulin treatment had no effect on this activity (Fig. 2D). Prolonged nursing delayed the appearance of alpha -1,2-fucoproteins in the brush-border membranes (Fig. 3), but insulin treatment of the rats that received prolonged nursing induced the appearance of some alpha -1,2-fucoproteins with a molecular mass (100-110 kDa) similar to those of the alpha -1,2-fucoproteins of the weaned rats. These results indicate that insulin treatment suppresses the delay caused by prolonged nursing in the evolution of the glycoprotein fucosylation process that naturally occurs at weaning and that insulin responsiveness is lost after weaning.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The aim of this study was to find out if insulin is involved in the regulation of the glycoprotein fucosylation process during rat postnatal development. To begin with, when we looked at alpha -1,2-fucosyltransferase activity and insulin levels in the small intestine of the rat between birth and adulthood, we found that they both increased just after weaning. We had already found (24) that an increase in fucosyltransferase activity at the time of weaning is accompanied by the appearance of fucoproteins in the brush-border membranes. Because fucosyltransferase activity may be regulated either by fuctinin or by substrate availability (23), in this study, we examined the different parameters involved in fucosylation regulation.

The treatment of young suckling rats with insulin increases alpha -1,2-fucosyltransferase activity and biosynthesis of the GDP-fucose substrate but does not affect the GDP-fucose degradation. Given the stability of the fuctinin level, the premature increase in fucosyltransferase activity is likely to be due either to higher substrate availability or to the regulation of fucosyltransferase expression at transcriptional level rather than to the control of fucosyltransferase activity by fuctinin. Moreover, the observed acceleration of intestinal maturation as the result of insulin treatment indicates correlated changes in fucose metabolism and in the fucosylation regulation process. Mahmood and Torres-Pinedo (19) did not find any modification of intestinal fucosylation after the treatment of suckling rats with insulin. Following the treatment conditions used by Mahmood and Torres-Pinedo (19) (i.e., subcutaneous administration once a day), we also observed (1) a nonsignificant increase of the fucosyltransferase activity. However, when the injections were given twice a day rather than once and by the intraperitoneal route, the circulating insulin level remained higher or close to that found in the adult rat for several hours after each injection. This method may thus be more effective in producing variations in the fucosylation regulation process. The fact that insulin seems to cause significant stimulation of the fucosylation process suggests that it may be a regulator of intestinal maturation, as well as for digestive enzymes, as previously found (9, 21, 27). It remains to be seen whether insulin modifies glycoprotein fucosylation in the crypts or the villi, given that Taatjes and Roth (28) have shown that variations in sialylation and fucosylation maturation are more commonly observed in specific cell types than in the position of cells along the crypt-to-villus axis. The results of the present study also show that immature intestinal cells from suckling rats respond to insulin, whereas mature intestinal cells from weaned rats (treated between the 24th and 28th days) do not. The mechanism by which insulin affects the maturation of the intestine is not well known. However, Buts et al. (11) indicate that the glycosidase response of immature enterocytes to insulin is mediated by the binding of the hormone to its receptors. The circulating insulin is low in the suckling rat, but it increases markedly just after weaning, whereas the concentration of insulin receptors follows an inverse ontogenic pattern (15). On the other hand, the binding capacity of the hormone on intestinal receptors decreases with age (15). These phenomena could explain the loss of sensitivity of the intestinal fucosylation process to insulin that was observed in the weaned rats.

Given that the level of circulating insulin increases at weaning time and that insulin levels may depend on dietary changes, we studied variations in the level of insulin and in the fucosylation process at the end of the third week of life in rats that were either normally weaned (high-carbohydrate diet) or received prolonged nursing (high-fat diet). In the small intestine of 22-day-old control rats that received prolonged nursing, alpha -1,2-fucosyltransferase activity was found to be significantly lower than in those of weaned age-matched rats, and fucoproteins (recognized with the UEA-I lectin, which is specific to fucose with linkage in alpha -1,2) were absent from the brush-border membranes of rats that received prolonged nursing, although they were relatively numerous in those of weaned rats. We studied the effect of insulin on 22-day-old rats that received prolonged nursing, whose circulating insulin level was lower than that of the age-matched weaned rats. After insulin treatment of the 22-day-old rats that received prolonged nursing, the alpha -1,2-fucosyltransferase activity was found to have increased significantly, although its level of activity did not quite reach that of the 22-day-old weaned rats. Moreover, this treatment resulted in the appearance in the brush-border membranes of alpha -1,2-fucoproteins with molecular mass similar to those observed in the membranes of the 22-day-old weaned rats. The other regulatory and enzymic parameters of the fucosylation process did not seem to be involved in its regulation by insulin. For example, fuctinin activity in the rats that received prolonged nursing was not significantly modified. Although the degradation of the GDP-fucose substrate was considerably lower in the weaned rats than in the rats that received prolonged nursing, insulin treatment of the latter rats did not have any effect on the GDP-fucose degradation process. However, the diet-related modifications of this process may explain the fact that alpha -1,2-fucoproteins that appeared in the brush-border membranes of the rats that received both prolonged nursing and insulin were not as intensely stained as the alpha -1,2-fucoproteins observed in the brush-border membranes of the weaned rats (weaned rats with or without insulin), perhaps due to a difference in the availability of endogenous GDP-fucose. These results indicate that insulin, whose level is sensitive to dietary modifications, and in any case naturally increases at the time of weaning, is involved in the regulation of the fucosylation of intestinal glycoproteins, essentially by the regulation of fucosyltransferase activity. Jaswal et al. (16) have shown that undernutrition during the suckling period delays the maturational development of the intestine, as shown by the increased sialylation and decreased fucosylation of the intestinal membranes, which are characteristic of immature tissue and by the fact that insulin treatment of undernourished pups delays the sialylation-fucosylation shift. We had already shown that intestinal fucosyltransferase activity as in adult rats was increased by a high-carbohydrate diet, compared with a control diet or a high-fat diet (2). In the present study, we show that the natural developmental increase in the insulin level is delayed in rats that receive prolonged nursing (high-fat diet) compared with age-matched weaned rats (high-carbohydrate diet). Blazquez and Lopez-Quijada (5) have also demonstrated that the administration of a high-fat diet toward the end of the weaning period produces a decrease in the circulating insulin. Moreover, Craig et al. (13) found that major changes in the macronutrient composition of the diet can affect the insulin receptors in the gastrointestinal tract, and, in particular, that there is a decrease in insulin binding in the intestine of adult rats fed a high-fat diet, compared with rats fed a high-carbohydrate diet. However, the hypothesis that insulin responsiveness may be related to the state of the receptor remains to be demonstrated.

In conclusion, regarding the parallel changes that take place in the circulating insulin level and the glycoprotein fucosylation process during postnatal development and the effect of insulin on the fucosylation process in young rats, our results indicate that insulin plays a regulatory role, causing the premature appearance of some developmental processes in the intestine of the suckling rat with the glycoprotein fucosylation process being particularly affected. In other words, given that the manipulation of dietary parameters at weaning time is observed to be accompanied by modifications in both the circulating insulin level and the glycoprotein fucosylation process, it may be that the insulin level is actually the factor that causes the modifications in the glycoprotein fucosylation process, perhaps via the number or sensitivity of insulin receptors.

    ACKNOWLEDGEMENTS

We thank A. Martin for helpful discussions.

    FOOTNOTES

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: P. Louisot, Unité Institut National de la Santé et de la Recherche Médicale U189-SDI Centre National de la Recherche Scientifique, Faculté de Médecine Lyon-Sud, BP 12, 69600 Oullins, France.

Received 20 April 1998; accepted in final form 10 July 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(5):G936-G942
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society