Nadine Dekeyser

Saccharomyces boulardii enhances rat intestinal enzyme expression by endoluminal release of polyamines.

Saccharomyces boulardii is a yeast widely used in humans for the prevention and treatment of infectious enteritis and Clostridium difficile-associated enterocolopathies. After oral administration to human volunteers or growing rats, S. boulardii enhances markedly the expression of intestinal enzymes as well as the production of the polymeric immunoglobulin receptor by mechanisms that remain unknown. We have analyzed the role of the yeast polyamines as potential mediators in the intestinal trophic response. In weanling rats (d 20 to d 30), a daily dose of 100 mg of lyophilized S. boulardii produced significant (p < 0.025) increases in sucrase (157%) and maltase (47%) activities. This dose corresponded to a total oral load of 678 nmol of polyamines per day (spermidine: 376 ± 32, sper-mine: 293 ± 26, putrescine: 9.5 ± 1.4 nmol/100 mg). Sper-mine, given orally to growing rats at doses nearly equivalent (500 nmol) to the load of polyamines provided by the yeast (678 nmol), reproduced similar enzymatic changes, including a 2.5-fold induction of sucrase, and enhanced maltase activity (+24%). Spermidine and spermine concentrations measured in the jejunal mucosa of treated rats were increased over matched controls by 21.4% (p < 0.005) and 21.9%, respectively (p < 0.002). After being centrifuged and filtered to discard residual yeast cells, 2-mL samples of jejunal and ileal fluid collected from 5. boulardii-treated rats by intestinal flushing contained higher levels of spermidine (48 and 60%) and spermine (150 and 316%) than did control rats. our data indicate that lyophilized S. boulardii exerts trophic effects on the small intestine that are likely mediated by the endoluminal release of spermine and spermidine.

Maturation of villus and crypt cell functions in rat small intestine. Role of dietary polyamines.

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 μmol/dose. Compared, to saline treated controls, spermine (5 μmol) 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 μmol) or ornithine (5 μmol) was without effect. Intestinal changes were found to be significant (P<0.05) for doses of spermine exceeding 1 μmol/day, which is in the range of the amount of polyamines provided by solid pellets at weaning (0.4 μmol/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 μmol/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 α-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 μg/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.

Polyamine profiles in human milk, infant artificial formulas, and semi-elemental diets.

Summary Using a sensitive high-performance liquid chromatography method, we quantified the concentration of polyamines (putrescine, spermidine, and spermine) in human milk as well as in a representative group of commonly used artificial infant formulas. Variations in polyamine levels were also analyzed in human milk during the immediate postnatal period. During the first week postpartum, putrescine levels in human milk remained very low and varied little, while spermidine and spermine concentrations rose markedly during the first 3 days, reaching plateau levels that were 12 and eight times higher, respectively, than the values measured on day 0. The mean total polyamine concentration was 557 ± 18 nmol/dl with the following profile: spermine, 313 ± 16; spermidine, 220 ± 20; and putrescine, 24 ± 3.5. In artificial powdered formulas, the polyamine concentration was 10 times lower than in human milk, with no difference in putrescine and spermine contents between first-age and second-age formulas. By contrast, semi-elemental diets prepared by hydrolytic procedures using crude extracts of pancreatic enzymes were shown to be major sources of polyamines with a profile similar to that of human milk. Compared with first-age formulas, mean concentrations in spermine and spermidine were 39 and six times higher, respectively, in these semi-elemental diets, whereas putrescine levels remained almost equivalent in all types of milk tested. These data indicate that human milk and some semi-elemental diets provide substantial amounts of spermine and spermidine to neonates and infants that could potentially modulate intestinal maturation.

Saccharomyces boulardii Produces in Rat Small Intestine a Novel Protein Phosphatase that Inhibits Escherichia coli Endotoxin by Dephosphorylation

Using a polyclonal antibody raised against a highly conserved sequence of 38 amino acids containing the activation site (VTDSAAGAT) common to mammalian and yeast alkaline phosphatases (AP), we identified in decapsidated Saccharomyces boulardii a protein phosphatase detected by autoradiography as a single signal (63 kD). Using an affinity chromatography column, the protein phosphatase could be concentrated 39.1-fold and presented as a doublet of two subunits. Compared with rat and bovine purified intestinal AP, the enzyme from S. boulardii had a greater ability to dephosphorylate the lipopolysaccharide (LPS) of Escherichia coli 055B5. When tested in vivo, intraperitoneal injection of intact LPS to rats produced, after 9 h, 100 ng/mL of circulating tumor necrosis factor-α with inflammatory lesions and apoptotic bodies in the liver and the heart, whereas rats injected with partially dephosphorylated LPS produced only 40 ng/mL tumor necrosis factor-α without organic lesions. In conclusion, S. boulardii is able to inhibit toxicity of E. coli surface endotoxins by the release of a protein phosphatase exhibiting a great capacity of dephosphorylation.

Intestinal development in the suckling rat: effect of insulin on the maturation of villus and crypt cell functions.

Abstract The influence of insulin on the postnatal development of intestinal functions linked to villus cells (sucrase, lactase, maltase and aminopeptidase) and crypt cells (secretory component of immunoglobulins, SC) has been studied in suckling and weanling rats. At 9 days of age, the animals received a daily injection of insulin 12·5 mU g‐1body weight day‐1for 4 days. Compared with saline‐treated controls, insulin had no effect on the development of the intestinal mucosal mass parameters determined in the jejunum, ileum and colon. A premature appearance of sucrase was noted in isolated jejunal villus and crypt cells, the level of activity reached by the enzyme being dependent of the amount of insulin injected. By 6 and 12 h after a single injection of the hormone (12·5 mU g‐1body weight), sucrase activity was detected in all the cell fractions along the villus‐crypt axis. In villus cells of insulin‐treated rats, maltase, lactase and aminopeptidase activities were significantly (P < 0·001) increased (+201%, +50%, +207%, respectively, vs. controls), whereas the concentration of SC measured by a sensitive immunoradiometric assay was enhanced over the controls by 75% in villus cells, 83% in crypt cells and 172% in the liver. Weanling rats treated from day 10 to day 20 postpartum with 12·5 mU insulin also exhibited a higher intestinal production of SC (+93%, P < 0·01) than did saline controls. The administration of Actinomycin D to 9‐day‐old suckling rats 1 h after insulin injection completely inhibited the adaptative process to the hormone; the activities of sucrase, lactase, maltase and aminopeptidase measured in rats treated with Actinomycin D being equivalent to those recorded in saline controls. Our data demonstrate that: (i) a premature increase of the circulating level of insulin accelerates the maturation of intestinal functions linked to both villus and crypt cells; (ii) the response to the hormone occurs rapidly over the entire villus‐crypt unit, whatever the degree of epithelial cellular differentiation; (iii) the stimulation of the synthesis of brushborder membrane enzymes by insulin appears to be regulated at the level of transcription.

Hormonal regulation of the rat small intestine: responsiveness of villus and crypt cells to insulin during the suckling period and unresponsiveness after weaning.

ABSTRACT: To further document the effect of insulin on intestinal maturation, suckling rats were treated either with exogenous insulin (12.5 mU · g body wt, intraperitoneally, twice daily) or with saline from d 8 to 12 postpartum. Sucrase activity in brush border membrane extracts was precociously induced by insulin, whereas the activities of other brush border membrane enzymes (maltase, aminopeptidase, and neutral lactase) were enhanced (+30 to +131%,p < 0.01 versus controls). The lysosomal enzyme, N-acetyl-β-glucosaminidase, which normally declines at weaning was significantly (p < 0.025) decreased in both villus (-51%) and crypt cells (-57%) isolated from the jejunum of insulin-treated rats. The microsomal enzyme, sulfatase C, and the cytosolic enzyme, lactate dehydrogenase, were also sensitive to insulin with decreases in activity ranging from -37 to -63% (p < 0.05) compared to salinetreated control rats. Insulin at doses of 0.5 or 12.5 mU did not influence plasma total corticosterone levels, which were about 9-fold lower in suckling than in 25-d-old weaned rats. In weaned rats (from d 25 to 32) insulin treatment (12.5 mU) failed to influence the activity of brush border membrane hydrolases or of lysosomal, microsomal, and cytosolic enzymes. The synthesis rate of mature sucraseisomaltase, measured in weaned rats (32 d) by the incorporation of 14C-leucine into the enzyme precursor protein, was equivalent in both groups. These data demonstrate that the immature enterocyte of the suckling rat is responsive to insulin, whereas the mature enterocyte of the weaned rat is unresponsive. The effect of insulin on the intestinal cell appears not to be mediated via an endogenous stimulation of corticosterone release.

Refeeding after starvation in the rat: comparative effects of lipids, proteins and carbohydrates on jejunal and ileal mucosal adaptation.

Abstract. To compare the tropic effect of different dietary nutrients on mucosal adaptation in the jejunum and ileum, adult rats were submitted to a 96‐h period of starvation and refed isocaloric liquid diets (1.5 kcal ml‐1) containing either protein (casein), carbohydrate (starch) or lipids. In the jejunum, 4 days of starvation caused mucosal hypoplasia, villus and crypt shortening and a decrease in the total activity of disaccharidases with the exception of lactase which was markedly enhanced. In contrast, mucosal hypoplasia was incomplete in the ileum which exhibited an increase in crypt depth and in the specific and total activities of disaccharidases and of aminopeptidase.

Saccharomyces boulardii enhances N-terminal peptide hydrolysis in suckling rat small intestine by endoluminal release of a zinc-binding metalloprotease.

Saccharomyces boulardii (S. boulardii), a biotherapeutic agent effective in acute and chronic enterocolopathies, produces trophic intestinal effects at least in part mediated by the endoluminal release of polyamines. However, the effects of the yeast on peptide hydrolysis have not yet been studied. The objectives of this study were to assess in suckling rats the endoluminal and mucosal aminopeptidase activities in response to S. boulardii treatment and to analyze their related mechanisms. Peptidase activities were assayed on yeast cells by using several l-amino acid-p-nitroanilide substrates in the pH range of 2 to 10. A marked hydrolytic activity was found for l-leucine-p-nitroanilide that peaked at pH = 8 (Km = 0.334 mM, Vmax = 44.7 μmol·min−1·g-1 protein). N-terminal peptide hydrolysis was confirmed using as substrate l-Leu-Gly-Gly (Km = 4.71 mM, Vmax = 18.08 μmol·min−1·g-1 protein). Enzyme reactions were inhibited in the presence of 1 mM Zn2+. Oral treatment of sucklings with S. boulardii significantly enhanced jejunal and ileal mucosal leucine-aminopeptidase activities by 24 and 34%, respectively, over controls. In concordance, aminopeptidase activity was enhanced in jejunal and ileal endoluminal fluid samples by 47 and 105%, respectively. By use of an IgG-purified antibody raised against the zinc-binding domain common to metalloproteases, the yeast aminopeptidase was immunoprecipitated and detected as an heteromeric enzyme of 108 and 87-kD subunits. S. boulardii, when given orally to suckling rats, is able to significantly enhance hydrolysis of N-terminal oligopeptides in both endoluminal fluid and intestinal mucosa by the endoluminal release of a leucine aminopeptidase that appears to be a zinc-binding metalloprotease belonging to the M1 family of peptidases.

Premature stimulation of rat sucrase-isomaltase (SI) by exogenous insulin and the analog B-Asp10 is regulated by a receptor-mediated signal triggering SI gene transcription

The mechanism(s) by which insulin enhance prematurely the activity of brush border membrane (BBM) hydrolases in rat immature intestine is unknown. Therefore, we have compared the responses of four BBM enzymes[sucrase-isomaltase (SI), maltase, lactase-phloridzine hydrolase (LPH), and aminopeptidase] with exogenous insulin, the analog B-Asp10, IGF-I, and antireceptor MAb [insulin-receptor (IR) MAb] given to preweaning pups. Low doses of insulin caused a precocious induction of SI and of SI mRNA and stimulated maltase activity without effect on LPH nor on aminopeptidase activities. IGF-I given at the same dose as that of insulin had no detectable effect on these enzymes. Administration to sucklings of IR MAb prevented the effect of endogenous insulin by inhibiting the expression of SI and maltase without effect on LPH activity. B-Asp10, an insulin analogue that exhibits in vitro a 3.5-fold increase in receptor affinity with sustained signaling of the receptor tyrosine kinase, caused an overexpression of SI by 3.5-fold and of maltase by 1.5-fold compared with equivalent doses of normal insulin. The premature increases in SI activity, SI mRNA, and maltase activity in response to insulin were dose-dependent and were associated with dose-dependent increases in intracellular spermine and spermidine concentrations. In conclusion, these data suggest that the premature induction of SI by insulin is mediated by a dose-dependent signal initiated by binding of the hormone to its intestinal receptor, which after transduction into the cell indirectly triggers the transcription of the SI gene, possibly by changes in intracellular polyamine concentrations.

Intestinal transport of calcium in rat biliary cirrhosis

The characteristics of intestinal calcium transport in chronic cholestasis remain largely unknown. Using an experimental model of biliary cirrhosis in the rat, we aimed to investigate changes in calcium transport at the jejunal and ileal levels. Two methods were used: 1) uptake of 45Ca in brush border membrane vesicles and 2) measurements of transepithelial fluxes of calcium in Ussing chambers. Thirty days postsurgery, cholestatic rats presented biliary cirrhosis, with normal growth, normal daily energy, and calcium intakes, but had depressed circulating levels of 25-(OH)-vitamin D2 and 1,25-(OH)-vitamin D3. Compared with sham-operated controls, 45Ca uptake ([Ca2+] = 0.03 mmol) measured in vesicles from cholestatic rats was decreased by 3-fold in the duodenojejunum, in concordance with a lower content in brush border membrane calmodulin. Other changes in brush border membrane composition included decreases in structural proteins, microvillous enzymes, and in triglyceride content. Transepithelial fluxes of calcium measured in the ileum([Ca2+] = 1.2 mmol) revealed in controls a net basal secretion flux(Jnet = -30.4 ± 8.1 mmol·h-1·cm-2) that was reduced by 3-fold (p < 0.05) in vitamin D-deficient rats(Jnet = -10.4 ± 4.8 mmol·h 1·cm 2). In response to 25-(OH)-vitamin D2 treatment, calcium uptake rates increased by 40% in the jejunum, whereas in the ileum, the secretion flux returned to basal control levels. Oral administration of taurocholate or tauroursodeoxycholate (50 mmol) depressed almost completely calcium uptake capacity in the duodenojejunum. By complexing free calcium, tauroconjugated bile acids inhibited in vitro calcium uptake proportionally to their concentration in the medium (0-40 mmol). Our data indicate that, in rat biliary cirrhosis, transport capacity of calcium in the duodenojejunum is markedly reduced in association with vitamin D deficiency and alterations in brush border membrane composition.