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 |
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
-1,2-fucosyltransferase activity and
with the appearance of
-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
-1,2-fucoproteins and the increase in activity of
-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
-1,2-fucosyltransferase activity reached a level close to that observed in age-matched weaned
rats, and several
-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 |
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 |
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
-1,2-linkage of
fucose to the galactose residue and the
-1,3-linkage of fucose to
the N-acetylglucosamine residue of the
galactose(
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
-1,2-fucosyltransferase activity (galactoside
2-
-L-fucosyltransferase, E.C.
2.4.1.69) was more specifically determined in the conditions described
above, but with phenyl
-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
-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
-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
-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 |
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
-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
-1,2-fucosyltransferase (as
determined using phenyl
-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.
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.
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
-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.
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.
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
-1,2-fucosyltransferase (with phenyl
-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
-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: -1,2-fucosyltransferase
(determined with phenyl
-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 -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.
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|
When rats that received prolonged nursing were treated with insulin,
the activity levels of fucosyltransferase (with asialofetuin) and
-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
-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
-1,2-fucoproteins with a molecular
mass (100-110 kDa) similar to those of the
-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 |
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
-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
-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,
-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
-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
-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
-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
-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
-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.
 |
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