Regulation of Dab2 expression in intestinal and renal epithelia by development

Disabled-2 (Dab2) is an intracellular adaptor protein proposed to function in endocytosis. Here, we investigate the intestinal and renal Dab2 expression versus maturation. Dab2 mRNA levels measured by RT-PCR are greater in the small than in the large intestine. Immunological studies localize Dab2 to the terminal web domain of the enterocytes and reveal the presence of a 96-kDa Dab2 isoform in the apical membrane of the jejunum, ileum, and renal cortex of the suckling and adult rat. A 69-kDa Dab2 isoform is only observed in the apical membranes of the suckling ileum. During the suckling period, the Dab2 mRNA levels measured in the enterocytes and crypts and those of the 96-kDa Dab2 isoform are greater in the ileum than in the jejunum. No segmental differences are observed in the adult intestine. In the intestine, the levels of Dab2 mRNA and those of the 96-kDa Dab2 isoform decrease to adult values at weaning, whereas in the kidney they increase with development. Weaning the pups on a commercial milk diet slows the periweaning decline in the levels of Dab2 mRNA in the crypts and of those of the 96-kDa isoform. This is the first report showing that the 96-kDa Dab2 isoform is expressed at the apical domain of rat small intestine, that ontogeny regulates Dab2 gene expression in intestine and kidney and that retarding weaning affects intestinal Dab2 gene expression.     

Short-term effects of spermine ingestion on the small intestine: a comparison of suckling and weaned rats

We have previously shown that spermine, shortly after its ingestion, can induce the alteration of the morphology of the small intestine of suckling rats. It was proposed that this alteration is due to polyamine accumulation inside the epithelial cells. This could also be related to the fact that the intestine of the suckling rat is in an immature state. To shed light on this issue, disaccharidase and alkaline phosphatase activity assays, protein, DNA and RNA content measurements and polyamine concentration analysis were performed on the small intestine of suckling and weaned Wistar rats treated with spermine. Spermine did not induce the same intestinal alterations in weaned rats compared to suckling animals. Indeed, in sucklings, spermine administration induced a decrease of the protein, DNA, putrescine and spermidine intestinal content, suggesting a cell loss. The cell loss impaired the activity of intestinal enzymes: lactase, maltase and alkaline phosphatase. In weaned rats, the same treatment did not alter these parameters. Exogenous spermine by itself is not sufficient to induce the alterations described here and previously. The maturity degree of the small intestine could be the basis of this process.     

Pre- and postnatal development of differentiated functions in rat intestinal epithelial cells

A panel of monoclonal antibodies to intestinal cell surface components has been used to compare the expression of differentiation-specific antigens in the epithelial cells of fetal, suckling, and adult rat small intestine. Indirect immunofluorescence staining, and immunopurification of detergent-solubilized membrane proteins, followed by single- and two-dimensional slab gel electrophoretic analysis, have demonstrated that fetal intestinal cells (at day 21 of gestation) express most differentiation-specific markers typical of adult absorptive villus cells. A marked heterogeneity in antigen expression was observed among different villus cell populations in suckling rat intestine, and three cell surface components were identified which are exclusively present during this period of intestinal development. Striking changes in the patterns of antigen expression in crypt and villus cells, and variations in the apparent isoelectric points for most luminal membrane components, were associated with the maturation of the intestinal mucosa at weaning. These changes could not be prematurely induced by cortisone injection in newborn rats, suggesting that factors other than glucocorticoids are responsible for the postnatal development of the intestinal epithelium. These results suggest that basic differences in biological properties and regulatory mechanisms exist among intestinal epithelial cells at different stages of pre- and postnatal maturation.     

Ontogenic and nutritional modifications in the intestinal fucosylation process at the weaning period. Influence of dietary fibers

In the rat small intestine, the glycosylation changes which normally take place at the weaning period are characterized by a shift from sialylation of fucosylation. The introduction of dietary fibers at weaning is one of the more striking nutritional modification so that some authors have suggested that the presence of fibers and the development of colonic fermentation might be important for the development of the small intestine, as for the colon. In order to define the respective contribution of ontogenic and nutritional factors to the intestinal glycosylation changes at this period, some aspects of the intestinal glycosylation were studied in five groups of rats (16-day-old suckling rats, prolonged nursing 23-day-old rats, 23-day-old rats weaned at day 19 with either a fiber-free, a cellulose or a pectin diet). Intestinal glycoproteins of suckling rats are characterized by a low fucose content and a high proportion of mannose. The amounts of the neutral sugars (fucose, mannose and galactose), expressed either per gram of intestine or for one intestine, are alwars higher in the fiber-fed groups than in the prolonged-nursing group or the group fed the fiber-free diet. Activities which promote fucosylation process (GDP-fucose production and fucosyltransferase activities) and those which are opposed to fucosylation (endogenous inhibitor of fucosyltransferase and GDP-fucose pyrophosphatase) are strongly modified in opposite ways at day 23 as compared to day 16. These modifications depend on the age of the animal (ontogenic factors) with additional modifications induced by the dietary factors. In particular, similar sugar contents and patterns are obtained with cellulose and pectin diests though the enzymatic activities of the fucosylation pathway are very different. No correlation was found between the caecal content of short chain fatty acids and any of the parameters under study. Thus, dietary fibers induce metabolic changes in the small intestine glycosylation in short-term experiments independently of colonic fermentation. Besides, these results point out that the consideration of fucosyl-transferase activities alone are not sufficient to predict glycoprotein fucose content and that other regulatory sites are involved. Dietary manipulations at the weaning period could represent a good model for the study of glycosylation regulation.     

Ontogeny of two vitamin A-metabolizing enzymes and two retinol-binding proteins present in the small intestine of the rat.

The patterns of expression of cellular retinol-binding protein (CRBP), cellular retinol-binding protein, type two [CRBP(II)], lecithin: retinol acyltransferase (LRAT), and microsomal retinal reductase were examined for rat small intestine during the perinatal period. CRBP was present (15 pmole per mg soluble protein) at the earliest time examined, the 16th day of gestation, declining by 70% by birth, maintained to adulthood. In contrast, CRBP(II) appeared 2-3 days before birth, rising to its highest level (500 pmole per mg soluble protein) by day 3 after birth, then declining by 50% during the late suckling period to the adult level. Immunohistochemistry revealed that CRBP(II) initially appeared in the epithelial cell layer in a patchy manner, resolving by birth into an even staining of all villus-associated enterocytes. In contrast, CRBP was evenly expressed in the epithelial cell layer at day 17/18 but was absent by birth. Intestinal LRAT activity increased rapidly in the 2 days prior to birth, then declined at weaning to the adult level. Microsomal retinal reductase was measurable in the intestine at birth, but not detected during the early suckling period, reappearing at day 21. Considerable increase was then observed coincident with weaning, when carotenes, from which retinal is derived, became an important source of vitamin A. The pattern of appearance of these elements appears to prepare the intestine for the necessary processing of vitamin A required after birth.     

Calcitonin secretion during postnatal development in the rat: beta-adrenergic agents and vitamin D3 metabolites

The possible contribution of catecholamines and vitamin D3 metabolites to the high plasma calcitonin (CT) levels in suckling baby rats is unknown. So, in vivo and in vitro (using a perifusion system) effects of beta-adrenergic agents and vitamin D3 metabolites on CT release were studied in the rat during the postnatal development. In 13-day-old rats, the increase in plasma CT levels induced by isoproterenol injection (0.1 micrograms/kg b.w.) was inhibited by a previous administration of propranolol. A significant decrease in plasma CT levels was observed after propranolol injection in baby rats (0.68 +/- 0.05 ng/ml vs. 0.93 +/- 0.01 ng/ml). A daily injection of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3; 25 pmoles/rat/day during 4 days) induced a marked rise in plasma calcium (16.1 +/- 0.2 mg/dl), and a great decrease in thyroidal CT contents (approximately 70% of control values) in 13-day-old rats while no change was noted with 24,25-dihydroxycholecalciferol (24,25-(OH)2D3). A negative correlation between plasma calcium and thyroidal CT stores was found in suckling and in weaning rats treated with different doses of 1,25-(OH)2D3, suggesting an indirect effect of 1,25-(OH)2D3 on CT secretion. The mobilization of the thyroidal CT content was greater in weaning than in suckling rats in response to a given hypercalcemia. In vitro, 5 X 10(-5) M isoproterenol induced a rapid increase in CT secretion rate while 1,25-(OH)2D3 inhibited the rise in CT release induced by 3.0 mM calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes in the activities of sialyl- and fucosyltransferases in rat small intestine

To study an enzymatic basis for the postnatal changes in intestinal glycosylation, the activities of sialyl- and fucosyltransferases were determined in the particulate fraction of mucosal cells prepared from rat small intestine of various ages. The results show that sialyltransferase activity was present in increased levels compared to adults during the preweaning period (1-2 weeks) and subsequently declined 5-fold to adult levels after weaning, while fucosyltransferase activity was decreased compared to adults in the first 3 weeks of life, rapidly increased at 4 weeks, and reached adult levels (10-fold) by 5 weeks. The changes in both sialyl- and fucosyltransferase activities were reflected by the membranous content of glycosidic-bound sialic acid and fucose, respectively. Cortisone injection precociously induced a decreased sialyltransferase activity and an increased fucosyltransferase activity in 2-week-old suckling rats. This study indicates that the activities of sialyl- and fucosyltransferases were reciprocally related and modulated by cortisone action in the developing intestine. These enzyme changes may be responsible for the previously noted shift from sialylation to fucosylation of the intestinal mucosa during maturation.     

Development of pyrroline-5-carboxylate synthase and N-acetylglutamate synthase and their changes in lactation and aging

Using newly developed assay procedures, we studied the development of pyrroline-5-carboxylate synthase (PCS) and N-acetylglutamate synthase (AGAS) activity in rat tissues. PCS in the small intestine of fetuses was 1/5 that of adults and reached an adult level as early as postnatal Day 1. The highest peak was observed at Day 14, and then activity decreased to the adult level. However, PCS in the brain was highest at birth and quickly inactivated in a few days. AGAS in the fetus small intestine was 1/3 that of adults and became higher than the adult level by 40% at Day 1 but was reduced to 1/2 that of adults at Day 3. Subsequently activity increased gradually to the adult level at Day 24. On the contrary, AGAS in the fetus liver was only 1/20 that of adults, and activity increased slowly up to 10 weeks and more. Pregnancy and lactation reduced liver AGAS markedly up to Day 8 and intestinal PCS considerably up to Day 14 after parturition. PCS in the small intestine of senescent rats was almost halved compared to young controls on a whole tissue basis. AGAS in the small intestine was also halved on a gram wet weight basis. Nonetheless the liver AGAS of 430-day-old rats was higher than that of the controls, although that of 630-day rats was lower. The results indicate that the arginine synthesizing enzymes in the small intestine are highly activated in suckling and weaning, and raise a question whether arginine remains fully dispensable in pregnancy, lactation, and senescence.     

Ileal VIP submucous neurons: confocal study of the area enlargement induced by myenteric denervation in weanling rats

Vasoactive Intestinal Peptide (VIP) neurons are maturing during suckling and weaning periods and the neuropeptide VIP is thought to be neurotrophic during ontogenesis. We have previously demonstrated that suckling rats with myenteric ablation have significantly higher mitotic index and an increase on villus height and crypt depth 15 days after treatment. In the current study, we measured the area of VIP neurons of submucous plexus in the ileum of weanling rats, in which myenteric neurons were ablated by serosal application of benzalkonium chloride (BAC). The area of VIP immunoreactive cell bodies, reconstructed under confocal microscope, was significantly increased in response to denervation. This result suggests that the myenteric plexus may have an inhibitory role over submucous plexus in the normal intestine. The enhanced production of VIP may be correlated with the increased epithelial proliferation induced by denervation in a critical period of life, from suckling to weaning time.     

Expression of galectin 4 in the rat small intestine during postnatal development

We determined the expression of an endogenous lectin, galectin 4, in the rat small intestine during postnatal development. The mRNA levels of galectin 4 did not change significantly between birth and adulthood. In contrast, the protein was present at higher levels after than before weaning, and the potential ligands for galectin 4 were more highly represented in the enterocyte microvilli of weaned than of suckling rats. These results suggest possible differences either in galectin 4 mRNA stability, in its translation regulation or in the protein stability.     

Intestinal neuraminidase activity of suckling rats and other mammals. Relationship to the sialic acid content of milk.

1. The neuraminidase activity of homogenates of the mucosa of the middle and distal thirds of the small intestine of rats increased about 5-fold between birth and 4 to 8 days of age, and then gradually declined to the much lower adult activity by 24 days. No comparable changes occurred in the proximal third. 2. In 8-day-old rats, the neuraminidase activity of the middle and distal thirds of the small intestine was about 10 times greater than that of the proximal third, 20 times greater than that of the colon and at least 100 times greater than that of the liver, brain, gastric mucosa or pancreas. 3. In all other species investigated (mice, rabbits, cats and guinea pigs), the neuraminidase activity of the middle and distal thirds of the small intestine was greater in suckling animals than in adults. 4. The sialic acid content of rat milk increased about 2-fold between birth and 8 days post partum and then declined. 5. There was a highly significant positive correlation between the intestinal neuraminidase activity of suckling animals of various species and ages and the sialic acid content of milk obtained from the corresponding species and stage of lactation. 6. It is suggested that the intestinal neuraminidase of suckling mammals functions primarily to remove sialic acid from various components of milk, thus providing sialic acid for the synthesis of sialoglycoproteins and gangliosides by the young.     

Effect of weaning terms and protein deficit in the rat pup nutrition on activities of digestive enzymes

Restriction of protein in nutrition of rat pups weaned at different terms has been found to produce changes in activities of digestive enzymes (maltase, alkaline phosphatase, aminopeptidase M, and glycyl-L-leucine dipeptidase) in the small and large intestine both at once after cessation of nutrition with low-protein diet for 10 days and 4 months later. In adult animals after the earlier or later weaning there are observed not only a decrease or increase of the enzyme activities, but also a different type of distribution of the alkaline phosphatase activity along the small intestine, which is more pronounced in the lately weaned rats. Thus, disturbance of metabolic programming of enzyme systems of the small and large intestine due to a change of quality of nutrition in early ontogenesis depends on terms of weaning of animals.     

Effect of weaning terms and protein deficit in rat pup nutrition on activities of digestive enzymes

Restriction of protein in nutrition of rat pups weaned at different terms has been found to produce changes in activities of digestive enzymes (maltase, alkaline phosphatase, aminopeptidase M, and glycyl-L-leucine dipeptidase) in the small and large intestine both at once after cessation of nutrition with low-protein diet for 10 days and 4 months later. In adult animals after the earlier or later weaning there are observed not only a decrease or increase of the enzyme activities, but also a different type of distribution of the alkaline phosphatase activity along the small intestine, which is more pronounced in the lately weaned rats. Thus, disturbance of metabolic programming of enzyme systems of the small and large intestine due to a change of quality of nutrition in early ontogenesis depends on terms of weaning of animals.     

Obesity and changes of alkaline phosphatase activity in the small intestine of 40- and 80-day-old rats subjected to early postnatal overfeeding or monosodium glutamate

To investigate the relationship between development of obesity and the small intestinal functions two experimental models of male Wistar rats were used in the present work: 1) early postnatally overfed rats, nursed from birth to weaning in small litters (SL, 4 pups/nest), and 2) neonatally monosodium glutamate treated rats (MSG 2 mg/g b.w. administered s.c. for 4 days after birth) submitted to the same early nutritional manipulation. After weaning, all animals had free access to a standard pellet diet and at 40 and 80 days of age their body weight, body fat content and food consumption as well as changes of the brush-border-bound duodenal and jejunal alkaline phosphatase (AP) activity were compared with parameters of the offsprings raised under normal feeding conditions (NL, 8 pups/nest). At 40 and 80 days of age the postnatally overfed pups from SL nests became heavier, displayed a significantly increased epididymal plus retroperitoneal fat pad weight (P<0.01) and significantly higher AP activity in both segments of the small intestine (P<0.01) in comparison with rats nursed in NL nests, although their mean daily food intake did not differ from that of non-obese rats during the postweaning periods examined. In contrast, the same treatment of MSG rats had only a small effect on late appearance of obesity, i.e. in early postnatally overfed and normally fed MSG rats a similar pattern of body weight, food intake, adiposity and AP activity was found after weaning. The effect of MSG-treatment was also accompanied by the appearance of normophagia, hypophagia and stunted growth on day 40 and day 80, respectively. Moreover, the size of fat depots and the increase of brush-border-bound AP activity in MSG rats belonging to the SL and NL groups was quantitatively similar to the values size of these parameters observed in SL obese rats subjected to early postnatal overnutrition. These results indicate that postnatal nutritional experience (overnutrition) may represent a predisposing factor in control rats from small litters for the development of obesity in later life. Permanently increased small intestinal AP activity observed after weaning in both models of obesity when hyperphagia is not present suggest that these functional changes and associated alterations in food digestion could be a component of regulatory mechanisms contributing to the maintenance of their elevated body fat weight.     

Mucosal IgA increase in rats by continuous CLA feeding during suckling and early infancy

The aim of this work was to establish the effect of the cis9,trans11 conjugated linoleic acid (CLA) isomer on mucosal immunity during early life in rats, a period when mucosal immunoglobulin production is poorly developed, as is also the case in humans. CLA supplementation was performed during three life periods: gestation, suckling, and early infancy. The immune status of supplemented animals was evaluated at two time points: at the end of the suckling period (21-day-old rats) and 1 week after weaning (28-day-old rats). Secretory IgA was quantified in intestinal washes from 28-day-old rats by ELISA technique. IgA, TGFbeta, and PPARgamma mRNA expression was measured in small intestine and colon by real time PCR, using Taqman specific probes and primers. IgA mucosal production was enhanced in animals supplemented with CLA during suckling and early infancy: in 28-day-old rats, IgA mRNA expression was increased in small intestine and colon by approximately 6- and 4-fold, respectively, and intestinal IgA protein by approximately 2-fold. TGFbeta gene expression was independent of age and type of tissue considered, and was not modified by dietary CLA. Gene expression of PPARgamma, a possible mediator of CLA's effects was also upregulated in animals receiving CLA during early life. In conclusion, dietary supplementation with CLA during suckling and extended to early infancy enhances development of the intestinal immune response in rats.     

Elevated iron absorption in the neonatal rat reflects high expression of iron transport genes in the distal alimentary tract

Intestinal iron absorption is extremely high in neonatal mammals but falls rapidly to adult levels following weaning. The aim of this study was to investigate the molecular basis of this elevated neonatal absorption using the rat as an experimental model. RNA was extracted from various sections of the intestine of 10-, 15-, 20-, 25-, and 300-day-old rats and the expression of the genes encoding DMT1 (Slc11a2), ferroportin (Slc40a1), Cybrd1 (Cybrd1), and hephaestin (heph) determined by ribonuclease protection assay. The hepatic expression of Hamp was studied at the same ages. Iron absorption was examined by following (59)Fe uptake in both whole animals and in isolated intestinal loops. Slc11a2, Slc40a1, and Cybrd1 mRNAs were highly expressed in all regions of the small intestine and colon studied in suckling rats. However, after weaning, when iron absorption declined significantly, strong expression was retained only in the duodenum. No change in hephaestin mRNA occurred in any part of the digestive tract. In the distal small intestine and colon, Slc40a1 expression most closely followed the change in absorption that occurred after weaning. Hamp expression was low during the neonatal period and increased to adult levels following weaning. Our results suggest that the distal small intestine and colon contribute significantly to the high intestinal iron absorption seen in neonatal animals and that this reflects increased expression of the iron transporters, particularly Slc40a1.     

A growth-stimulating activity derived from the proximal small intestine is associated with an adaptive response

Luminal nutrition is important for the maintenance of small intestinal structure and function. The equilibrium between crypt cell production and villous cell loss in the mucosal epithelium of the small intestine is altered under certain conditions such as after a small bowel resection. Immediately after resection, there is a marked increase in crypt cell proliferation giving rise to an adaptive hyperplasia in the remnant intestine and for this response luminal nutrition is a critical factor. We have previously demonstrated the presence of a growth-stimulating (GS) activity in a heat-stable acidic extract of the rat proximal intestine 24, 48, and 96 h after resection, which is coincidental with an increase in crypt cell proliferation as measured by thymidine kinase activity. Eight days after resection when the GS activity is no longer detectable, the thymidine kinase activity returns to control values. The molecular weights of the peptides associated with this GS activity are 4500 and 1500, as determined by Sephadex gel filtration. Of note is that the oral intake of food is necessary for the appearance of the GS activity postoperatively. The presence of the GS activity has also been demonstrated upon refeeding after a fast, as well as at weaning in the rat, two physiological situations known to be associated with increased proliferation in the small intestine. This GS activity in the proximal intestine first detected in the resection model may represent a general mechanism by which food controls the cell renewal pattern of the small intestine.