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The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5 + intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5 + ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5 + ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5 + ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

There are conflicting data from human studies regarding the ability of exogenous glucocorticoids to stimulate maturation of the small intestine. The discrepancies may relate to differences in hormone doses and age administered. To explore this general concept, we have used a mouse model to determine intestinal responsiveness to dexamethasone (DEX) at various times during development. We first showed that trehalase mRNA is a sensitive marker of intestinal maturation in the mouse; being undetectable (by Northern blotting) in the prenatal period, expressed at low levels during the first 2 postnatal weeks and then displaying a marked increase in the 3rd postnatal week. DEX was unable to elicit detectable trehalase mRNA in fetal mice, but caused significant increases in the postnatal period. The use of a range of DEX doses (0.0125-2.5 nmol/g BW per day) established that there is no change in sensitivity between the 1st and 2nd postnatal weeks, but there is a significant increase in maximal responsiveness of trehalase mRNA to the hormone. Similar results were obtained when sucrase-isomaltase mRNA was assayed in the same animals. Thus, in this rodent model, there appears to be at least three phases in the DEX responsiveness of the developing intestine: an early phase (prenatal) when DEX is unable to elicit intestinal maturation; then a phase (first postnatal week) of modest responsiveness; then a transition to increased responsiveness. These findings point to the need for careful attention to dose and age in analyses of glucocorticoid effects in human infants.

The synergistic effects of dexamethasone (DEX) and thyroxine (T4) on the postnatal maturation of the 13-d-old rodent small intestine has been studied. Previous studies have shown that hydrocortisone and T4 produced a synergistic response in enzyme maturation. However, T4 elevates corticosteroid-binding globulin, which reduces the clearance of hydrocortisone. Thus, the apparent synergy between T4 and hydrocortisone may have been due to increased glucocorticoid availability. DEX, which does not bind to corticosteroid-binding globulin, was given (d8-12) at 25 pmol (i.e. 0.01 micrograms)/g body wt/d as established by a dose-response study in which this dose of DEX induced one third the maximum response in sucrase activity. In this way, synergy with T4 (130 pmol/g body wt/d, i.e. 0.1 micrograms/g body wt/d, d 5-12) could still be observed. Glucoamylase, lactase, acid beta-galactosidase, alkaline phosphatase, and sucrase activities were determined in two regions of the small intestine. Overall, the results for the two hormones administered alone showed intestinal maturation to be not significantly affected in the T4 group and partially stimulated in the DEX group. When combined, DEX + T4 synergistically increased jejunal sucrase, ileal glucoamylase, and duodenal alkaline phosphatase, and lowered ileal acid beta-galactosidase. The striking exceptions to the general pattern were two brush border enzymes that normally decline during intestinal maturation, namely ileal alkaline phosphatase and jejunal and ileal lactase. For these enzymes, DEX alone did not elicit precocious maturation, and there was no evidence for a synergistic interaction of these two hormones. Serum corticosterone concentrations also were measured. When corticosterone concentrations were compared with enzyme activity, no correlation was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Milk can be a significant source of lead (Pb) for young mammals, including humans. Certain essential trace elements have previously been shown to be specifically associated with particular milk components and such associations often increase bioavailability. Thus, the first goal of this study was to determine the distribution of Pb in cream, casein, and whey fractions of various milks under various conditions using 203Pb as a tracer. In rat milk almost 90% of the Pb was found to be associated with the casein micelles, regardless of: 1) whether the milk was labeled in vivo or in vitro; b) whether the milk was fresh or frozen; and c) the added concentration of Pb (over the range 0.01-75 micrograms/ml). The remainder of the Pb was approximately equally distributed between cream and whey. A virtually identical pattern of Pb distribution was observed with bovine milk. Pb added to infant formula also associated predominantly with casein micelles, although the Pb content of this fraction was significantly less than with rat and bovine milks. The second goal of the study was to determine if Pb remained associated with casein as it traversed the gastrointestinal tract of infant rats. For this purpose, rat pups aged 15-16 days were gavaged with 203Pb-labeled rat milk, and lumenal contents from the stomach and small intestine were collected 2 h later. Differential centrifugation of the homogenized lumenal contents showed that in the stomach the Pb was associated primarily with the casein curd. By the time chyme reached the distal small intestine, Pb was found predominantly in a fraction that was not precipitable by high-speed centrifugation (thus, not intact casein micelles), but was nondialyzable. We conclude that Pb in milk is protein bound and remains this way as it traverses the stomach and proximal small intestine of the infant rat.

This study was designed to determine the critical difference between rat milk and rat chow with respect ot their effects on jejunal sucrase activity during the fourth postnatal wk. Rats were weaned onto special diets on postnatal day 17, and jejunal sucrase was assayed on day 28. A pelleted diet containing lactose as sole carbohydrate did not cause depression of sucrase activity. Sucrase values (micromoles/hr/mg protein) were 10.49 +/- 0.81 (n = 15) for the lactose chow and 6.65 +/- 0.29 (n = 16) for the sucrose chow. This indicates that the nature of the dietary carbohydrate does not account for the sucrase differences of weaned and nonweaned animals. Likewise, the physical consistency of the diet is unimportant because sucrase values were just as high on liquid diet (10.91 +/- 0.77 micromoles/hr/mg protein; n = 8) as on he solid diets. However, when the relative proportions of carbohydrate and fat in the diet were varied, there were significant effects on sucrase activity; values on a low carbohydrate diet (4.30 +/- 0.33 micromoles/hr/mg protein; n = 8) being less than one-half those on a high carbohydrate diet (10.91 +/- 0.77 micromoles/hr/mg protein; n = 8).

Infant rats exhibit a marked rise in serum concentrations of corticosterone between postnatal days 12 and 24. As this rise appears to be cued by L-thyroxine, the aim of the current study was to determine the physiological bases of the effects of both age and thyroid status on serum corticosterone. The in vivo response to either adrenocorticotropic hormone (60 mU/g body weight) or dibutyryl 3',5'-cyclic adenylate (0.3 mg/g body weight) increased between 10 and 16 days and was advanced and delayed by hyper- and hypothyroidism, respectively. In contrast, in vitro studies with adrenals from rats aged 10 and 16 days showed no effect of age on either basal or adrenocorticotropic hormone-stimulated production of corticosterone. Chronic administration of exogenous corticosterone to hyperthyroid animals resulted in significantly higher concentrations of serum corticosterone than did an identical administration to hypothyroid animals. As hyperthyroidism was associated with marked elevations in the concentration of corticosteroid-binding globulin and in the extent of binding of corticosterone, these results suggest that the effects of both age and thyroid status on serum concentrations of corticosterone may reflect changes in the metabolic clearance of the hormone.

Corticosterone plays an important role in the regulation of postnatal development in the rat. Basal concentrations of plasma corticosterone increase markedly during the 3rd wk of life. To date, however, the physiologic bases of this increase have remained unclear. To understand the determinants of circulating concentrations of corticosterone during this period, the plasma half-life of disappearance at steady state (t1/2), the apparent volume of distribution, and metabolic clearance rate were determined after injection of a tracer dose of 3H-corticosterone in rats at 12, 16, and 22 days of age. The t1/2 for total plasma corticosterone decreased with increasing age. The volume of distribution decreased even more steeply and, consequently, the MCR displayed a highly significant decline between 12 and 22 days of age. As plasma concentrations of corticosteroid-binding globulin are known to increase markedly during this period, the t1/2 of protein-bound corticosterone was measured and that of free corticosterone was computed. At all ages the t1/2 of bound corticosterone was less than that of free corticosterone. Protein binding of the injected 3H-corticosterone increased significantly with development. Thus, increased binding of corticosterone is associated with decreased t1/2. The increasing association of corticosterone with corticosteroid-binding globulin during this developmental period is the most likely explanation for the steep decline of volume of distribution and thus of the metabolic clearance rate for corticosterone. The latter provides, for the first time, an understanding of the basis of the developmental increase in plasma concentrations of corticosterone.

Studies of intestinal enzyme development and regulation relevant to the human infant require an animal model with a rate of maturation similar to that of the human infant. Hanford miniature pigs were weaned at 3 days of age to a standard swine weaning formula. At 1, 2, 3, 4, 5, and 6 wk of age, duodenal jejunal, and ileal segments were analyzed for protein content and lactase, sucrase, maltase, glucoamylase, and acid beta-galactosidase activities. Protein content of the small intestine changed significantly with age only in the ileum (p less than 0.05). Lactase activity fell significantly with age in all segments of the small intestine (p less than 0.001); activity was highest in the jejunum. Sucrase and maltase activities were present in all segments of the small intestine at 1 wk of age. Sucrase increased significantly (2-fold, p less than 0.02) with age only in the ileum and maltase increased significantly with age in the jejunum (by 50%, p less than 0.05) and the ileum (3-fold, p less than 0.001). Activities were highest in the jejunum. Glucoamylase activity was present at 1 wk of age and showed a small but significant increase with age only in the duodenum (p less than 0.005). Acid beta-galactosidase activity demonstrated small but significant decreases with age in all small intestinal segments. Glucoamylase and acid beta-galactosidase activities were similar in all segments. In the 6-wk-old pigs, activities of all the enzymes tested were similar to those found in young human infants.(ABSTRACT TRUNCATED AT 250 WORDS)