Sucrase-isomaltase ontogeny: synergism between glucocorticoids and thyroxine reflects increased mRNA and no change in cell migration.

During postnatal maturation of the rat small intestine, glucocorticoid hormones (GC) and thyroxine (T4) act synergistically to elicit a precocious increase of sucrase activity. The current work shows that the synergistic effect on sucrase activity is paralleled by increased steady-state levels of sucrase-isomaltase mRNA. The enhancing effects of T4 on dexamethasone (DEX)-induced sucrase activity was seen even after prolonged treatment (9 days). Moreover, when the location of sucrase-bearing cells was examined after 2 days of hormone treatment, there was distinctly stronger immunostaining of sucrase in the presence of T4, and the sucrase-bearing cells were located on the lower quarter of the intestinal villi regardless of whether the animals received DEX or T4 plus DEX. Thus, despite predictions from the literature, there was no evidence for increased migration in the presence of T4. Instead, we conclude that the synergism between the two hormones is due to greater accumulation of sucrase-isomaltase per epithelial cell.

Intestinal maturation in mice lacking CCAAT/enhancer-binding protein alpha (C/EPBalpha).

In rodents, there is a surge of intestinal expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) in the late fetal phase just before morphological maturation and the onset of expression of numerous epithelial genes. To investigate directly the hypothesis that C/EBPalpha plays a causal role in the latter phenomena, we have assessed both structural and functional maturation in neonatal intestine from C/EBPalpha-null mice and their littermates. No effects of C/EBPalpha genotype were observed on mucosal architecture or on the size of the proliferative zone in the intestinal crypts. Likewise, the mRNA levels for the glucose transporter 2 (GLUT2), intestinal and liver fatty acid-binding proteins, and apolipoprotein A-IV in newborn intestine were similar in all genotypes. Paradoxically, Na+/glucose co-transporter (SGLT1), lactase phlorizin-hydrolase and apolipoprotein B mRNAs were more abundant in the C/EBPalpha-deficient animals. In wild-type intestines, C/EBPbeta and C/EBPdelta mRNAs were detectable throughout the late fetal period and increased toward term in parallel with C/EBPalpha mRNA. In newborn intestine, there was no compensatory up-regulation of these isoforms in the C/EBPalpha-deficient mice. We conclude that C/EBPalpha has no essential role in morphological maturation of the intestine, the pattern of proliferation of the epithelium, or the onset of expression of this cluster of epithelial mRNAs. However, since other C/EBP isoforms are present in the developing intestine, it is possible that there is a generic requirement for a member of the C/EBP family.

The sucrase-isomaltase structural gene (Si-s) and a regulatory gene (Si-r) are closely linked to esterase-26 (Es-26) on mouse chromosome 3.

A cDNA clone of the rat sucrase-isomaltase (SI) structural gene detected two distinct patterns of DNA fragments on Southern blots of mouse DNA. Screening of 18 AKXL and 25 AKXD recombinant inbred (RI) strains revealed that all bands in each pattern co-segregated and there were no (0/43) recombinants with Es-26 on mouse Chromosome (Chr) 3. Since CBA/CaJ mice have approximately threefold less sucrase activity than other strains, we intercrossed them with SJL/J mice to map the previously identified SI regulatory gene, Si-r. Fifty-six mice from the F2 generation were assayed for sucrase activity, and the genotype of the murine SI structural gene locus, Si-s, was determined by Southern blot analysis. Nine animals (16%) were homozygous for the CBA/CaJ allele (C) and had an average sucrase activity (jejunum+ileum) of 1.51 mumoles/h/mg protein (SE = 0.067), 19 (34%) were homozygous for the SJL/J allele (S) and had an average sucrase activity of 5.95 mumoles/h/mg protein (SE = 0.267), and 28 (50%) were heterozygous (C/S) for Si-s with an average sucrase activity of 3.70 mumoles/h/mg protein (SE = 0.127). We conclude that Si-s and Si-r are closely linked on Chr 3.

Hormonal regulation of the mRNA for cysteine-rich intestinal protein in rat jejunum during maturation.

Cysteine-rich intestinal protein (CRIP) has been implicated as an important zinc-binding protein in the rat intestine. However, its specific role remains undefined. As an approach to the ultimate elucidation of the function of CRIP, we have explored the role of glucocorticoids and L-thyroxine (T4) in the increase of CRIP mRNA that occurs during postnatal development. Hydrocortisone administration on day 10 elicited a precocious increase of CRIP mRNA. The response to hydrocortisone was readily detectable 12 h after injection. Lack of endogenous glucocorticoids in rat pups adrenalectomized on day 9 impeded but did not prevent the normal rise of CRIP mRNA. Furthermore, injections of dexamethasone (DEX) on days 10, 16, and 18 led to a loss of responsiveness of CRIP mRNA as the pups matured. The administration of T4 alone resulted in a small increase of CRIP mRNA, whereas when combined, T4 and DEX synergistically raised the concentration of CRIP mRNA. All of these patterns of response to hormone manipulation indicate the possibility that CRIP is a mediator of glucocorticoid action on the developing intestine. They do not appear to support the hypothesis that CRIP plays a role in zinc transport during the postnatal period. The potent effects of glucocorticoids and T4 on CRIP mRNA levels should provide useful tools for further investigations in this area.

Development and tissue distribution of sucrase-isomaltase mRNA in rats.

Previous studies of sucrase-isomaltase (SI) activities have shown this complex to be absent in the suckling rat and to appear during the weaning period. We describe here the cloning of a heterologous SI cDNA and its use for the quantitation of SI mRNA as a first step toward understanding the molecular basis of SI development. A survey of RNA from 12 tissues of mature rats by Northern blot analysis showed a 6-kb band of SI mRNA only in the small intestine. Within the latter, both sucrase activity and SI mRNA peaked in the jejunum. Assay of jejunal tissue from developing rats showed sucrase activity and SI mRNA to be first detectable at 18 days, to rise in parallel through 24 days, and then to diverge a little (enzyme activity being lower) by 36 days. When glucocorticoid was administered to 10-day-old rats, neither sucrase activity nor SI mRNA was detectable 12 h later. Both parameters were readily detected 24 h postinjection, although the mRNA had risen relatively more than the enzyme activity. The two parameters increased in concert through 5 days postinjection and then plateaued. We conclude that, with respect to distribution along the intestine and to normal and precocious development, activities of SI in the rat are determined primarily by the abundance of its mRNA.

Kinetics of circulating corticosterone in infant rats.

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.

Circulating corticosterone in the infant rat: the mechanism of age and thyroxine effects.

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.

Duodenal uptake of lead by suckling and weanling rats.

The aim of the current study was to determine the site of absorption of Pb by suckling rats. When 203PbCl2 (carrier-free) plus Pb-acetate (50 micrograms/g BW) was intragastrically administered to rat pups aged 10 and 14 days, mucosal uptake of Pb, measured 2 h after intubation, was manyfold greater in the duodenum than in other regions of the small intestine. By 24 days of age, this duodenal uptake was no longer apparent. In suckling pups the duodenal content of Pb became undetectable by 24 h postintubation; in contrast, ileal uptake was minimal at 2 h but increased progressively through 24 h. The ileal uptake component was also age-dependent, having disappeared by 24 days of age. These findings suggest that it is the duodenum where Pb absorption occurs, whereas ileal uptake represents intestinal retention. To assess the role of the milk diet in the elevated Pb absorption by the duodenum of suckling animals, the effect of fasting following intubation was determined. The duodenal uptake of Pb was substantially higher in fasted pups than in suckled littermates. Correspondingly, blood Pb levels were more than 4-fold higher in fasted pups than in suckled pups. We conclude that the enhanced absorption of Pb in the suckling rat is due to duodenal absorption and that this absorptive process is even further enhanced in the absence of milk.

Effect of cortisone on intestinal uptake of lead in the suckling rat.

A previous study showed that there is extensive uptake of lead by the duodenum and the ileum of the suckling rat. At both sites, uptake declines with advancing age. This paper reports the effects of administration of cortisone acetate on in vivo lead uptake in 14- to 15-day-old rats. The results indicate that the duodenal uptake component is unaffected by hormone treatment whereas the ileal uptake component is markedly suppressed. There was no significant difference in blood lead concentrations in cortisone-treated pups as compared with vehicle-treated controls. We conclude that lead absorption in the suckling rat occurs primarily in duodenum and is not under glucocorticoid control. The ileal uptake of lead probably represents intestinal retention rather than absorption: its responsiveness to cortisone would be consistent with uptake via pinocytosis.

Hormonal control of postnatal development of ileal neuraminidase and acid beta-galactosidase.

This study examines the effect of changes of thyroid and glucocorticoid status on the development of ileal neuraminidase and acid beta-galactosidase. Thyroxine (T4) administration on postnatal days 6-13 had no effect on the activity of either enzyme. In contrast, a single injection of cortisone acetate on day 6 caused a precocious reduction of the activities of both enzymes. Hypothyroidism delayed the usual developmental decline of neuraminidase activity and prevented the decline of acid beta-galactosidase activity. This was probably due to an effect of T4 on endogenous glucocorticoids because cortisone acetate was just as effective as T4 in restoring enzyme activities to control levels. Adrenalectomy delayed the decline of both enzyme activities whereas glucocorticoid replacement in these same animals depressed enzyme activities to, or below, control levels. It is likely that glucocorticoids act as the primary cue in the maturation of these enzymes, but with T4 interaction necessary for the normal pattern to be elicited.

Coordinate loss of glucocorticoid responsiveness by intestinal enzymes during postnatal development.

Jejunal sucrase is known to display glucocorticoid responsiveness from birth through day 17 but not beyond that age. The aim of the current study was to determine whether this abrupt loss of responsiveness was shared by maltase, lactase, and acid beta-galactosidase. Glucocorticoid concentrations were manipulated by both adrenalectomy (ADX) and by administration of cortisone acetate (CA). Surgery or treatment was performed on each day from 16--22 days of age. Maltase activity was reduced by ADX at day 18 and earlier and was increased by CA at days 16 and 17. There were no effects at later ages. Acid beta-galactosidase was increased by ADX only at day 18 and earlier and was decreased by CA only at day 16. Lactase activity was increased by ADX at all ages up to and including day 20 but was reduced by CA only at days 16 and 17. Thus, we conclude that loss of glucocorticoid responsiveness at a relatively early stage of development is a common feature of both brush-border and lysosomal enzymes of the small intestine.