Spermine-Induced Alteration of Small Intestine in Suckling Rat: Involvement of Apoptosis or Zn2+ Enzymes?

Polyamines are of great importance in several physiological processes, such as cell proliferation and differentiation. The ingestion of spermine by suckling rats induces precocious maturation of their small intestine. Shortly after ingestion, spermine produces cell elimination at the villous top. The origin of this exfoliation was investigated to determine whether it was due to apoptosis. Wistar rats were orally treated with spermine. Apoptosis was analyzed in their small intestine by Tdt-mediated dUTP-fluorescein nick-end labeling reaction, caspase-3-like analysis, and DNA laddering. Polyamine content was measured by HPLC. The intestinal transitory alteration appeared as soon as 2 hr after spermine administration. Apoptosis events increased strongly at the same moment in the small intestine. They were evidenced by Tdt-mediated dUTP-fluorescein nick-end labeling analysis, DNA laddering, and caspase-3-like activity. Changes observed are consistent with apoptosis, but caspase inhibitor did not reduce intestinal alteration, as did Zn²⁺ chelator.     

Reversibility of spermine-induced intestinal maturation in the rat

In the present investigation, the reversibility of spermine-induced precocious intestinal maturation was studied. Neonatal rats received either saline or spermine (4 mol, twice daily) solution orally on the 11th and 12th postnatal day. They were killed on the 13th, 14th, 15th, 16th, and 17th postnatal days. After the small bowel was removed, it was either divided into three equal parts or prepared for electrophoretic analysis. Histological examination, protein content measurement, and disaccharidase activity estimation were performed on each part of the intestine. Spermine administration was shown to induce structural and mucosal enzyme changes characteristic of postnatal maturation. This phenomenon, which was generally clearly observed in 13- and 14-day-old rats, then became less apparent in 15- and 16-day-old animals. Differences were noted according to the segment of intestine or the biochemical parameter analyzed. When rats were 17 days old, no significant differences generally existed between control and spermine-treated rats. If the 140-to 150-kDa proteins, isolated by electrophoresis, are assumed to represent the subunits of the sucrase-isomaltase complex, the results obtained indicate that spermine induces a modification of the concentration of this complex. When compared to values obtained in adult rats, the concentration of the complex was approximately three times higher in spermine-treated 13-day-old rats, while no differences were found in spermine-treated 14-day-old rats. Further, similar concentrations were found in control and spermine-treated rats with an age of 17 days. These results suggest that spermine-induced precocious intestinal maturation is reversible when spermine treatment is stopped.This work was supported by grants from the F.R.S.M. (number 3.4535.88) and from la Loterie Nationale (1987).     

Cyclosporine A Inhibits Partially Spermine-Induced Differentiation But Not Cell Loss of Suckling Rat Small Intestine

The polyamines are of great importance in several biological processes, such as cell proliferation, and differentiation. The ingestion of spermine by suckling rats induces the precocious maturation of their small intestine. This phenomenon is preceded by a cell elimination at the villus tip. We hypothesize that these two phenomena could be mediated by the immune system and thus inhibited by an immunosuppressive agent such as cyclosporine A. Cyclosporine A inhibits, at least partially, the spermine-induced increase of the maltase- and sucrase-specific activities in the small intestine but failed to inhibit lactase-specific activity decrease and cell loss. Spermine does not act by the same mechanism in differentiation and in cell loss. Moreover, spermine acts in a different way on lactase-specific activity compared to maltase- or sucrase-specific activity. We hypothesize that spermine acts on differentiation by a T-cell/IL-2-dependent mechanism.     

Jejunal mucosal DNA content and maturation

Although pentagastrin has a tropic action on intestinal mucosa in suckling rat pups, and at weaning a rise in gastrin levels coincides with maturation of the intestinal mucosa, direct correlations of serum gastrin levels and intestinal maturation have yet to be made. Ten-day-old rats were subjected either to antrectomy to produce a 43% decrease in serum gastrin levels or to fundectomy to produce a 319% increase over gastrin levels in rumenectomized or normal animals. These changes were not associated with tropic or adaptive changes in jejunal or colonic mucosa as determined by jejunal and colonic DNA content, jejunal sucrase activity, jejunal villous height, or crypt depths in jejunum and colon at the beginning (day 15), middle (day 21), or end (day 27) of the weaning period. To the contrary, an inverse relation was found between serum gastrin levels and both jejunal mucosal DNA content and sucrase activity as an index of maturation.     

Role of interleukin-1β, interleukin-6, and TNF-α in intestinal maturation induced by dietary spermine in rats

In the present investigation, the authors aimed to evaluate the role of cytokines in intestinal postnatal maturation induced by dietary polyamines. Neonatal rats were administered either saline or spermine (8 μmol) orally. Spermine increased interleukin-1β (IL-1β), IL-6, and TNF-α plasma concentration. The maximum concentrations of IL-1β, IL-6, and TNF-α were, respectively, observed at 4, 4, and 8 h posttreatment. Intraperitoneal (ip) injection of IL-1β increased the specific activity of sucrase in whole small intestine, whereas the specific activities of maltase and lactase were significantly enhanced only in the jejunum. IL-6 elicited sucrase and increased maltase specific activity in the whole small intestine, but lactase specific activity was not affected. TNF-α had no effect on sucrase and maltase specific activity, but a slight augmentation of lactase specific activity was detected in the jejunum. Spermine and spermidine content in the intestine was increased by ip injection of IL-1β and IL-6. Corticosterone secretion was elevated by single ip injection of IL-1β, IL-6, or TNF-α. These findings suggest that spermine could induce postnatal intestinal development and corticosterone secretion through a cytokine-dependent mechanism.     

Modulatory effects of polyamines and GABA on rat ileal motility in vitro]

AIM: The aim of our work was to determine the effects of polyamines and GABA on rat ileum motility in vitro. METHODS: Longitudinal strips dissected from control ileum or ileum without myenteric plexus after benzalkonium chloride (BAC) treatment were placed in organ bath chambers. RESULTS: Spermine significantly inhibited spontaneous activity and nerve stimulation-induced response. Inhibition of spontaneous activity was not altered by BAC treatment or tetrodotoxin but was antagonized by BAY K 8644, a L-type calcium channel agonist. Spermine inhibitory effect on nerve-stimulation induced response disappeared after BAC treatment. GABA enhanced the response induced by nerve stimulation but did not antagonize spermine effects; its action was inhibited in presence of atropine and was mimicked by baclofen, a GABAB agonist. CONCLUSION: Polyamines and GABA can modulate rat ileum motility in vitro. GABA acted via neural GABAB receptors. We demonstrate for the first time that spermine exerts a dual action through different mechanisms on both smooth muscle cells and probably intramural neurons.     

Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis

It is estimated that the etiology of 20-30% of epithelial cancers is directly associated with inflammation, although the direct molecular events linking inflammation and carcinogenesis are poorly defined. In the context of gastrointestinal disease, the bacterium enterotoxigenic Bacteroides fragilis (ETBF) is a significant source of chronic inflammation and has been implicated as a risk factor for colorectal cancer. Spermine oxidase (SMO) is a polyamine catabolic enzyme that is highly inducible by inflammatory stimuli resulting in increased reactive oxygen species (ROS) and DNA damage. We now demonstrate that purified B. fragilis toxin (BFT) up-regulates SMO in HT29/c1 and T84 colonic epithelial cells, resulting in SMO-dependent generation of ROS and induction of γ-H2A.x, a marker of DNA damage. Further, ETBF-induced colitis in C57BL/6 mice is associated with increased SMO expression and treatment of mice with an inhibitor of polyamine catabolism, N(1),N(4)-bis(2,3-butandienyl)-1,4-butanediamine (MDL 72527), significantly reduces ETBF-induced chronic inflammation and proliferation. Most importantly, in the multiple intestinal neoplasia (Min) mouse model, treatment with MDL 72527 reduces ETBF-induced colon tumorigenesis by 69% (P < 0.001). The results of these studies indicate that SMO is a source of bacteria-induced ROS directly associated with tumorigenesis and could serve as a unique target for chemoprevention.     

The effect of a single dose of cyclophosphamide on the jejunum of specified pathogenfree and germfree rats.

After a single dose of cyclophosphamide (100 mg/kg) cell proliferation in the jejunum of the rat decreased within the first 24 h and returned to the initial level after 48 h. Under the influence of cyclophosphamide, an increased cell loss in germfree rats could be observed. Villus height and villus cell count tended to decrease. Changes in disaccharidase activity in mucosal scrapings with respect to protein and DNA content could not be demonstrated. An influence of presence or absence of bacterial flora could be observed.     

Maturation of villus and crypt cell functions in rat small intestine

To evaluate the role of dietary polyamines in maturation of the rat small intestine, spermine was given orally twice daily to suckling pups from day 10 to day 14 postpartum at different doses: 0, 0.2, 0.5, 1, 2.5, and 5 mol/dose. Compared, to saline treated controls, spermine (5 mol) produced significant increases in mucosal mass parameters (+12 to +57%,P<0.05), induced prematurely, an adult pattern of microvillous enzymes, and enhanced respectively, by 19- and 3.5-fikd (P<0.01 vs controls) the concentration of the secretory component ofp-immunoglobulins in villous and crypt cells. The response of microvillous enzymes (lactase, sucrase, maltase, and aminopeptidase) to spermine was dose-dependent and-specific since oral administration of arginine (5 mol) or ornithine (5 mol) was without effect. Intestinal changes were found to be significant (P<0.05) for doses of spermine exceeding 1 mol/day, which is in the range of the amount of polyamines provided by solid pellets at weaning (0.4 mol/g). However, intestinal changes were undetectable at the physiological amounts of polyamines consumed by pups from rat milk during the suckling period (less than 0.3 mol/day). Consistent with a direct effect of spermine on the intestinal cell, the cytosolic activity of ornithine decarboxylase was depressed by 27-fold (P<0.005 vs controls) in the jejunum, while inhibition of ornithine decarboxylase by -difluoromethylornithine did markedly decrease but did not suppress the cell response to spermine. Alternately, plasma corticosteronemia, which was virtually, absent by day 14 in controls, ranged between 1.4 and 4.6 g/dl in 60% (N=9) of the spermine-treated rats. These novel findings indicate that dietary polyamines exert direct and indirect trophic effects on the rat immature intestine and can trigger at a critical level of intake the adult expression of villus and crypt cell functions.     

Jejunal disaccharidase activity in maturity onset diabetes

Because intestinal disaccharidase activity is elevated in experimental diabetes of rats, we measured these enzymes in small bowel biopsies from a group of patients with maturity onset diabetes and a group of controls. There was no difference in disaccharidase activity between the two groups, and we conclude that in man maturity onset diabetes not requiring insulin therapy does not affect intestinal disaccharidase activity.     

Jejunal morphology and mucosal enzyme activity following intestinal shunt operation for obesity.

Jejunal biopsy in 33 patients before and after intestinal shunt operation for obesity has demonstrated that neither surface nor volume of the villi increase after surgery. Specific disaccharidase activity remained unchanged, and specific alkaline phosphatase activity increased slightly. There was a significant decrease in protein content in the postoperative biopsies. It is concluded that weight stabilization after the shunt operation is due to adaptive compensation in the ileal remnant.     

Effect of mouse epidermal growth factor/urogastrone on the functional maturation of rat intestine

Mouse epidermal growth factor/urogastrone (EGF/UG), administered sc in a dose of 0.1 microgram/g BW twice daily for 3 days, increased intestinal weight per unit length, lactase specific activity, and net calcium transport in normal 2-week-old suckling rats, but had no effect on maltase or sucrase specific activity. In normal 3-week-old weanling rats, the intestinal function of which is essentially fully mature, EGF/UG had no effect. These results suggest that EGF/UG, either secreted endogenously or ingested in breast milk, may have a role in both the morphological and functional maturation of the suckling rat intestine.     

Activation of polyamine catabolism in transgenic rats induces acute pancreatitis

Polyamines are required for optimal growth and function of cells. Regulation of their cellular homeostasis is therefore tightly controlled. The key regulatory enzyme for polyamine catabolism is the spermidine/spermine N(1)-acetyltransferase (SSAT). Depletion of cellular polyamines has been associated with inhibition of growth and programmed cell death. To investigate the physiological function SSAT, we generated a transgenic rat line overexpressing the SSAT gene under the control of the inducible mouse metallothionein I promoter. Administration of zinc resulted in a marked induction of pancreatic SSAT, overaccumulation of putrescine, and appearance of N(1)-acetylspermidine with extensive depletion of spermidine and spermine in transgenic animals. The activation of pancreatic polyamine catabolism resulted in acute pancreatitis. In nontransgenic animals, an equal dose of zinc did not affect pancreatic polyamine pools, nor did it induce pancreatitis. Acetylated polyamines, products of the SSAT-catalyzed reaction, are metabolized further by the polyamine oxidase (PAO) generating hydrogen peroxide, which might cause or contribute to the pancreatic inflammatory process. Administration of specific PAO inhibitor, MDL72527 [N(1),N(2)-bis(2,3-butadienyl)-1,4-butanediamine], however, did not affect the histological score of the pancreatitis. Induction of SSAT by the polyamine analogue N(1),N(11)-diethylnorspermine reduced pancreatic polyamines levels only moderately and without signs of organ inflammation. In contrast, the combination of N(1), N(11)-diethylnorspermine with MDL72527 dramatically activated SSAT, causing profound depletion of pancreatic polyamines and acute pancreatitis. These results demonstrate that acute induction of SSAT leads to pancreatic inflammation, suggesting that sufficient pools of higher polyamine levels are essential to maintain pancreatic integrity. This inflammatory process is independent of the production of hydrogen peroxide by PAO.     

The Effects of Iron Deficiency, Protein Deficiency and Worm Infestation upon Disaccharidase Activity in the Rat

The effect of iron deficiency anaemia, protein deficiency and worm infestation upon intestinal disaccharidase activity in the rat was assessed following the observation that these factors may have contributed to the premature onset of lactase deficiency in man. Adaptation of intestinal lactase occurred between eight and ten weeks of age in young rats fed a 10% lactose diet. Iron deficiency anaemia depressed jejunal and ileal lactase activity. Sucrase and maltase activities were not significantly affected by iron deficiency. Adaptation of intestinal lactase was prevented by both protein deficiency and combined iron/protein deficiency. Sucrase activity was not significantly depressed by either of these and in many instances activity was higher than the control group. Similar changes were noted with maltase. Worm infestation with Nippostrongylus brasi-liensis consistently depressed jejunal lactase and maltase activities, but had little effect on sucrase activity. It was concluded that intestinal lactase in particular was depressed by a number of environmental factors and adaptation of lactase thereby prevented.     

Increase of intestinal brush border hydrolases in mucosa of streptozotocin-diabetic rats

Summary Experimental diabetes mellitus in rats was induced by streptozotocin. Five days after administration of streptozotocin intestinal brush border hydrolases (maltase, sucrase, trehalase, lactase) and alkaline phosphatase were markedly elevated at all levels of the small intestine as measured in the total homogenate and in the isolated brush border preparation. Insulin treatment beginning 15 h after administration of streptozotocin was able to decrease the increased disaccharidase activity due to streptozotocin diabetes. In experimental diabetes mellitus of rats trransport as well as digestive functions of the intestinal mucosa are stimulated.This work has been presented at the meeting of the European Society for Clinical Investigation, Scheveningen, The Netherlands, April 1972 and has appeared in abstract form (1a)     

Ligation or external fistulation of the common bile duct in the rat. II. Intestinal disaccharidase activities.

72 h after ligation or external fistulation of the common duct the activities of maltase, sucrase and lactase in the homogenate of the small intestinal mucosa of the rat were determined. The experiments were performed in connexion with intestinal perfusion studies, and the disaccharidase activities were measured in unperfused intestinal segments as well as in intestinal loops which had previously been perfused with a sucrose-containing solution. After bile duct ligation, the sucrase and maltase activities in a previously perfused intestinal loop were not different from those in sham-operated animals, the lactase activity was diminished. In a nonperfused segment, the sucrase activity was greater, the maltase activity was unchanged, and the lactase activity was lower than in control animals. After bile duct fistulation, the sucrase, maltase and lactase activities in a perfused segment were lower than in sham-operated rats. In a nonperfused loop, the sucrase activity was greater, the maltase activity was unchanged, and the lactase activity was lower then in the corresponding control group. These data suggest that bile is a factor which influences the total mucosal disaccharidase activities, and, probably, the intracellular enzyme distribution.     

Ontogenesis of the intestinal transport of biotin in the rat

Developmental aspects of the intestinal transport of biotin were examined in suckling (16 day old) and weanling (24 day old) rats using the everted sac technique. The results were compared with those of adult rats previously reported by us using the same intestinal preparation. Transport of biotin was linear for 20 min of incubation in all age groups. Transport of biotin was significantly (p less than 0.05) lower in the jejunum than the ileum of suckling rats but was not significantly different in the jejunum and the ileum of weanling rats. In adult rats, biotin transport was significantly (p less than 0.01) higher in the jejunum than the ileum. In all age groups, transport of biotin in the jejunum was saturable at low concentrations (less than 10 microM) but linear at high concentrations. The apparent Km and Vmax of the saturable process showed a progressive increase from suckling to weanling to adult rats (apparent Km of 0.63, 2.49, and 3.37 microM; Vmax of 18.3, 44.7, and 124.4 pmol/g.min, respectively). On the other hand, the rate of transport by the nonsaturable process showed a progressive decrease with maturation (143.8, 111.6, and 87.5 pmol/g.min for suckling, weanling, and adult rats, respectively). Transport of biotin in suckling and weanling rats was similar to that of adult rats in that it was Na+-, energy-, and temperature-dependent and inhibited by structural analogues. These results demonstrate that biotin transport undergoes clear maturational changes. These changes include a decrease in the affinity and an increase in the activity (and/or the numbers) of the transport carrier, a decrease in the rate of transport by the nonsaturable process, and a change in the preferential site of transport.     

Effect of epidermal growth factor on the development of rat gastric mucosa

Epidermal growth factor (EGF) has been shown to stimulate the growth of adult rat gastric mucosa and to increase DNA synthesis of mouse small and large intestinal mucosa. This study examines whether EGF affects the functional and structural development of the rat gastric mucosa. Rats were injected with 20 micrograms/kg EGF three times/day for 5 days beginning on the 10th day after birth. A control group of animals received saline injections of identical volume. All rats were killed on day 15. EGF significantly increased the weight of the whole stomach and the DNA, RNA, and protein content of the oxyntic gland mucosa, but had no effect on the RNA/DNA ratio, or antral and serum gastrin levels. Two groups of similarly treated rats, were anesthetized with ether, pylorus-ligated, and injected with either saline or pentagastrin (250 micrograms/kg) after they had recovered from anesthesia. EGF-treated rats had significantly higher rates of basal acid secretion and pentagastrin-stimulated acid secretion than the saline-treated controls. EGF, however, did not alter basal or pentagastrin-stimulated pepsin secretion nor did it change mucosal pepsinogen content. These results indicate that EGF stimulates oxyntic mucosal growth in unweaned rats but that it does not lead to precocious maturation or functional development.     

The role of the suckling stimulus in regulating pituitary prolactin mRNA in the rat

Prolactin (PRL) gene expression and the synthesis and secretion of PRL were examined in ovarian-intact lactating rats suckling eight pups on 10 days postpartum. Plasma samples were assayed for PRL concentrations, and pituitary glands were analyzed for total PRL content and PRL mRNA levels. We found that suckling-induced hyperprolactinemia was associated with very high levels of plasma PRL and a doubling in pituitary PRL mRNA levels, whereas pituitary PRL content was not changed. Removal of the suckling pups decreased plasma PRL concentrations 15-fold within 24 h. This decrease in PRL secretion was not accompanied by any significant change in pituitary PRL content. Evidently, both synthesis and secretion of PRL were decreased in the pituitary gland within 24 h following cessation of suckling, as pituitary PRL mRNA content had returned to diestrous levels at this time. To determine whether or not ovarian steroids might have contributed to the changes in PRL synthesis and secretion during lactation and after withdrawal of the suckling stimulus, the experiments were repeated in lactating rats ovariectomized (OVX) on day 2 postpartum. The results in these OVX rats were qualitatively similar to those described in ovarian-intact rats. We concluded from these findings that the stimulus of suckling induces increases in PRL mRNA levels in the pituitary which provides for the increased PRL synthesis accompanying increased PRL secretion. The cessation of suckling led to prompt decreases in PRL synthesis and secretion within 24 h.     

Implication of insulin and nutritional factors in the regulation of intestinal galactosyltransferase activity during postnatal development

In the rat small intestine, galactosyltransferases are the enzymes implicated in the biosynthesis of glycoproteins of the brush-border membranes and mucins. During postnatal development, the circulating insulin level increased at weaning in parallel with the activities of intestinal galactosyltransferases on O-glycans and N-glycans. This study deals with the role of insulin in the regulation of galactosyltransferase activities during postnatal development. The treatment of immature suckling rats with insulin induced a precocious increase in the activities of the O-glycan and N-glycan galactosyltransferases, partly reproducing the increase in galactosyltransferase activity normally found at weaning, since the O-glycan galactosyltransferase activity increased more quickly than the N-glycan galactosyltransferase activity. The sensitivity of the two galactosyltransferase activities to insulin disappeared after weaning, a period when drastic diet changes occur. In 22-day-old rats submitted to prolonged nursing (high-fat diet), the activities of the O-glycan and N-glycan galactosyltransferases were lower than those found in age-matched normally weaned rats (high-carbohydrate diet), indicating a delay in the maturation of the intestine of prolonged-nursing rats. The circulating insulin level of these animals stayed lower than that of the age-matched weaned rats. When the prolonged-nursing animals were treated with insulin, the O-glycan and N-glycan galactosyltransferase activities reached levels similar to those of the weaned rats. These observations suggest that insulin is one of the maturation factors for intestinal glycoprotein galactosylation and may be partly responsible for the natural enhancement of intestinal galactosyltransferase activities observed during postnatal development in relation to the dietary changes at weaning.