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*
Université Claude Bernard Lyon 1, BP 12, 69600;
The Institut National de la Santé et de la Recherche Médicale (Unité INSERM 189);
**
the Centre National de la Recherche Scientifique (SDI CNRS), Département de Biochimie, Faculté de Médecine Lyon-Sud, Oullins, France
2To whom correspondence should be addressed. E-mail: biol{at}lyon-sud.univ-lyon1.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • small intestine • polyamines • galactosyltransferase • glycoprotein • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and digestive
adaptation to nutritional changes (a decrease in lactase activity,
rises in sucrase, maltase and aminopeptidase activity)
(2)
.
In the intestine, glycoproteins play important functional roles.
Mucins, which are mucosal surface factors secreted by the goblet cells
as constituents of the mucus, are involved in intestinal permeability
(3)
and in the barrier function (4)
, whose
characteristics change during postnatal development. On the other hand,
most of the enzymes in enterocyte brush border membranes are
glycoproteins (lactase, sucrase, maltase, aminopeptidase and alkaline
phosphatase) whose activities are also considerably modified during
postnatal development to enable the animal to cope with the solid diet
of adulthood (1)
. Changes in intestinal glycoprotein
glycosylation have been demonstrated during postnatal development.
Studies have revealed a shift from high sialylation (before weaning) to
high fucosylation (after weaning) in the brush border membrane
glycoproteins (5
6
7)
and the mucins (8)
in
parallel with changes in the activity of the glycosyltransferases
involved in fucosylation and sialylation (9
10
11
12)
. Fucose
and sialic acid are generally linked to the nonreducing termini of the
external sugars in the glycan chains (often to galactose residues). Two
galactosyltransferases responsible for galactose linkage to
glycoproteins have been found in rat small intestine, i.e., a
O-glycan: GalNAc-ß1,3-galactosyltransferase and a
N-glycan: GlcNAc-ß1,4-galactosyltransferase
(13
,14)
. Both galactosyltransferase activities in the
small intestine have been found to increase at the end of the 3rd wk of
life (15
,16)
at the same time as fucosyltransferase
activity. However, the effect of the glycan chains on the biological
activity of glycoproteins is largely unknown. Differences in mucin
composition (especially that of their glycan chains)
(8)
between neonatal and mature rats may affect the
barrier function (4)
. During postnatal development, the
active form of lactase observed before weaning is sialylated, and the
inactive form observed after weaning is fucosylated
(17
,18)
. It has been proposed that the structure of the
glycan chains (particularly that of the complex chains) may be
important for the transport of lactase and/or sucrase and for their
integration into the apical membrane of the enterocyte
(17
,19)
.
The nature of the signals that regulate small intestine maturation are
still not fully understood. Numerous studies report that the postnatal
maturation of the digestive tract is controlled by hormonal factors
(20
21
22
23
24
25)
, but maturation factors, such as polyamines, are
also involved in this phenomenon. Polyamines are vital for the
functioning and renewal of the gut epithelium. Whether derived from
extracellular sources, particularly from the diet (26)
, or
from the biosynthesis pathway, polyamines play a role in such
phenomena. The oral administration of polyamines to suckling rodents
can induce most of the morphological and biochemical modifications that
characterize the intestinal maturation process that is observed at
weaning (27
28
29
30
31
32)
, which suggests that polyamines are
involved in the natural maturation process. Polyamines induce
precocious variations in the activities of some glycoproteinic
digestive enzymes in suckling rats (27
28
29
30)
, but their
involvement in the regulation of intestinal glycoprotein glycosylation
is not well known. In a previous article (33)
, we showed
that spermine, administered to suckling rats, can induce a precocious
increase in fucosyltransferase activity, indicating that some
polyamines might be involved in the regulation of glycoprotein
glycosylation during postnatal development.
Our objective in this study was to find out whether the natural rise in polyamine levels observed in the small intestine at weaning could explain the large variations in galactosylation that occur during this period. To this end, polyamines were administered orally to immature rats to reproduce levels of intestinal polyamines similar to those of weaned rats and to study their effects on the galactosyltransferase activity and the galactosylation of glycoproteins in the brush border membranes. The effects of different diets (containing various levels of polyamines) on the galactosylation process were studied at weaning.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Submaxillary gland mucin, fetuin, UDP-galactose and most of the other chemical products were obtained from Sigma-Aldrich Chimie (Saint Quentin Fallavier, France), UDP-14C-galactose from New England Nuclear Life Science Products (Boston, MA), digoxigenin-labeled lectins of Galanthus nivalis and Arachis hypogaea from Roche Diagnostics (Meylan, France), biotin-labeled lectin of Ricinus communis from Vector (Burlingame, CA), and alkaline phosphatase-labeled antidigoxigenin antibodies and alkaline phosphatase-labeled streptavidin from Roche Diagnostics.
Animals and treatments.
One day after birth, the pups [Sprague Dawley strain (IFFA CREDO,
L’Arbresle, France)] were divided into groups of 10 suckling males
and were maintained at controlled temperature of 21°C on a 12:12-h
light-dark cycle. The dams were fed a solid commercial diet (Ico;
UAR, Villemoisson sur Orge, France). The rats were weaned to the same
diet after the age of 19 d. The reason for using only male rats in
the experiments was to avoid excessive differences in body weights
(females being generally smaller than males) and the possible influence
of differences in hormonal status. From 10 to 14 d of age, pairs
of pups were given 0.4 µmoles of polyamine/g body (putrescine,
spermidine or spermine) or of the polyamine precursor (ornithine) in
water, orally once each day. In each group, a pair of rats (the control
group) received water alone in the same way. To avoid stress, the
products were not administered with a gastric canula but directly by
mouth using a syringe fitted with a soft nozzle tip. For the experiment
on 22-d-old rats fed different diets, three groups of rats were
studied. One group of eight pups (milk 14) was suckled between d 1 and
d 14 of life by a dam with a lactation time similar to the age of the
rats. When pups were 14 d of age, the dam was replaced by another,
whose lactation time was 7 d; they were suckled until the d 22 of
life so that the dam’s lactation time was only 14 d when the
suckling rats were 22 d. A second group of eight pups (milk 22)
was suckled between d 1 and d 22 of life by a dam with a lactation time
similar to the age of the rats, so that the dam’s lactation time was
22 d when the suckling rats were 22 d. For these two groups,
the rats were kept in special cages where they could not attain the
food given to the dam (11)
. A third group of eight pups
(Ico) was suckled between d 1 and d 18 of life by a dam with a
lactation time similar to the age of the rats, after which they were
abruptly weaned to a commercial diet (Ico) until they were 22 d
old. The experimental protocols were approved by the French Ministry of
Agriculture and Forests, Veterinary Department (Permit No. 69000581).
Milk collection.
Milk was obtained from anesthetized dams injected with 5 mU oxytocin/g
of body to stimulate milk recovery. Milk samples (250–700 µL) were
collected manually as quickly as possible (
15 min) in cryotubes
maintained at 4°C during the collection time to avoid as much as
possible the polyamine degradation by the milk polyamine oxidase. They
were immediately stored at -180°C pending the polyamine assays.
Cell fractionation.
The rats were killed by decapitation and their small intestines
removed, flushed with cold 9 g/L NaCl and opened. For each control and
assay group, the mucosae of two small intestines from 14-d-old rats or
one small intestine from a 22-d-old rat were harvested with a glass
slide and homogenized in 10 mmol/L Tris-HCl, 10 mmol/L KCl, 10
mmol/L MgCl2, 250 mmol/L sucrose (pH 7.4) buffer with a
Potter-Elvehjem homogenizer (Merck-Eurolab, Strasbourg, France)
(9 mL/g of wet tissue). Microsomal pellets were prepared by
centrifuging the homogenate at 30,000 x g (JA14
rotor) for 30 min in a J2-21 centrifuge (Beckman Instruments, Palo
Alto, CA), followed by centrifuging the supernatant at 140,000 x g (R70Ti rotor) for 1 h 30 min in a L7-65
centrifuge (Beckman Instruments) and quickly stored at -20°C. The
brush border membranes were prepared by the CaCl2
precipitation technique of Kessler et al. (34)
.
Polyamine determination.
Small intestines were quickly removed, washed with 9 g/L NaCl at 4°C, and intestinal mucosae (maintained at 4°C) were scraped with a glass slide and quickly homogenized in cold deionized water; the homogenates were immediately frozen in liquid nitrogen (to avoid polyamine degradation by diamine and polyamine oxidases) and stored at -180°C pending the polyamine determinations. Just before polyamine determination, the samples of milk, commercial food and the intestinal mucosae were conveniently diluted in deionized water at 4°C, one volume of cold 10% trichloracetic acid was immediately added, and 4 µmol/L of 1,6-hexanediamine was added as an internal standard. The mixture was maintained at 4°C and the proteins were discarded after precipitation for 15 min and centrifugation at 4000 x g for 10 min at 4°C. The supernatants were neutralized at pH 7.0 with 1.2 volumes of 1 mol/L sodium borate (pH 8.5) at 4°C. Derivatization of polyamines was performed with 1 mmol/L 9-fluorenylmethyl chloroformate for 45 s at room temperature, and stopped by the addition of 5 mmol/L glycine. The polyamines were quantified by HPLC after separation on a reverse-phase C18 column (Ultrasphere-ODS, 150 x 4.6 mm, 5 µm; Beckman Instruments) with a precolumn packed with the same support (45 x 4.6 mm) on a 625LC HPLC system (Waters, Milford, MA). A polyamine elution was performed for 3.5 min by a first isocratic phase with 75% of solvent A [70% of 50 mmol/L acetic acid (pH 4.2), 30% of acetonitrile] and 25% of solvent B (acetonitrile), followed by a nonlinear gradient for 21.5 min to 38% solvent A, 62% solvent B, then a linear gradient for 2.5 min to 100% solvent B and an isocratic phase for 5 min in the same solvent. A return to the initial state was obtained by a linear gradient for 2.5 min. The flow rate was 1.5 mL/min. Fluorescence was monitored using a Waters 474 scanning fluorescence detector at an excitation wavelength of 260 nm and an emission wavelength of 315 nm. The polyamines were identified by their retention times and routinely compared with standards. They were quantified by comparison with concentration curves determined for each standard polyamine, using the internal standard.
Chemical determinations.
Protein levels were determined by the method of Schaffner and
Weissmann (35)
.
Determination of galactosyl-transferase activity.
The activity of the glycoprotein: galactosyltransferases was determined
with exogenous glycoproteinic substrates, respectively, asialomucin for
ß-1,3-galactosyltransferase, which links galactose to the
N-acetylgalactosamine residue in
O-glycans, and asialoagalactofetuin for
galactosyltransferase, which can link galactose to the terminal
N-acetylglucosamine of N-glycans either
by ß-1,3 or ß-1,4 linkages. The glycoproteinic substrates were
prepared according to the method of Ko and Raghupathy (36)
from bovine submaxillary gland mucin and fetuin. Microsomal pellets
were suspended in 10 mmol/L Tris-HCl, 10 mmol/L KCl, 10 mmol/L
MgCl2, pH 7.4 buffer (protein concentration 1.5–2.0 g/L).
The reaction mixture (in 250 µL) contained 400–500 µg of
microsomal proteins, 0.8 mg/mL asialomucin or asialoagalactofetuin, 1
mmol/L MnCl2, 10 mmol/L adenosine 5'-monophosphate,
0.25% Triton X-100 and 50 µmol/L UDP-14C-galactose
(specific activity 12.0 GBq/mmol). The incubation was for 30 min at
37°C, and the reaction was found to be linear up to 45 min. To
determine the effect of polyamines in vitro, ornithine, putrescine,
spermidine or spermine was added to the reaction mixture to obtain
final concentrations of between 10-10 and
10-4 mol/L. The reactions were stopped by
precipitation of the proteins with 20% trichloracetic acid. The
radioactive glycoproteins were collected on GF/B fiberglass filters
(Whatman, Maidstone, UK), and the radioactivity was determined using
Toluene Scintillator (Packard Instruments, Groningen, The Netherlands).
The glycosyltransferase activity was calculated after subtraction of
the endogenous activity (determined without an exogenous acceptor).
Detection of galactose residues in the glycoproteins of the brush border membrane.
Proteins in the intestinal brush border membranes were resolved by
electrophoresis on a 0.1% sodium dodecyl sulfate, 7.5% gel
acrylamide, then electrotransferred onto a nitrocellulose membrane
(Schleicher & Schüll, Dassel, Germany). The glycoprotein
oligomannosidic chains were detected as previously described
(11)
, in 50 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween 20
(pH 7.5) buffer using a digoxigenin-labeled lectin (1.5 mg/L) from
G. nivalis. The galactose residues
preferentially linked in ß1,3 to GalNAc, and those
preferentially linked in ß1,4 to GlcNAc, were detected using,
respectively, digoxigenin-labeled lectin (5 mg/L) of A.
hypogaea, and biotin-labeled lectin (2 mg/L) of R.
communis. Then, digoxigenin was recognized with 750 U/L of an
alkaline phosphatase-labeled antidigoxigenin antibody and biotin
with 1 kU/L of alkaline phosphatase-labeled streptavidin, followed
by revelation of alkaline phosphatase with 1.8 mmol/L nitro-blue
tetrazolium and 0.5 mmol/L 5-bromo-4-chloro-3-indolylphosphate.
Statistical analysis.
All numeric data are expressed as means ± SEM. To make comparisons among several groups, the results were subjected to a one-way ANOVA. When the F test indicated a significant effect, the differences among the means were analyzed by the Newman Keuls test, with significance set at P < 0.05. For comparisons between pairs of groups, Student’s t test was used. All calculations were carried out using Instat (GraphPad, San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polyamines in the diet.
In rats, natural weaning takes place progressively between 18 and
21–22 d of life. Polyamines are nutritional components, which are
present both in milk and in the commercial diet given to our rats at
weaning, and the levels of some polyamines rose at this time. In the
milk (whose average density is near 1.05), the levels of
spermidine were actually higher than those of putrescine and spermine
(Fig. 1
), and the levels of all polyamines were similar between birth and d 14
of lactation (data not shown). As seen in Figure 1
, when expressed as
nmol/g of wet food ingested, the spermidine level in the milk by the
end of lactation on d 22 (milk 22) had approached that of the wet
commercial diet (Ico) and was higher than in the milk at the beginning
of lactation, on d 14 (milk 14). The spermine level in the milk at
22 d of lactation was between its levels in the milk at 14 d
of lactation and in the commercial diet. For putrescine, the levels
were unchanged during lactation, but the level was much higher in the
commercial diet than in the milk.
|
shows that, in terms of nmoles of polyamine/g of wet food
ingested by the rats, the commercial diet Ico, given from weaning time
until adulthood, contained polyamine levels higher than in the milk at
d 14 of lactation. However, the dry matter was largely lower in the
milk (14.2 ± 3.9%, n = 8) than in the commercial
diet (81.4 ± 1.2%, n = 7). The 14- and the
28-d-old rats ate similar quantities (in weight) of food, when
expressed per g of body weight [0.2 g of wet milk or of wet
commercial diet/(g body·d)]. If we assume that the 14-d-old suckling
rats (with an average body weight of 30 g) consumed 6 mL milk/d
[0.2 g of milk/(g body·d)], as shown by Godbole et al.
(37)
and indicated by others (30
,38)
, their
daily average consumption of polyamines would be in the range of 0.7
nmol putrescine/g body, 12 nmol spermidine/g body and 0.5 nmol
spermine/g body. At 28 d of age, because the weaned rats (with an
average body weight of 90 g) consumed 18–20 g of solid food per
day [0.20–0.23 g of food/(g body·d)], polyamine
consumption per day was, therefore, 48–54 nmol putrescine/g body,
18.5–20.5 nmol spermidine/g body and 3.2–3.5 nmol spermine/g body.
Thus, the 28-d-old weaned rats’ consumption of polyamines/g body was
higher than that of the 14-d-old suckling rats: on average 60- to
70-fold for putrescine, 1.5- to 1.7-fold for spermidine and 6.5- to
7.0-fold for spermine. Polyamine content of the small intestines of suckling and weaned rats. In the small intestine mucosae of the suckling and weaned rats, the concentration of spermidine was always higher than that of putrescine and spermine. In the suckling rats, the levels of putrescine, spermidine and spermine did not change significantly between 7 and 18 d of age. The spermidine and spermine levels rose considerably after weaning on d 22, then remained relatively stable until adulthood, whereas the putrescine levels fell after weaning. For instance, the spermidine and spermine levels were higher in the mucosae of the 28-d-old weaned rats (respectively, 18.6 ± 2.0 nmol spermidine/mg protein and 5.0 ± 0.6 nmol spermine/mg protein, n = 10) than in those of the 14-d-old suckling rats (respectively, 9.3 ± 1.0 nmol spermidine/mg protein, n = 10, P < 0.001, and 3.4 ± 0.4 nmol spermine/mg protein, n = 10, P < 0.050). In contrast, the putrescine level was lower in the mucosae of the weaned rats (1.1 ± 0.2 nmol/mg protein, n = 10) than in those of the suckling rats (2.2 ± 0.2 nmol/mg protein, n = 10, P < 0.001).
Galactosyltransferase activity in the small intestines of suckling
and weaned rats.
The maturation of galactosyltransferase activity takes place naturally
in the small intestine at weaning time. In the present study, the
activities of O-glycan:
ß1,3-galactosyltransferase and N-glycan:
ß1,4-galactosyltransferase were low in the suckling rats, but
increased between weaning (at 22 d of life) and adulthood, as
shown in Table 1
, where the enzyme activities of the 14-d-old suckling rats were
compared with those of the 28-d-old weaned rats.
|
). In contrast, neither the level of putrescine nor that of the other
polyamines was significantly affected by putrescine treatment.
Treatment with ornithine had no effect on the polyamine content of the
mucosa.
|
). In contrast, treatment with the same amount of putrescine or
ornithine did not significantly affect either type of
galactosyltransferase activity.
|
for spermine.
|
, and B
), whereas putrescine (Fig. 4A
, and B
) and ornithine (results not shown) had no
effect. Two proteins containing ß-1,3-galactose residues (molecular
mass: 95 and 100 kDa) were strongly detected after spermidine and
spermine treatment (Fig. 4A
), and four proteins containing
ß-1,4-galactose residues (molecular mass: 65, 105, 110 and 200
kDa) were more strongly detected after spermidine and spermine
treatment (Fig. 4B
). These results are consistent with the
fact that the number of oligomannosidic chains, detected with the
G. nivalis lectin, was lower in membranes from the
spermidine- and spermine-treated rats than in those from the
control rats (Fig. 4C
), indicating the appearance of a high
number of complex chains.
|
. The O- and
N-glycan: galactosyltransferase activities were
significantly lower in the intestines of the 22-d-old rats suckled with
the milk whose polyamine levels were the lowest (milk 14) than in the
intestines of the 22-d-old rats suckled with the milk whose polyamine
levels were the highest (milk 22; Fig. 5
). These results indicate a possible role of the increase in the milk
polyamine concentration in the maturation of the galactosylation
process, because the rats were the same age. The two types of
galactosyltransferase activity were found at a higher level in the
intestines of the 22-d-old rats fed the commercial diet after weaning
than in the two groups of rats fed milk (milk 14 and milk 22). In terms
of nmol polyamines/g of wet food, the commercial diet contained the
same level of spermidine as the milk on d 22 of lactation but a higher
level of spermine (Fig. 1)
. In regards to the presence of the galactose
residues in the glycoproteins of the brush border membranes, no
significant difference was observed between the intestines of the rats
fed the commercial diet and the milk containing the highest level of
polyamines (milk 22), but the detection of the galactose was weaker in
the rats fed the milk containing the lowest level of polyamines (milk
14).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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28
29
30
31
32)
, and
play a role in the natural maturation process of the small intestine.
The rat small intestine is immature until the end of wk 3 of life,
which corresponds to weaning. The natural maturation of glycoprotein
galactosylation is expressed by a rise in galactosyltransferase
activity and in the more complex glycan chains of glycoproteins that
appear in the small intestine at this period. This study, like others,
showed that the intestinal levels of spermidine and spermine increased
at the end of wk 3 (39)
in proportion to the food intake
of spermidine and spermine (38
,40)
, which is higher in the
weaned rats than in the 14-d-old suckling rats (in terms of nmol
ingested/g body). On the contrary, there was no relationship between
the amount of putrescine ingested by the rats and its level in the
intestine.
Our aim in this study was to reproduce, in suckling rats, intestinal
levels of spermidine and spermine similar to those observed in weaned
rats to find out whether these polyamines might be responsible for the
maturation of glycoprotein galactosylation that occurs naturally at
weaning. We found that the ingestion of spermidine or spermine by
14-d-old suckling rats induced an increase of both polyamines in the
small intestine to the levels observed in the intestines of weaned
rats, as also found by Dufour et al. (27)
, whereas the
ingestion of putrescine and ornithine did not induce any change in
intestinal polyamine levels. The rises in spermidine and spermine in
the enterocytes of suckling rats treated with spermidine or spermine
were probably due solely to the intake of these polyamines at the
apical pole of the epithelial cells and not to changes in their
biosynthesis, because the activity of the key enzyme of the
biosynthetic pathway, ornithine decarboxylase, has not been observed to
be increased by such treatments (27
,41)
. We found that
treatment of suckling rats with spermidine or spermine induced a
precocious increase in the O-glycan:
ß-1,3-galactosyltransferase activity and of the N-glycan:
ß-1,4-galactosyltransferase activity to the levels observed in weaned
rats, whereas neither putrescine nor ornithine treatment induced any
change. In parallel, an increase in the number of galactose residues
linked in ß-1,3 or in ß-1,4 in the complex chains of glycoproteins
was observed in the brush border membranes of the spermidine- and
spermine-treated rats, as was confirmed by the observed decrease in
the oligomannosidic chains. Treatments with spermidine (unpublished
data) and spermine (33)
were also found to induce a
precocious maturation of the fucosylation of glycoproteins in suckling
rats. The mechanism by which spermine and spermidine induce precocious
maturation of the intestinal glycosylation process in suckling rats is
still poorly understood. It seems improbable that these two polyamines
have a direct effect on the enzyme proteins, because we have shown that
they have no effect in an acellular medium in vitro, although the
action of such molecules on the cell membrane environment (and, thus,
on the active sites of the enzymes) cannot be ruled out. The effect of
spermidine and spermine on the biosynthesis of galactosyltransferases
is possible because spermine interacts with DNA. However, some authors
have discussed the indirect effect of polyamines acting via secondary
effectors, e.g., corticoids and insulin (41
,42)
,
gastrointestinal hormones (42)
or a cytokine-dependent
mechanism (43)
. Spermidine and spermine are important
maturation factors for many glycoproteins in the brush border membranes
in terms of enzyme activity (as described for lactase, sucrase and
maltase (27
28
29
,31)
, but also in terms of maturation of
their complex glycannic chains (galactosylation and fucosylation
(33)
). However, these mechanisms stay to be enlightened
with more precision.
Contrary to our observations on spermidine and spermine, we found no
relationship between the levels of putrescine in the intestine and in
the diet. Indeed, putrescine level in the intestine was lower in the
weaned than in the suckling rats, despite a higher level in the solid
food than in the milk. In contrast, its level in the intestine of the
suckling rats was not affected by 4 d of treatment with
putrescine. This surprising result could be due to differences in the
uptake of the polyamines by the enterocytes, decomposition or
conversion of the polyamines. Putrescine and spermidine/spermine have
different carriers (44)
, and the uptake characteristics of
putrescine at the apical membrane of the intestinal cells may differ
from those of spermidine and spermine. The lack of effect of treating
the suckling rats with putrescine for 4 d on the intestinal
putrescine content (determined 24 h after the last ingestion) is
consistent with the absence of any precocious maturation of the
galactosylation process. These results were probably due to a quick
decomposition of putrescine, since we have found that 2 h after
the ingestion of putrescine by the suckling rats, its level in the
intestine temporarily increased, and did not undergo any apparent
conversion into spermidine or spermine (data not shown). Bardocz et al.
(26)
have shown that polyamines from extracellular sources
are partly metabolized during the absorption process and that
putrescine is metabolized more than spermidine or spermine. One hour
after putrescine administration, 80% of putrescine was converted into
nonpolyamines metabolites, and especially amino acids, whereas 70–80%
of spermidine and spermine remained in their original forms after
spermidine and spermine administration. We have also shown that
putrescine may be destroyed quickly in the putrescine-treated rats,
whose diamine oxidase activity was twice as high as in the spermidine-
and spermine-treated rats (unpublished data). In contrast,
putrescine (despite the fact that its level increased temporarily in
the putrescine-treated suckling rats) might have no effect on the
intestinal maturation process. Yuan et al. (45)
have shown
that putrescine by itself is not sufficient for the migration and
growth of IEC-6 cells originating in the intestine, unlike spermidine
and spermine. On the other hand, the involvement of ornithine in the
amino acid biosynthetic pathway and the low level of activity of
ornithine decarboxylase in the suckling rat intestine (39)
may be the reasons why ornithine has no effect on intestinal polyamine
content.
The direct effect of the levels of spermine and spermidine ingested by
the rats at the end of wk 3 may partly explain the galactosylation
maturation observed at this period, because in the 22-d-old rats fed
two different milks, galactosyltransferase activity was higher in the
intestines of rats fed milk containing a high level of the two
polyamines (milk 22) than in those of rats fed a low level (milk 14).
However, galactosyltransferase activities did not attain the levels
found in the 22-d-old rats fed the commercial diet containing higher
levels (expressed in nmol/g wet food) of polyamines than the milk at
the end of lactation (except for spermidine). The higher quantity of
spermine in the commercial diet could be partly responsible for this
difference, but probably is not sufficient to explain it. Other
differences between the composition of the milks and the commercial
diet (which was rich in carbohydrates) may also have induced changes in
the intestinal microflora (which produces an increase in exogenous
polyamines in the intestinal lumen) and the circulating insulin, whose
level was increased by the commercial diet, and which is also able to
increase glycoprotein glycosylation (25
,16)
. The role of
some hormonal factors, such as insulin, during this period is already
known (16
,25)
and it is possible that there are
interrelations between insulin and polyamines because Buts et al.
(46)
have demonstrated the effect of insulin on spermine
uptake and on spermidine and spermine levels in the intestine. Thus,
the maturation of the intestinal galactosylation of glycoproteins could
be a multifactorial event in which spermidine and spermine are
involved.
In conclusion, increases in the intestinal levels of spermidine and spermine after their oral ingestion by immature suckling rats appears to induce a precocious maturation of the glycoprotein galactosylation process similar to that observed at weaning, unlike the ingestion of putrescine or its precursor (ornithine), which induce no change. Spermidine and spermine, ingested in higher quantities by rats at the end of lactation or after weaning than by rats at the beginning of lactation, could be at least partly responsible for the postnatal intestinal maturation of glycoprotein galactosylation observed around the weaning.
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FOOTNOTES |
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Manuscript received October 15, 2000. Initial review completed January 8, 2001. Revision accepted April 10, 2001.
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