www.elsevier.com / locate / livprodsci
Intestinal maturation induced by spermine in young animals
*
O. Peulen, P. Deloyer, C. Grandfils, S. Loret, G. Dandrifosse
Department of Biochemistry and General Physiology, Institute of Chemistry (B6c), Liege University, Sart-Tilman, 4000 Liege, Belgium
Abstract
In suckling rats, it has been established that oral administration of spermine, a dietary polyamine, at appropriate doses, induces all the modifications in the digestive tract that occur at weaning, namely, (a) in the intestine: variations in the specific activities of disaccharidases and peptidases, gene expression, the level of receptors to polymeric immunoglobulins (RPIs), tissue histology, and the intestinal permeability to macromolecules; (b) maturation of the intestinal immunological system, an observation confirmed in mice; (c) an increase in the specific activity of enzymes contained in the pancreas; (d) a change in the growth rate and biochemical properties of the liver, e.g., in the synthesis of RPIs. The mechanism of spermine action is partly understood. In the intestine, two phases of events have been recorded. The first phase consists of the desquamation of the epithelium resulting from an activation of apoptosis, probably induced by the entrance of spermine into the enterocytes. The second phase concerns a hormonal cascade in which adrenocorticotrophic hormone, cytokines, bombesin and corticosterone intervene. A general hypothesis taking into account all the results obtained until now is presented as well as other observations recorded on the effect of exogenous spermine on the intestinal maturation of vertebrates other than the rat.
2000 Elsevier Science B.V. All rights reserved.
Keywords: Polyamine; Spermine; Small intestine; Maturation; Rat
1. Introduction polyamines, especially spermine, on intestinal matu-ration.
Maturation of the intestine is known to occur in Marine fish larvae undergo major changes in the first few postnatal weeks in many vertebrates. As morphology and functionality of their digestive tract the objective of the present review is to report the during the first 5 weeks of life, i.e., until they have importance of spermine in this phenomenon, we will achieved their development. This process has been first give a short description of postnatal intestinal well described (for example, Henning, 1987; Cahu maturation in the animals where the role of this and Zambonino Infante, 1995). It essentially consists substance is proven, i.e., in the sea bass (Dicentrach- of modifications in digestive enzyme activity. us labrax), the mouse and the rat. Thereafter, we will In the mouse and rat (Henning, 1981), the small report on the observations concerning the effects of intestinal mucosa undergoes many modifications during the second or the third postnatal week. Among others, the large supranuclear vacuoles con-*Corresponding author. Tel.: 132-4-3663-577; fax: 1
32-4-tained in the enterocytes of the distal immature 3662-887.
E-mail address: [email protected] (G. Dandrifosse). intestine disappear (Veress and Baintner Jr., 1971), a
significant decrease in the intestinal permeability maturation in suckling rats and, thus, could play a towards proteins (‘‘gut closure’’) is observed and role in normal intestinal development.
some mucosal enzyme specific activities (SAs) are In mice, it was also demonstrated that spermine modified. For example, maltase and sucrase SAs induces the maturation of the small intestine and the increase in the third postnatal week. Lactase which is associated immune system (Ter Steege et al., 1997).
´ essential for lactose hydrolysis during the first two In the sea bass, data confirm this assertion (Peres et postnatal weeks, and lysosomal enzymes, which al., 1997). A diet enriched in spermine can accelerate intervene in the digestion of chyme molecules, lose a the appearance of the adult enzymatic profile in the part of their activity and concentration. Some of intestine and in the pancreas of this fish species. these phenomena are also observed in babies. This
suggests that mice and rats could represent good
models to understand biochemical and physio– 2. Polyamines in milk
pathological mechanisms taking place during
de-velopment of the human small intestine, and to Since intestinal maturation occurs in the rat for understand or correct causes and consequences of doses of spermine equal to or higher than 1 mmol / different dysfunctions of the intestine. day, a likely role of dietary polyamines in normal Luk et al. (1980) and Luk (1990) have shown that maturation of the intestine was proposed for this ornithine decarboxylase (ODC) SA and polyamine animal.
concentrations increase in the intestinal mucosa In order to find out whether ingested polyamines during weaning in rats. They established that a- can intervene in the normal development of the rat difluoromethylornithine (DFMO), an inhibitor of intestine, the concentration of these substances in rat ODC, delays the intestinal maturation process. milk and food was estimated. As shown by Pollack Therefore, further research to better evaluate the role et al. (1992) and Romain et al. (1992), the con-played by polyamines in the intestine maturation was centration of putrescine and spermine is low (less undertaken. than 2.5 nmol / ml) and does not vary much during Previous observations, taking into account that the lactation period, while the spermidine concen-polyamines are substances playing an important role tration is higher and seems to increase during in tissue proliferation and differentiation, led Dufour lactation (from 9 to .20 nmol / ml). Rat food et al. (1988) to test the effect of dietary polyamines contains approximately 150-times more putrescine on intestinal maturation. They orally administered and spermine, and approximately 30-times more spermine and spermidine to rats, which were 7 or 11 spermidine than rat milk. Thus, solid rat food days old, at doses from 3 to 8mmol twice a day for 3 represents a substantial increase in the exogenous days. They showed that these treatments induce contribution of polyamines at weaning in the rat. changes in intestinal enzyme activity similar to those Consequently, the polyamines contained in the occurring during normal maturation. These results milk at the end of suckling and in the solid-rat food were confirmed by Buts et al. (1993), Wild et al. could play a role in the postnatal maturation of the (1993), Harada et al. (1994) and Dorhout et al. rat intestine or in maintaining a functional stage in (1996). Georges et al. (1990) extended these ob- the bowel. This possibility is reinforced by the fact servations to the ultrastructure of the cell epithelium that bacterial flora, which constitute another source
´
in rats. Later on, Wery et al. (1992) and Osman et al. of exogenous polyamines, grow with the administra-(1998) proved that these spermine treatments induce tion of solid food (Osborne and Seidel, 1990). ‘‘gut closure’’ in these animals. Buts et al. (1993) Therefore, it appears that further investigation on the demonstrated that the concentration of the secretory intestinal maturation induced by spermine is fun-component of polymeric immunoglobulins was in- damental.
al., 1995), goat (Poszaj et al., 1997) and human which was never observed after weaning. Neverthe-female (Sanguansermsri et al., 1974; Pollack et al., less, irreversibility of normal intestinal maturation 1992; Romain et al., 1992; Buts et al., 1995). In has never been proven, maybe because adult rat diets general, goat’s colostrum and milk are rich in always contain high polyamine levels as compared polyamines, and concentrations are higher than those with rat milk (Pollack et al., 1992; Romain et al., in milk of the other mammals. Polyamine concen- 1992) (see later) and because intestinal microflora trations vary with the diet, species, time of lactation, synthesise polyamines (Osborne and Seidel, 1990). total litter weight, offspring number and milking Thus, it may be suggested that dietary polyamines time. Although there are no data demonstrating a are furnished at such an amount after weaning that role of dietary polyamines in maturation of the postnatal maturation cannot return to its suckling digestive tract (except maybe in babies), there is also state.
no data disproving this possibility. Therefore, experiments were performed in order to reduce, where possible, all sources providing poly-amines to the intestine of adult rats (Deloyer et al.,
3. Role of dietary spermine in intestinal 1996b). By using germ-free animals, the polyamine
development supply from intestinal microflora was avoided. Diet-ary polyamines were reduced by giving a specially As spermine could play an important role in the defined diet to the rats. Endogenous synthesis of intestinal maturation occurring at weaning, it is polyamines was limited by administration of DFMO useful to carefully analyse its morphological and and methylglyoxal bis guanylhydrazone (MGBG), an biochemical effects, and its mode of action. inhibitor of S-adenosylmethionine decarboxylase and
Peulen et al. (1998b, and unpublished results) of plasma membrane permeability to polyamines. confirmed, in the rat, that during the first 3 days of This treatment allowed Deloyer et al. (1996b) to spermine treatment (see earlier), the evolution of the observe that: (1) rats with intact intestinal microflora following parameters was similar in both natural and had higher SAs of maltase and higher amounts of in spermine-induced maturation of the intestine: spermidine and spermine, but lower lactase SA, than proteins, RNA and DNA concentrations of the pathogen-free animals; (2) a low polyamine diet intestinal mucosa, SAs of brush border disaccharid- given to germ-free rats had little effect on the ases, such as lactase, maltase and sucrase, morpholo- functional variables analysed (sucrase, maltase and gy of small intestine mucosa and ultrastructure of lactase SAs) and did not modify the intracellular enterocytes, as well as alkaline phosphatase SA, amounts of polyamines; (3) DFMO and / or MGBG TNF-a plasma concentration and gene expression administered to germ-free rats receiving a low (cdx-1, cdx-2, L-FABP and APO A4 ). Consequently, polyamine diet induced modifications in most of the it was proposed that spermine-induced intestinal variables studied; (4) body weight and wet weight of maturation is identical to the normal intestinal matu- the proximal and distal intestine decreased, disac-ration which occurs at weaning. charidase SA decreased and polyamine amounts
However, other observations do not agree with changed according to the inhibitor used.
aspect of distal enterocytes) were never observed. spermine orally is an important observation, giving Consequently, at weaning or just after this period, researchers an excellent model in order to investigate irreversible modifications of gene expression could the development of the intestine.
occur in the crypt stem cells, probably as a result of a biological clock effect or of the new hormonal status of the animals (see later).
4. Evolution of different intestinal variables
Another experiment, undertaken with other
re-after the ingestion of a single dose of dietary
search goals in mind, was performed in which
germ-spermine
free rats were fed for 2 months with a synthetic diet containing a very low amount of polyamines
(De-In order to investigate the role and effects of loyer et al., 1998). The conclusions obtained
con-spermine administered orally to suckling rats, the firmed the previous ones. They allow us to conclude
evolution of intestinal variables after the ingestion of that the intestine is an organ which is well protected
´ a single dose of spermine was studied (Wery and against such experimental conditions, in contrast to
´
Dandrifosse, 1993; Kaouass et al., 1996; Wery et al., other organs.
1996a). The effect seems to evolve in two phases: On the other hand, as postnatal maturation induced
some intestinal variables change during the initial by spermine ingestion appears to be reversible, it
few hours of the experiment whereas other intestinal may be suggested that a spermine treatment lasting
variables are clearly modified about 30 h after more than 3 days could maintain the adult stage of
spermine administration. the enterocytes until the time of weaning, and that
this stage would be irreversible. This possibility was
also investigated (Deloyer et al., unpublished re- 4.1. Initial hours of the experiment sults). Indeed, when rats ingested spermine for more
than 3 days (for doses, see earlier), intestinal wet As early as 4 h after spermine ingestion, the weight was greater compared with the control rats. In weight of intestine per cm of length decreased in the same way, lactase SA remained lower in the parallel with a decrease in DNA weight per cm of intestine of the spermine-treated rats. In contrast, length. This resulted from a loss of villus epithelium sucrase and maltase SAs in the jejunum and in the cells: optical microscopy showed that villus height ileum of the spermine-treated rats decreased when also decreased drastically. These modifications the duration of the treatment was longer than 3 days occurred simultaneously to a decrease in lactase and and finally returned to control values after 6 days. maltase SAs, especially in the ileum. The ileum was Furthermore, histological studies showed that large more affected than the jejunum by spermine adminis-supranuclear vacuoles progressively reappeared in tration. This observation could be due to the well the ileum of the rats that were ingesting spermine for known difference in the enterocytes ultrastructure more than 3 days. and function of these two parts of the small intestine
These experiments, along with other data, suggest (Baintner and Veress, 1967).
that the state of differentiation of enterocytes, rather The oral administration of spermine to suckling than being fixed and irreversible, could be dynamic rats had no effect on DNA synthesis and had only a and could require continuous control by environmen- slight effect on protein synthesis 3 h after the tal factors. beginning of treatment. Thus, the growth arrest of In conclusion, it appears that dietary spermine can intestinal cells can be excluded as the cause of the induce all the studied variables characterising post- cell loss observed 4 h after spermine administration. natal intestinal maturation; thus, it is likely to play a Furthermore, at 5 h post-treatment, the incorporation
14
role in normal intestinal maturation although, by of C-leucine into proteins isolated from the je-itself, it is unable to maintain the stability or junum increased, indicating that protein synthesis irreversibility of intestinal development in the rat. was enhanced at that time.
that spermine accumulation in cells can induce death. The SA of maltase increased as for sucrase SA and Recently, Stefanelli et al. (1998) demonstrated that also reached adult values. The spermine and sper-spermine can directly activate some caspase and, in midine levels in the intestine of the spermine-treated this way, induce apoptosis. Furthermore, it is well rats were generally higher than that of the control known that apoptosis occurs in the small intestine in animals.
order to regulate cell numbers (Hall et al., 1994). Thus, the early gut atrophy which occurred after Thus, this phenomenon could explain the observed the oral administration of spermine was rapidly cell loss (see later). This fact was proven by Peulen reversed, taking about 24 h in the jejunum and 48 h et al. (1997,1998a) by using terminal deoxynu- in the ileum. The reversal is due to new cell cleotidyl transferase-mediated deoxyuracyl triphos- differentiation. However, simple reversal can be phate-fluorescein nick end labelling (TUNEL), trans- obtained in suckling rats after indomethacin (Ka-mission electronic microscopy, DNA laddering and ouass et al., unpublished data) or muscimol (El caspase-3-like activity measurement. These authors Khefif et al., unpublished data) treatment.
have also obtained data indicating that this phenom- To explain the results obtained above, especially enon is probably not induced by a cytokine secretion those showing that spermine ingestion induces a but rather by spermine per se after its accumulation desquamation of the villus cells, it may be argued in the enterocytes (Peulen and Dandrifosse, 2000). that the effect of exogenous spermine on intestinal The effect of spermine ingestion on the SAs of variables is due to polyamine toxicity. Such a intestinal disaccharidases was also investigated in possibility can be excluded for the following reasons: cells coming from different parts of the crypto–villus (1) the spermine dose ingested corresponds to the
´ ´
axis (Wery and Dandrifosse, 1993; Wery et al., amount found in the daily solid food eaten by the 1996a). Sucrase SA was undetectable in all the 25-day-old rats (Pollack et al., 1992; Romain et al., categories of isolated enterocytes. Two hours after 1992), (2) the amount of spermine administered to spermine ingestion, lactase SA increased in the the animals is not cytotoxic (Rosenthal and Tabor, epithelial cells at the top of the villi and decreased in 1956), and (3) when spermine is administered to the epithelial cells at the bottom of the crypts. The suckling rats at nephrotoxic doses, the animals died maltase SA increased in all the epithelial cells, within 24 h (Buts et al., personal communication). except in those at the bottom of the crypts. Furthermore, the concentration of spermine, which
alone seems to be responsible for the desquamation 6. Dietary spermine and hormonal secretion
observed.
It may also argued that, in mammalian cells, Modifications of mature intestinal functions and spermine is converted to acetylspermine and that this enzyme expression during weaning are dependent is rapidly oxidised by polyamine oxidase (PAO), upon genetic, dietary and hormonal factors (Lee and yielding spermidine and cytotoxic products. The Lebenthal, 1983). The question as to whether or not inhibition of PAO by MDL72527, used in order to dietary polyamines act to evoke these changes should slow down the metabolisation of spermine and thus be addressed. To answer this question, adrenalec-the synadrenalec-thesis of acetamidopropanal, did not avoid adrenalec-the tomy was performed (Kaouass et al., 1994b). It gut atrophy induced by the ingestion of spermine reduced the changes in the SAs of disaccharidases (Kaouass et al., 1996). Moreover, the intestine of induced by dietary spermine. Thus, the intestinal rats treated simultaneously with spermine and postnatal maturation promoted by spermine could be MDL72527 appeared more mature than that of rats mediated, at least in part, by the adrenal glands. receiving spermine alone. Nevertheless, adrenalectomy did not completely These findings prove that spermine itself, without eliminate the spermine-induced appearance of suc-being metabolised to the toxic products mentioned rase and the increase in the SA of maltase (Kaouass above, is responsible for both cell loss observed at 8 et al., 1994b). This may be due to the direct effect of h post-treatment and intestinal maturation observed 3 spermine on the enterocytes (see earlier) and / or to days later. The physiological state of the cell could factors secreted by tissues other than the adrenal be important in this process since the spermine dose glands. Furthermore, the spermine-induced decrease used to induce intestinal maturation in suckling rats in the SA of lactase was not affected by adrenalec-is without effect on the speed of desquamation of the tomy. Thus, adrenal glands are not involved in the enterocytes in adult animals (Peulen et al., unpub- spermine-induced decrease of lactase SA observed in lished data). in vivo studies. Thyroxine and bombesin could be
implicated in this phenomenon (see later).
Since parenteral spermine injection had no effect
5. Direct effect of spermine on the enterocytes on intestinal postnatal maturation (Kaouass et al., 1994a) and since the adrenal gland is involved in this Previous results show that dietary spermine acts in phenomenon, it may be possible that spermine given two phases on intestinal properties in suckling rats. orally induces intestinal secretion of one or more To know whether these effects are direct (i.e., substances which, in turn, stimulate the secretion of concern the enterocytes only) or indirect (i.e., in- endogenous local factors and / or hormones. Results volve hormones, neurotransmitters or local factors), have confirmed this possibility (Kaouass et al., different experiments have been performed. It was 1994b). The ability of spermine to increase corticos-found that: (1) in organ culture, spermine had no terone output was maximal 5 and 6 h after ingestion, maturational effect on intestinal explants from suckl- a period during which maximal stimulation of adre-ing rats (Kaouass et al., 1994a), (2) spermine no-corticotropic hormone (ACTH) output was re-induced maturation in enterocytes isolated from the corded. This observation suggests that there must be proximal intestine of suckling rats and cultivated in a link between the digestive tract and the pituitary–
´
synthetic media (Wery et al., unpublished results), adrenal axis. This link is not spermine: parenteral and (3) spermine accelerated the differentiation of spermine administration is ineffective in inducing CaCo-2 cells (Deloyer et al., unpublished results). intestinal maturation and corticosterone secretion Although inconclusive, these observations indicate (Kaouass et al., 1994a). It could be a gastrointestianl that spermine could have a direct effect on the (GI) hormone, a cytokine and / or the gut nervous enterocytes but do not exclude an indirect effect (see system (see earlier).
intraperitone-ally injected, were tested (Kaouass et al., 1997a). plasma. Nevertheless, a direct action of these cyto-None of these polypeptides affected brush-border kines on the enterocytes is not excluded: receptors enzyme SA. Thus, these GI hormones do not seem to for IL-6 are present on the human intestinal epithelial be involved in spermine-induced intestinal matura- cells (Shirota et al., 1990).
tion. From another point of view, there are several There is increasing evidence for a link between the reports indicating that IL-1b, IL-6 and TNF-a ad-neuroendocrine and immune systems (Rothwell, ministration increases ACTH and corticosterone 1991). Interleukin-1b (IL-1b), interleukin-6 (IL-6) plasma concentrations in adult animals (Rivier et al., and tumour necrosis factor alpha (TNF-a), poly- 1989; Besedovsky et al., 1991; O’Grady et al., peptides predominantly produced by monocytes and 1993). Such observations were also made in suckling macrophages, have been reported to activate the rats, although IL-1b induced a more marked effect pituitary–adrenal axis by stimulating the corticot- than did IL-6 and TNF-a. Assuming that this effect ropin releasing-factor (CRF)-containing cells in the is mediated in the same way as in adult animals, i.e., hypothalamus. In order to assess the possible role of through increasing CRF release, these results show IL-1b, IL-6 and TNF-a as a link between the that the pituitary–adrenal system is already mature ingestion of spermine and the activation of the enough in suckling rats to respond to the stimulatory pituitary–adrenal axis, different experiments were effect of immunological factors.
undertaken. These cytokines also increased the intracellular The ingestion of spermine produced increases in polyamine concentration (Kaouass et al., 1997b). As plasma concentrations of IL-1b, IL-6 and TNF-a IL-1b activates the ODC contained in intestinal (Kaouass et al., 1997b). The peak responses of IL-1b epithelial cells in vitro, it is possible that the increase and IL-6, appearing within 6 h after spermine of polyamine content induced by IL-1band IL-6 is a administration, corresponded to the peaks in plasma consequence of a direct action of these cytokines on corticosterone and ACTH concentrations. This the intestinal ODC activity. Nevertheless, it cannot ACTH peak could be a result of IL-1b action be excluded that an indirect action of IL-1band IL-6 (Uehara et al., 1987). The increases in plasma on the intestinal polyamine concentration occurs concentrations of IL-1b, IL-6 and TNF-a were all through an increase in plasma concentration of observed at the same time; as it has been shown that corticosterone. The latter is known to be an activator IL-1b and IL-6 can induce TNF-a release (Wong of ODC.
and Clark, 1988), it cannot be excluded that the The indirect action of spermine, via factors other action of spermine on TNF-a release was mediated than corticosterone, on the enterocytes is not ex-by IL-1b and / or IL-6. cluded: the administration of IL-1b to the rats Thus, it may be suggested that spermine ingestion induced release of insulin and prostaglandins (Ue-by unweaned rats stimulates the secretion of IL-1b, hara et al., 1987), both of which are involved in IL-6 and / or TNF-a, from the gut-associated- postnatal intestinal maturation. Cytokines other than lymphoid-tissue (GALT) system. These substances IL-1band IL-6 could also play a role in this effect. then appear in the general blood circulation and
could increase corticosterone secretion, thus
trig-gering the intestinal maturation process. This possi- 7. Effect of dietary spermine on the nervous
bility is highly probable because data (Kaouass et al., system
1997b) show that i.p. administration of IL-1bor IL-6
stimulated growth of the small intestine (i.e., in- Indeed, injection of hormones as corticosterone creased intestinal weight and length, and intestinal (Martin and Henning, 1982), insulin (Buts et al., protein and DNA contents). It also induced appear- 1988), prostaglandins (Neu et al., 1983) or insulin-ance of sucrase, an increase in maltase SA and a like growth factor-I (IGF-I) (Young et al., 1990), decrease in lactase SA. usually enhances the SA of intestinal lactase in The mechanism by which bombesin affects intesti- suckling animals. These findings suggest that, like nal development is unknown. Specific receptors to thyroxine, bombesin can be involved in the decline this neuropeptide are present on intestinal en- of lactase SA during the period of weaning. terocytes (Moran et al., 1988). Thus, a direct action Other arguments confirm that that dietary sper-of bombesin on enterocyte metabolism cannot be mine could affect the nervous system of the gut. excluded. Bombesin could also modify intestinal Putrescine can be metabolised to g-aminobutyric functions via endogenous growth factors or hor- acid (GABA) and the endogenous phosphorylation of mones, such as polyamines (Grillo, 1985) and cor- the GABA type A receptor is modulated by spermine ticosterone (Henning, 1981). Indeed, plasma con- (Bureau and Laschet, 1995). In preliminary experi-centrations of corticosterone and intestinal levels of ments (El Khefif et al., unpublished results), we intracellular polyamines were enhanced when bom- showed that spermine modified intestinal motility in besin was administered to the animals (Kaouass et suckling and adult rats, and that muscimol, a GABA al., 1997a). Adrenalectomy in bombesin-treated rats agonist, produced an intestinal desquamation similar generally decreased the disaccharidase SA. to the type caused by spermine but followed by As the effect of bombesin on corticosterone regeneration without the appearance of adult forms secretion could be mediated in the same way in adult of enterocytes.
animals, i.e., through an increase in ACTH release Consequently, dietary spermine could act indirect-(see earlier), it was questioned whether or not blood ly on the enterocytes, by way of the nervous system, and intestinal bombesin concentrations are modified at the first stage of its action in the small intestine. by spermine treatment. It was shown that this
polyamine had no apparent action on the
concen-tration of bombesin in plasma (Kaouass et al., 8. Effect of dietary spermine on the immune
1997a). However, the intestinal content of bombesin system
was significantly decreased. Since this change in
intestinal bombesin coincides with the increase in The immune system could be influenced by sper-plasma corticosterone concentration, produced by mine ingestion as suggested by Kaouass et al. spermine ingestion, the existence of a relationship (1997b) and by Peulen and Dandrifosse (2000) in between the two phenomena, by the way of the rats (see earlier: the effects of interleukins on post-nervous system, was suggested. The more plausible natal intestinal maturation). This was confirmed by hypothesis is that spermine stimulates the secretion studies performed in suckling mice. Indeed, Ter of bombesin from enteric nerves in the synaptic cleft Steege et al. (1997) have proven that the percentage where bombesin could act directly on the enterocytes of intra-epithelial lymphocytes expressing the T-cell and / or on a postsynaptic nervous receptor (with, at receptor ab (TCRab), clusters of differentiation the end, activation of the pituitary–adrenal axis). (CDs)-4, -5 and -54, as well as the levels of Afterwards, this neuropeptide would be rapidly expression of these antigens, increased similarly after catabolised (‘‘inactivated’’). spermine ingestion to that seen with normal
matura-Based on these findings, the effect of spermine on tion.
medium which supports in vitro proliferation of all the modifications observed in the pancreas after human T cells stimulated by the same mitogen, feeding spermine to suckling rats are similar to those spermine induced proliferation of these cells. It also observed in normal pancreatic maturation. Thus, it acted on the differentiation of the latter. Indeed, may be proposed that spermine treatment induces when spermine was added to culture media, a maturation of the pancreas in suckling rats as it does decrease in the expression of the IL-2 receptor for the intestine.
(CD25 marker) and of the transferrin receptor (CD71 The effect of spermine on intestinal maturation marker) was observed, whereas the CD4 / CD8 ratio could involve at least the activation of ACTH and was not modified. Under the same experimental corticosterone secretion. As the latter could have an conditions, the expression of the CD23 marker of B effect on pancreas or liver maturation, in the experi-lymphocytes was not affected. ments described below, hepatic properties were
analysed after feeding spermine to suckling rats. Putrescine and spermidine contents were higher in
9. Effect of dietary spermine on postnatal the livers of pups than of adults, while liver spermine
maturation of the pancreas and liver content increased slowly with age. After spermine treatment, the putrescine or spermidine content de-As reported above, during the third postnatal creased in pup liver until it was close to that week, many modifications appear in the digestive measured in adult liver. Administration of spermine system of the rat. They seem to be an adaptation to for 3 or 5 days to suckling rats induced an almost the progressive transition from a milk regime (high two-fold increase in liver spermine content which fat, low carbohydrate diet) to a solid regime (high was higher than that found in adult liver. In rats, carbohydrate, low fat diet). In the pancreas, modi- during the normal maturation, the liver spermidine / fications occur spontaneously during the first 3 spermine ratio decreased progressively with age until weeks of postnatal life (for references, see Snook, it stabilised at about 1:1 in adult animals. The oral 1971). They are probably under a hormonally-con- administration of spermine to unweaned rats pro-trolled ‘‘biological clock’’ but could also be depen- duced a significant decrease in this ratio, which dent on food intake. The following modifications reached a value close to 1:1.
occur in rats aged between 15 and 30 days: (1) an Vaerman et al. (1989) detected only a trace of RPI increase in pancreatic weight (proportionally higher in the liver of suckling rats but observed that the than the body weight increase) and (2) an increase of amounts of RPI started to increase in rats from the acinous cell number compared with Langerhans-islet age of 20 days, and reached adult values in rats older cells. From a biochemical point of view, normal than 40 days. An increase (almost two-fold) in this maturation of the rat pancreas occurs mainly during variable was recorded after 3 days of treatment with the fourth week after birth. It is characterised by an spermine, although the value obtained did not reach
´
increase in a-amylase, trypsin and lipase activities. the adult one (Wery et al., 1996b).
Morisset and Grondin (1987) have shown that ODC The enzymatic activity of ornithine aminotrans-activity and polyamines intervene in the pancreatic ferase (OAT) is a typical marker of postnatal liver growth of neonatal rats, and results are supported by maturation and it markedly increases during weaning those obtained by Romain et al. (1998). (Greengard, 1970). Spermine given orally to suckl-Romain et al. (1998) treated suckling rats with ing rats induced a significant increase in liver OAT
10. Molecular mechanism of spermine action on esis, resulting from established observations, is that spermine stimulates the secretion of bombesin from
intestinal maturation
enteric nerves in synaptic clefts; the bombesin then acts directly on the enterocytes and / or on postsynap-In the rat, the most recent findings (see earlier)
tic nerve receptors with, eventually, activation of the allow us to present a working hypothesis explaining
pituitary–adrenal axis. Besides bombesin, 6, IL-the effect of spermine on IL-the maturation of IL-the GIT.
1b and maybe other not yet identified cytokines This particularly depends on results obtained at the
could also indirectly affect this axis. As the nervous level of the intestine. In this organ, two phases
system and cytokine secretions are stimulated, the characterise the effect of spermine. A desquamation
c-fos gene is activated, and ACTH and corticosterone of the intestinal epithelium is followed by a
hormon-are secreted. Corticosterone increases insulin and al cascade.
thyroxine secretion, and, since insulin affects the In the early phase, spermine first enters the
permeability of the intestinal cells to spermine and enterocytes where it induces apoptosis. This latter
since the effect of polyamine on the DNA expression phenomenon does not seem to be influenced by
is well known, it may be inferred that another pattern cytokines, even if the secretion of these substances
of enterocyte differentiation occurs, which is similar increases. Secondly, the high permeability of the
to the adult stage. The increase in the spermine intestinal epithelium, characteristically observed in
intracellular concentration results also from a direct suckling rats, allows spermine to enter into the
entrance of spermine into the enterocytes and from intercellular space where it reacts with different
an indirect effect of corticosterone on the ODC receptors located in the nervous system, in the
activity of the stem cells. immune system and on the endothelial cells at the
The molecular mechanism at the basis of the capillary ends. The nervous system target is not yet
maturation of the pancreas and of the liver after identified but we know that spermine can be
trans-spermine ingestion originates mainly from the hor-formed into GABA and can act directly on the
monal cascade reported above. GABA receptor. Results also suggest that bombesin
When normal maturation occurs, the same phe-neurosecretion is affected. Thirdly, as a consequence,
nomena take place (Peulen et al., 1998b). They are intestinal motility is decreased, as observed in vitro
less marked and difficult to show, except for the in rat pups and as reported in adult animals.
Fourth-increase in spermine, the secretion of several hor-ly, a stagnation of biliary salts in the lumen of the mones (corticosterone, TNF-a, insulin), the variation intestine appears, speeding up the desquamation of in polyamine concentration in the enterocytes, the the cells at the top of the villi. Fifthly, spermine and modification of gene expression, apoptosis and maybe aldehydes, derived from polyamines by me- oedema (Peulen et al., 1997, 1998a; unpublished tabolism, act in the immune system or on epithelial results). The irreversibility of the normal process is cells, where they induce the secretion of various unclear but could be due either to the biological cytokines such as IL-1b, IL-6 and TNF-a. This clock (often cited as the basis of the intestinal could provoke, among other things, the production of maturation process) or to the changing hormonal nitric oxide and some oedema (noted at the top of the status observed after weaning.
villi), a phenomenon subsequently reinforced by an inhibition of ATPase activities by spermine. This
oedema provokes the start of inflammation (as has Acknowledgements been observed) but this phenomenon is antagonised
by spermine. Although not yet conclusively demon- This work was supported by FRFC-IM (No. 455-strated, the oedema and inflammation seem to be 338) and by FRSM (No. 3-4531-96) grants. side effects of spermine action since neither
anti-IL-1b and anti-TNFa antibodies nor cyclosporin A
prevent the desquamation of the intestinal epithelium References (Peulen and Dandrifosse, 2000).
Besedovsky, H.O., del Rey, A., Klusman, I., Furukawa, H., tion of cell number in mammalian gastrointestinal tract: the importance of apoptosis. J. Cell Sci. 107, 3569–3577. Monge Arditi, G., Kabiersch, A., 1991. Cytokines as
Harada, E., Hashimoto, Y., Syuto, B., 1994. Orally administered modulators of the hypothalamus–pituitary–adrenal axis. J.
spermine induces precocious intestinal maturation of macro-Steroid Biochem. Mol. Biol. 40, 613–618.
molecular transport and disaccharidase development in suckl-Bureau, M., Laschet, J., 1995. Endogenous phosphorylation of
ing rats. Comp. Biochem. Physiol. Physiol. A 109, 667–673. distinct g-aminobutyric acid type A receptor polypeptides by
Henning, S.J., 1981. Postnatal development: coordination of Ser / Thr and Tyr kinase activities associated with the purified
feeding, digestion, and metabolism. Am. J. Physiol. 241, receptor. J. Biol. Chem. 270, 26482–26487.
G199–G214. Buts, J.P., De Keyser, N., Dive, C., 1988. Intestinal development
Henning, S.J., 1987. Functional development of the digestive tract. in the suckling rat: effect of insulin on the maturation of villus
In: Johnson, L.R. (Ed.), Physiology of the Gastrointestinal and crypt cell functions. Eur. J. Clin. Invest. 18, 391–398.
Tract, 2nd Edition. Raven Press, New York, pp. 285–300. Buts, J.P., De Keyser, N., Kolanowski, J., Sokal, E., Van Hoof, F.,
Kaouass, M., Deloyer, P., Romain, N., Dandrifosse, G., 1994a. 1993. Maturation of villus and crypt cell functions in rat small
Intestinal development in suckling rats: direct or indirect intestine. Role of dietary polyamines. Dig. Dis. Sci. 38, 1091–
spermine action? Digestion 55, 160–167.
1098. Kaouass, M., Sulon, J., Deloyer, P., Dandrifosse, G., 1994b.
Buts, J.P., De Keyser, N., De Raedemaeker, L., Collette, E., Sokal, Spermine-induced precocious intestinal maturation in suckling E.M., 1995. Polyamine profiles in human milk, infant formulas, rats: possible involvement of glucocorticoids. J. Endocrinol. and semi-elemental diets. J. Pediatr. Gastroenterol. Nutr. 21, 141, 279–283.
44–49. Kaouass, M., Deloyer, P., Gouders, I., Dandrifosse, G., 1996. Cahu, C.L., Zambonino Infante, J.L., 1995. Maturation of the Analysis of structural and biochemical events occurring in the pancreatic and intestinal digestive function in sea bass (Di- small intestine after dietary polyamine ingestion in suckling centrachus labrax): effect of weaning with different protein rats. Dig. Dis. Sci. 41, 1434–1444.
sources. Comp. Biochem. Physiol. A 109, 213–222. Kaouass, M., Deloyer, P., Dandrifosse, G., 1997a. Involvement of Deloyer, P., Nollet, N., Dandrifosse, G., 1996a. Effect of pro- bombesin in spermine-induced corticosterone secretion and longed spermine treatment on intestinal maturation in the intestinal maturation in suckling rats. J. Endocrinol. 153, 429– suckling rat. In: Proceedings of the International Symposium 436.
on Polyamines, Tokyo, pp. 228–229. Kaouass, M., Deloyer, P., Gouders, I., Peulen, O., Dandrifosse, G., Deloyer, P., Dandrifosse, G., Bartholomeus, C., Romain, N., 1997b. Role of interleukin-1 beta, interleukin-6, and TNF alpha
´
Klimek, M., Salmon, J., Gerard, P., Goessens, G., Eyssen, H.J., in precocious intestinal maturation induced by dietary spermine 1996b. Polyamines and intestinal properties in adult rats. Br. J. in suckling rats. Endocrine 6, 187–194.
Nutr. 76, 627–637. Kelly, D., King, T.P., Brown, D.S., Mc Fadyen, M., 1991. Deloyer, P., Peulen, O., Klimek, M., Remacle, R., Focant, C., Polyamine profiles of porcine milk and of intestinal tissue of
Dandrifosse, G., 1998. Effects of a long-term low-polyamine pigs during suckling. Reprod. Nutr. Dev. 31, 73–80. diet on biochemical parameters and polyamine content in Lee, P.C., Lebenthal, E., 1983. Early weaning and precocious different organs of the germ-free rat. In: Bardocz, S., White, A. development of small intestine in rats: genetic, dietary or (Eds.), Biogenically Active Amines in Food. Metabolic Effects hormonal control. Pediatr. Res. 17, 645–650.
of Biologically Active Amines in Food, Vol. II. Office for Luk, G.D., 1990. Polyamines in intestinal growth. Biochem. Soc. Official Publications of the European Communities, pp. 130– Trans. 18, 1190–1191.
135. Luk, G.D., Marton, L.J., Baylin, S.B., 1980. Ornithine decarbox-Dorhout, B., van Beusekom, M., Huisman, M., Kingma, A.W., de ylase is important in intestinal mucosal maturation and
re-Hoog, E., Boersma, E.R., Muskiet, F.A.J., 1996. Estimation of covery from injury in rats. Science 210, 195–198.
twenty-four hour polyamine intake from mature human milk. J. Martin, G.R., Henning, S.J., 1982. Relative importance of cor-Pediatr. Gastroenterol. Nutr. 23, 298–302. ticosterone and thyroxine in the postnatal development of Dufour, C., Dandrifosse, G., Forget, P., Vermesse, F., Romain, N., sucrase and maltase in rat small intestine. Endocrinology 111,
Lepoint, P., 1988. Spermine and spermidine induce intestinal 912–918.
maturation in the rat. Gastroenterology 95, 112–116. Moran, T.H., Moody, T.W., Hostetler, A.M., Robinson, P.H., El Khefif, N., Peulen, O., Dandrifosse, G., 1995. [Spermine Goldrich, M., McHugh, P.R., 1988. Distribution of bombesin induces human lymphocyte proliferation]. Arch. Physiol. Bio- binding sites in the rat gastrointestinal tract. Peptides 9, 643–
chem. 103, D116. 649.
Georges, P., Dandrifosse, G., Vermesse, F., Forget, P., Deloyer, P., Morisset, J., Grondin, G., 1987. Implication of ornithine decarbox-Romain, N., 1990. Reversibility of spermine-induced intestinal ylase and polyamines in pancreatic growth of neonatal rats. maturation in the rat. Dig. Dis. Sci. 35, 1528–1536. Pancreas 2, 303–311.
Greengard, O., 1970. The development formation of enzymes in Motyl, T., Poszaj, T., Wojtasik, A., Kukulska, W., Podgurniak, M., rat liver. In: Litwack, G. (Ed.). Biochemical Actions of 1995. Polyamines in cow’s and sow’s milk. Comp. Biochem. Hormone, Vol. 1. Academic Press, New-York, pp. 53–87. Physiol. B 111, 427–433.
O’Grady, M.P., Hall, N.R., Menzies, R.A., 1993. Interleukin-1 Rothwell, N.J., 1991. Functions and mechanisms of interleukin 1 beta stimulates adrenocorticotropin and corticosterone release in the brain. Trends Pharmacol. Sci. 12, 430–436.
¨
in 10-day-old rat pups. Psychoneuroendocrinology 18, 241– Sanguansermsri, J., Gyorgy, P., Zilliken, F., 1974. Polyamines in
247. human and cow’s milk. Am. J. Clin. Nutr. 27, 859–865.
Osborne, D.L., Seidel, E.R., 1990. Microflora-derived polyamines Shirota, J., Ledug, L., Yuan, S.Y., Yothy, S., 1990. Interleukin 6 modulate obstructive-induced colonic mucosal hypertrophy. and its receptor are expressed in human intestinal epithelial Am. J. Physiol. 258, 6576–6584. cells. Virchows Arch. B Cell Pathol. 58, 303–308.
¨
Osman, N.E., Westrom, B., Wang, Q., Persson, L., Karlsson, B., Snook, J.T., 1971. Effect of diet on development of exocrine 1998. Spermine affects intestinal in vitro permeability to pancreas of the neonatal rat. Am. J. Physiol. 221, 1388–1391. different-sized molecules in rats. Comp. Biochem. Physiol. C Stefanelli, C., Bonavita, F., Stanic, I., Mignani, M., Facchini, A., 120, 211–216. Pignatti, C., Flamigni, F., Caldarera, C.M., 1998. Spermine ´
Peres, A., Cahu, C.L., Zambonino Infante, J.L., 1997. Dietary causes caspase activation in leukaemia cells. FEBS Lett. 437, spermine supplementation induces intestinal maturation in sea 233–236.
bass (Dicentrachus labrax) larvae. Fish Physiol. Biochem. 16, Ter Steege, J.C., Buurman, W.A., Forget, P.P., 1997. Spermine 479–485. induces maturation of the immature intestinal immune system Peulen, O., Muller, P., Defresne, M.P., Dandrifosse, G., 1997. in neonatal mice. J. Pediatr. Gastroenterol. Nutr. 25, 332–340. Spermine induced apoptosis in suckling rat small intestinal Uehara, A., Gottschall, P.E., Dahl, R.R., Arimura, A., 1987. mucosa. Arch. Physiol. Biochem. 105, 252–253. Interleukin 1 stimulates ACTH release by an indirect action
´
Peulen, O., Muller, P., Gerard, P., Dandrifosse, G., 1998a. which requires endogenous corticotropin releasing factor. Spermine-induced apoptosis in the small intestine of suckling Endocrinology 121, 1580–1582.
rats. Arch. Physiol. Biochem. 106, I29. Vaerman, J.P., Buts, J.P., Lescoat, G., 1989. Ontogeny of secretory Peulen, O., Pirlet, C., Klimek, M., Dandrifosse, G., 1998b. component expression in rat liver. Immunology 68, 295–299. Comparison between the natural postnatal maturation and the Veress, B., Baintner, Jr. K., 1971. Absorption of colostrum in the spermine-induced maturation of the rat intestine. Arch. Physiol. rat and the cat. A comparative electron microscopic study. Acta Biochem. 106, 46–55. Morphol. Acad. Sci. Hung. 19, 335–347.
´
Peulen, O., Dandrifosse, G., 2000. Cyclosporine A inhibits Wery, I., Dandrifosse, G., 1993. Evolution of biochemical parame-partially spermine-induced differentiation but not cell loss of ters characterising the proximal small intestine after orally suckling rat small intestine. Dig. Dis. Sci. 45, 750–754. administered spermine to unweaned rats. Endocr. Regul. 27, Pollack, P.F., Koldovsky, O., Nishioka, K., 1992. Polyamines in 201–207.
´
human and rat milk and in infant formulas. Am. J. Clin. Nutr. Wery, I., Romain, N., Dandrifosse, G., 1992. [Precocious intesti-56, 371–375. nal maturation induced by spermine in rat: kinetic and intesti-Poszaj, T., Ryniewicz, Z., Motyl, T., 1997. Polyamines in goat’s nal permeability]. Arch. Int. Physiol. Biochim. 101, A135.
´
colostrum and milk. Comp. Biochem. Physiol. B 118, 45–52. Wery, I., Deloyer, P., Dandrifosse, G., 1996a. Effects of a single Poulin, R., Pelletier, G., Pegg, A.E., 1995. Induction of apoptosis dose of orally-administered spermine on the intestinal develop-by excessive polyamine accumulation in ornithine decarbox- ment of unweaned rats. Arch. Int. Physiol. Biochem. 104, ylase-overproducing L1210 cells. Biochem. J. 311, 723–727. 163–172.
´
Rivier, C., Chizzonite, R., Vale, W., 1989. In the mouse, the Wery, I., Kaouass, M., Deloyer, P., Buts, J.P., Barbason, H., activation of the hypothalamic–pituitary–adrenal axis by a Dandrifosse, G., 1996b. Exogenous spermine induces matura-lipopolysaccharide (endotoxin) is mediated through interleukin- tion of the liver in suckling rats. Hepatology 24, 1206–1210. 1. Endocrinology 125, 2800–2805. Wild, G.E., Daly, A.S., Sauriol, N., Bennett, G., 1993. Effect of Romain, N., Dandrifosse, G., Jeusette, F., Forget, P., 1992. exogenously administered polyamine on the structural matura-Polyamine concentration in rat milk and food, human milk and tion and enzyme ontogeny of the postnatal rat intestine. Biol. infant formulae. Pediatr. Res. 32, 58–63. Neonate 63, 246–257.
Romain, N., Gesell, M.S., Leroy, O., Forget, P., Dandrifosse, G., Wong, G.G., Clark, S.C., 1988. Multiple actions of interleukin 6 Luk, G.D., 1998. Effect of spermine administration on pan- within a cytokine network. Immunol. Today 9, 137–142. creatic maturation in unweaned rats. Comp. Biochem. Physiol. Young, G.P., Taranto, T.M., Jonas, H.A., Cox, A.J., Hogg, A., A 120, 379–384. Werther, G.A., 1990. Insulin-like growth factors and the Rosenthal, M., Tabor, C.W., 1956. The pharmacology of spermine developing and mature rat small intestine: receptors and
