| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 University of Liege, Department Biochemistry and General Physiology, Immunology Center, Institute of Chemistry B6c, B-4000 Liege (Sart-Tilman), Belgium
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
(Received 14 July 2005;
accepted after revision 31 August 2005; first published online 5 September 2005)
Corresponding author G. Dandrifosse: Department of Biochemistry and General Physiology, Institute of Chemistry B6c, B-4000 Liege (Sart-Tilman), Belgium. Email: g.dandrifosse{at}ulg.ac.be
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
A time course analysis of the biochemical and histological modifications occurring after the ingestion of a single dose of spermine showed that from 4 to 10 h after administration, spermine drastically altered the integrity of the mucosa without disrupting the epithelium (Kaouass et al. 1996). The function of the mucosa was also impaired by a reduction in the lactase and maltase specific activities (SA; Wery et al. 1996) owing to cell loss by apoptosis (Peulen et al. 2001). The intestinal weight was significantly reduced by spermine ingestion (Wery et al. 1996). Between 30 and 40 h after spermine administration, intestinal weight and maltase SA recovered, and sucrase SA appeared in the jejunum and ileum. These parameters increased until 70 h after spermine administration. Lactase SA remained low between 10 and 60 h after spermine ingestion (Wery et al. 1996). Histological study showed that 48 h after spermine treatment, the mucosa had totally regenerated. The large supranuclear vacuoles (LSV) disappeared from ileum (Kaouass et al. 1996). The same modifications were observed when spermine was administered twice a day for 3 days (Dufour et al. 1988).
These observations suggest that spermine-induced intestinal maturation is a two-step phenomenon. The first step, fast and transient, is the elimination of immature enterocytes localized at the tip of the villus. The second step, longer than the first one, is the replacement of preceding immature cells by adult-type enterocytes.
Previous study has also shown that the spermine-induced maturation was reversible when administration was stopped after 2 days. Clear reappearance of immature intestinal features occurred about 2–3 days after the end of treatment (Georges et al. 1990).
This study was undertaken in order to check whether time extension of the polyamine administration after the appearance of the spermine-induced maturation could retain the mature state of the small intestine.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Wistar rats, housed in an air-conditioned room at 23°C with a 12 h:12 h light:dark cycle, were used throughout the study. They were fed with A03 10 mm pellets (Pavan Service, Brussels, Belgium) and had access to water ad libitum. The litters were reduced to 10 pups per lactating mother with free access between mother and pups. The day of birth was designated as day 0. In our experiments, there was no difference in body weight between male and female pups, and no distinction between genders was made. Since it is well known that the experimental values vary from one litter to another, the comparisons of results were always made between animals from the same litter. All animal experiments were approved by the Animal Welfare Committee of the University of Liege and of the Fonds de la Recherche Scientifique Médicale (FRSM).
Chemicals
All chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA), from Merck (Darmstad, Germany) or from Roche Applied Science (Basel, Switzerland).
Experimental procedure
Five litters, each of nine or 10 pups, were used throughout the study. The initial age of the pups was 8 days. In each litter, five animals received spermine per os (0.4 µmol (g body weight)–1) as previously described (Dufour et al. 1988). The remaining pups were used as control animals and received vehicle. Treatment was repeated daily until the animals were killed by cervical dislocation. Litters were killed 3, 4, 5, 6 or 7 days after the beginning of the treatment. Pups were then, respectively, 11, 12, 13, 14 or 15 days old. The small intestines were harvested and divided in two pieces of equal length designated jejunum (proximal part) and ileum (distal part). A small piece from each part was prepared for histological analysis (Bouin's fixative, and Haematoxylin and Eosin staining). The remaining pieces of the small intestine were homogenized in water (5 ml g–1) with an Ultra-Turrax disperser. Homogenates were kept frozen at –70°C until analysis.
Enzymatic analysis
Sucrase (EC 3.2.1.48 [EC] ), maltase (EC 3.2.1.20 [EC] ) and lactase (EC 3.2.1.23 [EC] ) activities were assayed according to Dahlqvist (1964, 1968). Enzyme activities were expressed as micromoles substrate hydrolysed per minute and per gram of intestinal proteins (specific activity; SA).
Protein analysis
The protein content of the homogenates was estimated by Bradford's method (Bradford, 1976) using bovine serum albumin as protein standard.
Statistical analysis
The results are reported as means ± S.E.M. Statistical analysis was performed using one-way ANOVA for comparison between groups. Kruskall-Wallis test was used when heteroscedasticity was suspected. Heteroscedasticity was assayed by Levene's test. P < 0.05 was considered as statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Intestinal weight
The changes in intestinal weight of 11- to 15-day-old rats is shown in Fig. 1. Animals, 8 days old at the beginning of the treatment, received spermine per os for 3–7 days. The weight of the jejunum and ileum of spermine-treated rats was significantly greater than that of control animals except after 5 days of treatment.
|
In control rats, at any time point, lactase SA was high, maltase SA was low and sucrase SA was undetected (Fig. 2). In spermine-treated rats, disaccharidase SA changed with time. On day 11, 3 days after the beginning of spermine administration, sucrase SA in the jejunum was significantly higher than in control rats. Time extension (for 4–6 days) of spermine administration led to a reduction, with a time-dependent pattern, of the amplitude of the sucrase SA increase. However, this SA remained significantly higher than in control rats. When spermine treatment lasted for 7 days, an increase in sucrase SA was observed; the SA appeared significantly higher than after 6 days of treatment (P < 0.001) but lower than after 3 days of treatment (P < 0.001). Sucrase SA in the ileum and maltase SA in the jejunum changed according to the same pattern. In the ileum, on day 11, 3 days after the beginning of spermine administration, maltase SA was significantly higher than in control rats. At days 4–7 of spermine administration, maltase SA returned to control values. Concerning lactase, spermine-treated rats showed, at any time, a reduced SA in comparison with control rats.
|
The jejunum of animals treated with spermine for 3–7 days was more developed (larger diameter, thicker and with more numerous villi) than in control rats (results not shown). This observation is consistent with the increased intestinal weight observed (Fig. 1).
Figures 3–5 show the histological evolution of ileum mucosa when spermine was administered to suckling rats over 3–7 days. In control rats, at any time, we observed a suckling-type mucosa characterized by LSV-containing enterocytes. Spermine administration, once a day for 3 days, induced the disappearance of the LSV. Time extension (for 4–7 days) of spermine administration led to a gradual reappearance of the LSV in enterocytes (Figs 4 and 5). This reappearance was very obvious after 7 days of treatment.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The physiological significance of the spermine-induced small intestine maturation is supported by the concentration of this molecule in milk throughout the lactation period. In rat milk, spermine and putrescine concentrations are low (generally less than 2.5 µM for putrescine and less than 1 µM for spermine; Romain et al. 1992). The spermidine concentration is higher and seems to increase during lactation. Moreover, the rat food contains more polyamines than the rat milk, suggesting that polyamines contained in rat food could play an important role in postnatal maturation of the rat intestine. All human foods contain some polyamines (up to 300 nmol g–1), although the concentrations in different individual food components are variable (Bardocz et al. 1993). Food appears to constitute the major source of polyamines for humans and animals. In the jejunum of adult rats, the spermidine concentration is about 0.1 mM and the spermine concentration is about 0.5 mM (Hinuma et al. 1992). In young humans, the duodenal fluid contains about 95 µM spermidine and 46 µM spermine (McEvoy & Hartley, 1975). In adult humans, the jejunum concentration is about 86 µM for putrescine, 25 µM for spermidine and 3.75 µM for spermine (Benamouzig et al. 1997). All the natural polyamines (putrescine, spermidine and spermine) seem to be efficient in inducing gut maturation (Dufour et al. 1988; Dorhout et al. 1997; Peulen et al. 2000).
In order to follow the evolution of spermine-induced maturation when this molecule is administered for more than 3 days, we treated 8-day-old rats, once a day for 7 days, with 0.25–0.4 µmol (g body weight)–1 of spermine. The eighth postnatal day was chosen to begin the treatment in order to avoid interference between spermine-induced maturation and weaning-associated maturation beginning on day 17 postnatal.
Our results showed that intestinal weight in spermine-treated rats was higher than in control rats (Fig. 1), indicating that the trophic effect of spermine, observed after 3 days of ingestion, was maintained when treatment with this polyamine was continued for a longer time. Hyperplasy and/or hypertrophy could explain this trophic effect. Our results do not exclude either of these possibilities. Spermine is well known for its effect on cell proliferation (Basu et al. 1989), but also for its effect on the appearance of Na+,K+-ATPase in the small intestine (Wild et al. 1993), an enzyme involved in the control of cell volume.
After 3 days of spermine administration, disaccharidase SA were comparable to those observed in adults, as already reported (Dufour et al. 1988). It was suggested that oral intake of spermine by suckling rats induces an increase of ACTH followed by an increase in plasma corticosterone concentration by activation of the hypothalamic–pituitary–adrenal (HPA) axis (Kaouass et al. 1994b). It is well known that corticosterone induces precocious intestinal maturation at least partly by increasing sucrase and maltase specific activities (Martin & Henning, 1982). In consequence, as early as 8 days after birth, the HPA axis could be triggered by dietary spermine.
Long-lasting spermine treatment (more than 3 days) could not retain the adult enzymatic pattern. Sucrase and maltase SA decreased and became as low as in the control rats. Maltase and sucrase SA increases are, at least partly, under the control of the HPA axis after 3 days treatment with spermine (Kaouass et al. 1994b). In consequence, a reduction of the SA of these enzymes leads us to suggest an interruption of intestinal cell stimulation by corticosterone. This interruption could happen in several ways, as follows. (1) Spermine-induced adult-like enterocytes (or the spermine-induced adult-like epithelium) might become impermeable to polyamines. In these conditions, spermine could be unable to induce the production of soluble factors (cytokines) responsible of the stimulation of the HPA axis. (2) The mucosa might become insensitive to spermine, unlike the situation in immature cells, and thus would not stimulate secretion of HPA-activating factor. (3) The age of the pups might be a factor. The HPA axis might become unresponsive to spermine (or to spermine-induced factors) with increasing age. (4) The ‘mature’ state of the mucosa might make it unresponsive to corticosterone.
Luminal spermine uptake by enterocytes has already been documented in detail (Milovic, 2001). Polyamines are initially bound to the apical membrane of enterocytes and subsequently transported across this lipid bilayer via specific carriers. In the case of the transport of polyamines across the basolateral membrane of enterocytes, the mechanism seems to be carrier mediated. In unweaned rats, spermine is quickly taken up by enterocytes (Wery & Dandrifosse, 1993; Wery et al. 1996). In weaned rats, spermine administration leads to an increase of mucosal spermine concentration (Peulen et al. 2004), indicating an uptake of the spermine or a modification of the polyamine metabolism. Results obtained recently with rats between 11 and 31 days old support the second hypothesis (Peulen et al. 2004).
As already mentioned, we observed in 11- to 18-day-old rats, 6 h after a single dose of spermine, a decrease of disaccharidase SA, indicating cell loss. This decrease was not observed when rats were more than 18 days old, suggesting an age-dependent sensitivity to spermine. This sensitivity could be related to the expression of metallo-enzymes as collagenase (Peulen et al. 2001) or meprin (Peulen et al. 2004).
The HPA axis could be insufficiently developed to ensure an irreversible maturation of the small intestine. Indeed, basal plasma corticosterone concentration changes with postnatal time. A significant increase is observed 14 days after birth (Henning, 1978). From this time, plasma corticosterone concentration increases daily until the day 24 postnatal. The increase observed at day 14 acts as a signal for the development of sucrase SA (Henning, 1978). The spermine-induced increase of plasma corticosterone concentration (Kaouass et al. 1994b) should play the same role. However, the basal plasma corticosterone concentration could be insufficient to retain a ‘mature’ type epithelium. Moreover, in weaned rats, the activity of the HPA axis follows a circadian rhythm (Beam & Henning, 1978; Barbason et al. 1995). This rhythmicity is not achieved before weaning (Barbason et al. 1974).
According to our last hypothesis, intestinal ‘mature’ cells might be insensitive to corticosterone. Indeed, in mice from day 17 postnatal, epithelial cells are unresponsive to corticosterone (Henning & Sims, 1979; Henning & Leeper, 1982). This characteristic could be achieved precociously due to spermine ingestion. In this case, in the context of the weaning-associated intestinal maturation, the ‘mature’ state of the epithelium would be maintained by a corticosterone-independent mechanism. It could be linked to cell–cell or cell–matrix interactions (Menard & Calvert, 1991; Kedinger et al. 1998), or to a peculiar molecular state of the DNA in progenitor cells as histone acetylation or DNA methylation (Cerny & Quesenberry, 2004).
Lactase SA changes during long-lasting spermine treatment did not follow the same pattern of cell renewal as proposed to explain the changes in maltase and sucrase SA. The SA of this enzyme remained low throughout the treatment, even when the mucosa resumed a suckling-like state (low maltase SA, low sucrase SA and reappearance of LSV). On the one hand, the results support an HPA-independent control mechanism for this enzyme (Freund et al. 1990, 1991; Krasinski et al. 1994). On the other hand, the control of lactase SA may be influenced by interleukin-2 (Peulen & Dandrifosse, 2004) or gastrointestinal (GI) hormones such as bombesin (Kaouass et al. 1997a). These GI hormones or interleukin could be secreted without interruption during the spermine-induced maturation process.
In conclusion, our results show that there are at least two different mechanisms involved in the spermine-induced maturation of the small intestine. The use of a long-lasting spermine administration should allow us to study the differences between the lactase SA control and the sucrase–maltase SA control.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Barbason H, Van Cantfort J & Houbrechts N (1974). Correlation between tissular and division functions in the liver of young rats. Cell Tissue Kinet 7, 319–326.[Medline]
Bardocz S, Grant G, Brown DS, Ralph A & Pusztai A (1993). Polyamines in food – implications for growth and health. J Nutr Biochem 4, 66–71.[CrossRef]
Basu
HS, Feuerstein
BG, Deen
DF, Lubich
WP, Bergeron
RJ, Samejima
K
&
Marton
LJ (1989). Correlation between the effects of polyamine analogues on DNA conformation and cell growth. Cancer Res
49, 5591–5597.
Beam HE & Henning SJ (1978). Development of the circadian rhythm of jejunal sucrase activity in the weanling rat. Am J Physiol 235, E437–E442.[Medline]
Benamouzig
R, Mahe
S, Luengo
C, Rautureau
J
&
Tome
D (1997). Fasting and postprandial polyamine concentrations in the human digestive lumen. Am J Clin Nutr
65, 766–770.
Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]
Buts JP, De Keyser N, Kolanowski J, Sokal E & Van Hoof F (1993). Maturation of villus and crypt cell functions in rat small intestine. Role of dietary polyamines. Dig Dis Sci 38, 1091–1098.[CrossRef][Medline]
Cerny J & Quesenberry PJ (2004). Chromatin remodeling and stem cell theory of relativity. J Cell Physiol 201, 1–16.[CrossRef][Medline]
Dahlqvist A (1964). Method for assay of intestinal disaccharidases. Anal Biochem 7, 18–25.[CrossRef][Medline]
Dahlqvist A (1968). Assay of intestinal disaccharidases. Anal Biochem 22, 99–107.[CrossRef][Medline]
Dorhout B, van Faassen A, van Beusekom CM, Kingma AW, de Hoog E, Nagel GT, Karrenbeld A, Boersma ER & Muskiet FA (1997). Oral administration of deuterium-labelled polyamines to sucking rat pups: luminal uptake, metabolic fate and effects on gastrointestinal maturation. Br J Nutr 78, 639–654.[CrossRef][Medline]
Dufour C, Dandrifosse G, Forget P, Vermesse F, Romain N & Lepoint P (1988). Spermine and spermidine induce intestinal maturation in the rat. Gastroenterology 95, 112–116.[Medline]
Freund JN, Duluc I, Foltzer-Jourdainne C, Gosse F & Raul F (1990). Specific expression of lactase in the jejunum and colon during postnatal development and hormone treatments in the rat. Biochem J 268, 99–103.[Medline]
Freund JN, Foltzer-Jourdainne C, Duluc I, Galluser M, Gosse F & Raul F (1991). Rat lactase activity and mRNA expression in relation to the thyroid and corticoid status. Cell Mol Biol 37, 463–466.[Medline]
Georges P, Dandrifosse G, Vermesse F, Forget P, Deloyer P & Romain N (1990). Reversibility of spermine-induced intestinal maturation in the rat. Dig Dis Sci 35, 1528–1536.[CrossRef][Medline]
Harada E, Hashimoto Y & Syuto B (1994). Orally administered spermine induces precocious intestinal maturation of macromolecular transport and disaccharidase development in suckling rats. Comp Biochem Physiol A Mol Integr Physiol 109, 667–673.
Henning SJ (1978). Plasma concentrations of total and free corticosterone during development in the rat. Am J Physiol 235, E451–E456.
Henning SJ & Leeper LL (1982). Coordinate loss of glucocorticoid responsiveness by intestinal enzymes during postnatal development. Am J Physiol 242, G89–G94.[Medline]
Henning SJ & Sims JM (1979). Delineation of the glucocorticoid-sensitive period of intestinal development in the rat. Endocrinology 104, 1158–1163.[Abstract]
Hinuma K, Maghsoudloo M, Murphy G & Dowling RH (1992). Dietary and intestinal polyamines in the rat: in vitro transport studies. In Polyamines in the Gastrointestinal Tract, ed. Dowling RH, Fölsch UR & Löser C, pp. 463–472. Kluwer Academic Publishers, London.
Kaouass M, Deloyer P & Dandrifosse G (1994a). Intestinal development in suckling rats: direct or indirect spermine action? Digestion 55, 160–167.[CrossRef][Medline]
Kaouass M, Deloyer P & Dandrifosse G (1997a). Involvement of bombesin in spermine-induced corticosterone secretion and intestinal maturation in suckling rats. J Endocrinol 153, 429–436.[Abstract]
Kaouass M, Deloyer P, Gouders I, Peulen O & Dandrifosse G (1997b). Role of interleukin-1 beta, interleukin-6, and TNF-alpha in intestinal maturation induced by dietary spermine in rats. Endocrine 6, 187–194.[Medline]
Kaouass M, Deloyer P, Wery I & Dandrifosse G (1996). Analysis of structural and biochemical events occurring in the small intestine after dietary polyamine ingestion in suckling rats. Dig Dis Sci 41, 1434–1444.[CrossRef][Medline]
Kaouass M, Sulon J, Deloyer P & Dandrifosse G (1994b). Spermine-induced precocious intestinal maturation in suckling rats: possible involvement of glucocorticoids. J Endocrinol 141, 279–283.[Abstract]
Kedinger M, Lefebvre O, Duluc I, Freund JN & Simon-Assmann P (1998). Cellular and molecular partners involved in gut morphogenesis and differentiation. Philos Trans R Soc Lond B Biol Sci 353, 847–856.[CrossRef][Medline]
Krasinski SD, Estrada G, Yeh KY, Yeh M, Traber PG, Rings EH, Buller HA, Verhave M, Montgomery RK & Grand RJ (1994). Transcriptional regulation of intestinal hydrolase biosynthesis during postnatal development in rats. Am J Physiol 267, G584–G594.
McEvoy FA & Hartley CB (1975). Polyamines in cystic fibrosis. Pediatr Res 9, 721–724.[Medline]
Martin GR & Henning SJ (1982). Relative importance of corticosterone and thyroxine in the postnatal development of sucrase and maltase in rat small intestine. Endocrinology 111, 912–918.[Abstract]
Menard D & Calvert R (1991). Fetal and postnatal development of the small and large intestine: patterns and regulation. In Growth of the Gastrointestinal Tract: Gastrointestinal Hormones & Growth Factors, ed. Morisset J & Solomon E, pp. 159–174. CRC, Boca Raton.
Milovic V (2001). Polyamines in the gut lumen: bioavailability and biodistribution. Eur J Gastroenerol Hepatol 13, 1021–1025.
Peulen O & Dandrifosse G (2004). Spermine-induced maturation in Wistar rat intestine: a cytokine-dependent mechanism. J Pediatr Gastroenterol Nutr 38, 524–532.[Medline]
Peulen O, Deloyer P & Dandrifosse G (2004). Short term effects of spermine ingestion: comparison between suckling and adult Wistar rats. Reprod Nutr Dev 44, 353–364.
Peulen O, Denis G, Defresne MP & Dandrifosse G (2001). Spermine-induced alteration of small intestine in suckling rat: involvement of apoptosis or Zn2+ enzymes? Dig Dis Sci 46, 2490–2498.[Medline]
Peulen O, Grandfils C & Dandrifosse G (2000). Maturation of the small intestine is induced by spermine but not by other similar amines. Pflugers Arch 440, R253–R254.
Peulen O, Pirlet C, Klimek M, Goffinet G & Dandrifosse G (1998). Comparison between the natural postnatal maturation and the spermine-induced maturation of the rat intestine. Arch Physiol Biochem 106, 46–55.[CrossRef][Medline]
Romain N, Dandrifosse G, Jeusette F & Forget P (1992). Polyamine concentration in rat milk and food, human milk, and infant formulas. Pediatr Res 32, 58–63.[Medline]
Wery I & Dandrifosse G (1993). Evolution of biochemical parameters characterizing the proximal small intestine after orally administered spermine in unweaned rats. Endocr Regul 27, 201–207.[Medline]
Wery I, Deloyer P & Dandrifosse G (1996). Effects of a single dose of orally-administered spermine on the intestinal development of unweaned rats. Arch Physiol Biochem 104, 163–172.[Medline]
Wild GE, Daly AS, Sauriol N & Bennett G (1993). Effect of exogenously administered polyamine on the structural maturation and enzyme ontogeny of the postnatal rat intestine. Biol Neonate 63, 246–257.[Medline]
|
Acknowledgements |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and spermine-treated rats (•) 
