INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

ILEAL BILE ACID BINDING protein (IBABP, also previously referred to as ileal lipid binding protein) is a 14-kDa cytosolic protein believed to be involved in the intestinal absorption of bile acids because of its specific distribution in the ileum and its ability to bind conjugated bile acids in vitro (7, 13, 31, 39). In rodent ileum the abundance of IBABP increases markedly during the third postnatal week (15, 43). This pattern of protein expression appears to reflect pretranslational regulation because there are parallel ontogenic changes in the levels of IBABP mRNA (8, 14, 39). The developmental surge of IBABP expression coincides with that of the apical sodium-bile acid cotransporter (41, 43), which mediates the initial step in the active transport of bile acids. The functional significance of the parallel changes of the transporter and the binding protein is that the onset of active absorption of bile acids in the rat ileum occurs during this time period (3, 5, 35, 43). Although there are several studies on the ontogenic regulation of the overall process of bile acid transport (2, 24, 25, 42), the regulation of its components such as IBABP has received little attention (14, 16). Such regulation is in turn an important aspect of the maturation of the enterohepatic circulation of bile acids (26).

The second and third postnatal weeks of rat development are notable for hormonal changes that suggest possible regulators of IBABP mRNA expression. A dramatic increase in circulating glucocorticoid (principally corticosterone) begins on day 12, peaks by day 20, and then modestly declines (19). This increase in serum corticosterone is preceded by a rise in serum thyroxine that starts at approximately day 5 and plateaus on about day 12 (19). Because these surges of both corticosterone and thyroxine begin before the detection of a significant increase in IBABP mRNA levels, they are logical candidates in regulating IBABP mRNA expression. To date it is apparent that pharmacological doses of glucocorticoid can elicit precocious induction of IBABP mRNA (14, 16). However, there have been no studies with lower glucocorticoid doses in the physiological range. Moreover, the potential role of thyroxine in the developmental regulation of IBABP expression has not been investigated. Thus the goals of the current study were to examine the influence of glucocorticoids and thyroxine on IBABP mRNA expression by administering physiological doses of these hormones to rats during the second postnatal week.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals. Rats of the Sprague-Dawley strain [Crl:DC(SD)BR] were obtained from Charles River Laboratories (Portage, MI). Adult male rats were used to generate pooled reference standards for mRNA and protein. The experiments utilized timed-pregnant dams. Animals were maintained in our facility using a 12:12-h light-dark cycle with lights on at 0600 daily. Adults received deionized water and Rodent Chow 5001 (Purina) ad libitum. The day of birth was designated as day 0. Litters were culled to nine pups on day 1. Hormones were administered by subcutaneous injections using doses and timing as indicated in individual experiments. All animal protocols were reviewed and approved by our Institutional Animal Care and Use Committee.

Normal ontogeny. To study the normal ontogeny of IBABP mRNA expression, three litters of untreated animals were used. Within each litter, pups were randomly assigned for use at postnatal ages 4, 7, 10, 13, 16, 19, 22, 25, and 28 days. Dams were removed from the remaining pups at 21 days. At each age indicated, the entire ileum (defined as the distal one-half of the small intestine between the ligament of Treitz and the ileocecal valve) was collected, flushed with ice-cold normal saline, and rapidly placed in liquid nitrogen. Preliminary studies in adult rats revealed that although there was more IBABP mRNA in the distal ileum, there was an appreciable amount in the proximal portion as well (data not shown). Therefore to evaluate the regulation of IBABP mRNA, the entire ileum was analyzed unless otherwise specified.

Dexamethasone dose response. Four litters of animals were utilized. Pups received various doses of dexamethasone (Dex) or 0.9% NaCl (vehicle) daily on days 10-13. By pair-weighing, each litter had 1-2 pups assigned to each treatment group. Dex (Sigma; St. Louis, MO) was chosen as the glucocorticoid because it does not bind to corticosteroid-binding globulin and thus is not subject to extraneous influences on its circulating concentration (21, 40). A stock solution was made in 100% ethanol at a concentration of 4 mg/ml and then diluted with sterile normal saline so that the ethanol content was <5%. Whole ileum was collected on day 14 as described in Normal ontogeny.

To calculate a "physiological" range for the Dex doses, three sources of information were used. First, for the natural glucocorticoid of the rat (corticosterone), it has been shown that a daily dose of 10 µg/g body wt can recapitulate normal developmental changes in disaccharidase activity in adrenalectomized suckling rats (32). Second, the notion that this represents a physiological dose of corticosterone is confirmed by the fact that when corticosterone pellets are implanted into adrenalectomized suckling rats, the size of pellet that yields normal circulating concentrations of corticosterone can be calculated to be releasing ~10 µg corticosterone · g body wt¹ · day¹ (34, 40). Finally, the relative potency of Dex is known to be 70 times that of corticosterone (18). Thus if 10 µg/g body wt represent a physiological dose of corticosterone, then the equivalent dose of Dex would be 0.1 µg · g body wt¹ · day¹. To encompass this, the Dex doses used were 0.005, 0.02, 0.1, and 0.4 µg · g body wt¹ · day¹.

Dex response at later age. Animals from two litters received either no treatment or Dex at a saturating dose of 0.1 µg/g body wt (determined from the Dex dose response study) on days 12-15. Whole ileum was collected on day 16. After flushing with ice-cold normal saline, the entire ileum was sectioned into 1-cm pieces before placing into liquid nitrogen. Alternating sections were designated for either Northern or Western blot analysis.

Time course of Dex response. Animals from four litters were injected daily with Dex at a dose of 0.1 µg/g body wt starting on day 12. Within each litter, pups were randomly assigned to be killed at time 0 or 12, 24, 48, and 72 h after start of Dex treatment. Whole ileum was collected as described in Normal ontogeny.

Normal and Dex-induced ileal distribution. Within each of five litters, animals were randomly assigned to one of the following groups: day 14 control, day 14 Dex, day 16 control, day 16 Dex, day 19 control, and day 28 control. Control animals were untreated. Dex animals received a dose of 0.1 µg/g body wt on days 12-15. After being flushed with ice-cold normal saline, the entire ileum was divided into proximal and distal one-half portions before placement into liquid nitrogen.

Thyroxine study. Rat pups from two litters were placed into four treatment groups: vehicle, Dex, thyroxine, and simultaneous Dex plus thyroxine. Pair weighing was done so that each litter had two pups assigned to each treatment group. Pups received daily injections on days 8-13. A stock of L-thyroxine (Sigma) was made in 5 mM NaOH. A volume of this solution was diluted with sterile saline to a concentration of 0.3 mM NaOH. L-Thyroxine was administered at a dose of 0.1 µg/g body wt, which yields circulating concentrations approximately threefold higher than in normal rats of this age range (10). Dex was given at 0.01 µg/g body wt, a dose that is submaximal from the prior study and thus would allow detection of synergism with thyroxine. Whole ileum was collected on day 14 as described in Normal ontogeny.

Northern blot analysis. RNA was isolated from the collected tissues by the guanidine isothiocyanate-cesium chloride method (36). Northern blotting was done from 0.8 or 1.0% formaldehyde agarose gels loaded with 20 µg of total RNA per lane. To assess for equal loading of RNA samples, ethidium bromide staining of the ribosomal bands was compared. The fractionated RNA was transferred to a Magna NT nylon membrane (MSI; Westboro, MA). Membranes were hybridized with the linearized plasmid of mouse IBABP cDNA donated by Dr. Sherrie Hauft (8). This ³²P-labeled probe was generated by random-primed oligolabeling (12). Membranes were hybridized and washed as described previously (4) or by a modification of the Church and Gilbert (6) protocol. Blots were exposed to Kodak XAR-5 film at 70°C with one screen. Exposure times were typically in the range of 5-20 h. Some of the membranes were stripped and reprobed with the linearized plasmid bearing the cDNA for rat sucrase-isomaltase (4). If the ethidium bromide staining appeared unequal in the gels, then Northern blots were reprobed with the cDNA for the constitutive marker -actin (1).

Western blot analysis. Tissue was homogenized in the following protease inhibitor cocktail: 50 mM mannitol, 2 mM Tris (pH 7.1), 25 µg/ml leupeptin, 5 µg/ml aprotonin, 40 µg/ml phenylmethylsulfonyl fluoride, 50 µg/ml benzamidine, and 0.5 µg/ml pepstatin. Protein concentrations of the homogenates were assessed using the Bio-Rad Protein Assay with bovine gamma globulin as the reference. In a preliminary experiment, varying amounts of the pooled standard from adult ileums were analyzed to determine the linear range of IBABP detection (data not shown). Total ileal homogenates were separated in 15% SDS-polyacrylamide gels. All gels were run with a pooled adult ileal standard and low-molecular weight protein markers (Amersham Life Science; Arlington Heights, IL). Based on the results of the standard curve, 5 µg of protein were loaded for each sample. Gels were transferred to nitrocellulose membranes (Bio-Rad Transblot 0.2-µm membrane; Hercules, CA) by electroblotting. Membranes were blocked overnight in the following solution: 20 mM Tris · HCl (pH 7.5), 2% bovine serum albumin, 150 mM NaCl, 0.1% Tween 20, and 10% Carnation nonfat-dry milk. Membranes were incubated with rabbit polyclonal anti-IBABP sera donated by Dr. Sherrie Hauft (8) at a 1:2,000 dilution for 2 h and then washed before a secondary incubation with goat anti-rabbit antibody conjugated to horseradish peroxidase (Sigma) at a 1:2,000 dilution for 1 h. Protein signal was detected by chemiluminescence using the ECL kit (Amersham Life Science).

Quantitation. Adult pooled mRNA and protein samples were included on each Northern and Western blot, respectively, as standards. IBABP mRNA hybridization signals were quantitated by phosphorimaging. Protein levels were quantitated by densitometry performed on the exposed film. For both mRNA and protein, values for each sample were expressed as a percentage of the adult standard from the same blot. If loading correction was necessary on any Northern blot, then the IBABP signal was expressed as a ratio with the -actin signal from the same lane before being expressed as a percentage of the standard (also expressed as a ratio). Data are expressed as means ± SE. Statistical analysis was performed by one- or two-way ANOVA, depending on the experiment. If significance was detected (P < 0.05), then further post hoc comparisons were performed by Fishers least-significant difference (LSD) test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Normal ontogeny. The developmental pattern of rat IBABP mRNA expression is one in which there is little or no mRNA during the suckling period, with a dramatic increase occurring in the third postnatal week. Because prior studies had shown only a single value for IBABP mRNA at each age (8, 14, 39), to set the stage for our hormonal studies we thought that it was important to assess the animal-to-animal variation and to include statistical analysis of the developmental rise. Figure 1 shows that after a 5-h exposure of the autoradiogram, IBABP mRNA was first detectable on postnatal day 19. A 33-fold longer exposure detected the mRNA on day 16 (data not shown) but not at earlier ages. Quantitation of the data (Fig. 1B) revealed an abrupt rise in IBABP mRNA during the third postnatal week, with levels peaking on day 25 followed by a decline to adult levels. Results of one-way ANOVA verified that there were significant differences in IBABP mRNA levels related to age (P < 0.001). The post hoc Fishers LSD test showed that by day 22 IBABP mRNA levels were not significantly different from those at day 37, i.e., the adult plateau had been reached.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Normal ontogeny of rat ileal bile acid binding protein (IBABP) mRNA. A: representative Northern blot shows IBABP mRNA from various postnatal ages (days). B: quantitative data generated from all samples in study. IBABP mRNA is expressed as percentage of pooled adult standard. Values are means ± SE for 3 animals at each age. Absence of error bars at days 16 and 37 reflects SE smaller than symbol.

Dex dose response. To assess the role of glucocorticoid hormones in the developmental increase of IBABP mRNA, suckling rats received Dex at various doses calculated to be within the physiological range (see METHODS for details of the calculation). Figure 2A shows that the daily administration of Dex at doses of 0.005-0.4 µg/g body wt on days 10-13 precociously induced IBABP mRNA in day 14 rats. Saline-injected animals did not exhibit any IBABP mRNA, whereas IBABP mRNA was detected in Dex-treated animals even at the lowest dose. One-way ANOVA supports a proportional relationship between Dex dose and IBABP mRNA levels (P < 0.001). As seen in Fig. 2B, plateau levels of IBABP mRNA were reached with the dose of 0.1 µg/g body wt. When the signal obtained at the plateau was compared with the limit of detection, the maximal induction of IBABP mRNA by Dex was at least 200-fold compared with the saline-injected group.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Dexamethasone (Dex) dose response in day 14 rats. A: representative Northern blot shows IBABP mRNA from duplicate animals receiving different doses of Dex on days 10-13. Numbers on top represent Dex dose given in µg/g body wt. V, vehicle injected; S, pooled adult standard. B: quantitative data generated from all samples in study. IBABP mRNA is expressed as percentage of pooled adult standard. Values are means ± SE for 5-8 animals per dose. Absence of error bars reflects SE smaller than symbol.

Although a 200-fold induction is clearly a robust effect, the plateau levels of IBABP mRNA were only 15-20% of adult values (Fig. 2B). In this regard, the response of IBABP mRNA to Dex is unlike that previously reported for sucrase-isomaltase mRNA in suckling rat jejunum, where induced expression reached 100% of adult levels (36). To ensure that the discrepancy between IBABP and sucrase-isomaltase induction was due to genuine differences in glucocorticoid responsiveness and not to lack of activity of administered Dex in the current experiment, the Northern blots used in Fig. 2 were reprobed with the cDNA for rat sucrase-isomaltase. This showed a dramatic induction of sucrase-isomaltase mRNA to 300% of adult levels at the highest Dex dose (data not shown). These results verify that the Dex used in the experiment was fully active and that, although it precociously induced IBABP mRNA, the levels achieved did not fully recapitulate the normal developmental increase.

Dex response at later age. To address the possibility of increased glucocorticoid responsiveness in older animals, we examined IBABP mRNA levels induced by a saturating dose of Dex given on days 12-15 (rather than days 10-13 as in Fig. 2). The results (Figs. 3, A and B) show that Dex treatment on days 12-15 was still able to precociously increase IBABP mRNA levels compared with day 16 controls (P < 0.01). Dex treatment at this older age resulted in IBABP mRNA reaching 20.4 ± 4.3% of adult levels, whereas Dex given at the earlier age yielded 16.4 ± 2.8% of adult levels (see Fig. 2). The difference between the two ages was not statistically significant (P = 0.49). Thus these data indicate that there is not an increase in the glucocorticoid responsiveness of IBABP mRNA with increasing age.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effect of Dex on IBABP mRNA and protein in day 16 rats. A: Northern blot from control and Dex-treated triplicate animals. The latter received Dex at dose of 0.1 µg/g body wt on days 12-15. B: quantitative data from Northern blot shown in A. IBABP mRNA is expressed as percentage of pooled adult standard. Values are means ± SE (n = 3). C: Western blot of total ileal protein from the same animals. D: quantitative data from Western blot analysis. IBABP is expressed as percentage of pooled adult standard. Values are means ± SE (n = 3).

Prior reports have indicated that IBABP increases in parallel with IBABP mRNA during normal development (15, 43). Because the studies above have shown that even at saturating doses glucocorticoid induction of IBABP mRNA reached only 20% of adult levels, it was of interest to determine the magnitude of the increase in protein levels. A strong 14-kDa protein signal corresponding to IBABP was detected in each of the Dex-treated animals on day 16 (Fig. 3C). Quantitation of the Western blot (Fig. 3D) showed that the effect of Dex was highly significant (P = 0.002) and that the resulting IBABP reached ~25% of adult levels, which is similar to the amount of mRNA induced in the same animals.

Time course of Dex response. In the suckling rat, glucocorticoid induction of mRNAs for brush-border hydrolases is characterized by a lag of ~24 h (36, 37) and thus has been presumed to reflect an indirect action of these hormones. However, this may not be the case for IBABP mRNA because the IBABP gene has been noted to include at least one glucocorticoid response element (8). This then raises the possibility of a direct and rapid induction of IBABP mRNA. To determine how quickly induced levels of IBABP mRNA are achieved, various time points after the onset of Dex treatment at a saturating dose were examined. Figure 4A shows a detectable mRNA signal 48 h after the start of Dex treatment. An ~25-fold longer exposure revealed a faint signal at 12 and 24 h (data not shown). Quantitation of the data (Fig. 4B) confirmed the slow response of IBABP mRNA to Dex treatment, with appreciable levels being first seen after 24 h. Reprobing of the blots showed that sucrase-isomaltase mRNA displayed an identical time course but with higher absolute values (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Time course of Dex response of IBABP mRNA in suckling rats. A: representative Northern blot shows IBABP mRNA at various time points after receiving Dex at a dose of 0.1 µg/g body wt starting on day 12. Numbers represent hours after the onset of Dex treatment. B: quantitative data generated from all samples in study. IBABP mRNA is expressed as percentage of pooled adult standard. Values are means ± SE for 4-7 animals per time point.

Although the main purpose of this study was to distinguish between a rapid response and a delayed one, the 72-h time point also allows inference regarding the ultimate levels of IBABP mRNA induced by Dex. Specifically, Fig. 4 shows that the mean value observed after 3 days of treatment (28.1 ± 3.4% adult) was not significantly different from that previously seen in Fig. 3 after 4 days of Dex treatment (20.4 ± 4.3% adult) at the same dose and same ages. Thus we conclude that despite the slow onset, the effects of Dex on IBABP mRNA plateau after 3 days of treatment.

Normal and Dex-induced ileal distribution. In mouse intestine, the developmental surge of IBABP expression is characterized by distinct regional patterns of IBABP localization along the length of the ileum (8). These patterns of expression have not been studied at the mRNA level. To assess whether there is a change in the proportion of IBABP mRNA expressed in the proximal and distal ileum during normal development and whether glucocorticoids recapitulate this pattern, IBABP mRNA levels were determined separately in the proximal and distal ileums of normal control and Dex-treated animals. As expected, there was no mRNA signal detected in the tissues from day 14 controls, whereas a faint signal was seen only in the distal ileums of day 16 controls (data not shown). Quantitation of the mRNA levels in the older control animals (Fig. 5A) confirmed that the developmental rise of IBABP expression in the distal ileum precedes that in the proximal ileum, with levels at day 19 being 60-fold greater in distal ileum compared with proximal ileum. At later ages there was increased expression in the proximal ileum attaining ~50% of that in distal ileum by day 28.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Ileal distribution of IBABP mRNA in rats during normal development and after precocious induction with Dex. A: IBABP mRNA levels in proximal ileum (Pi) and distal ileum (Di) in untreated animals at days 19 and 28. B: IBABP mRNA levels in proximal and distal ileums of rats aged 14 and 16 days, which received Dex at a dose of 0.1 µg/g body wt starting on day 12. In both panels IBABP mRNA is expressed as percentage of pooled adult standard and values are means ± SE for 7 or 8 animals at each age.

The data in Fig. 5B show that, just as in normal development, the precocious appearance of IBABP mRNA after Dex administration was more prominent distally. Thus, at day 14 (2 days after the start of Dex treatment), IBABP mRNA was detected only in the distal ileum. By day 16, IBABP mRNA began to be seen in the proximal ileum as well. This indicates that precocious glucocorticoids induce IBABP mRNA expression in a manner that duplicates the retrograde pattern seen in normal development.

Thyroxine study. Because circulating concentrations of thyroxine also increase during the postnatal period, the next experiment examined the possibility that thyroxine, acting alone or in concert with glucocorticoids, is capable of eliciting expression of IBABP mRNA. As shown in Fig. 6, administration of thyroxine for 6 days did not cause a significant increase in IBABP mRNA as assessed by balanced two-way ANOVA (P = 0.68). In contrast, the Dex response was highly significant (P < 0.001) and was comparable to that seen earlier (Fig. 2). There was no significant interaction of the variables, indicating that there was no synergism between glucocorticoids and thyroxine (P = 0.97). In view of the lack of effect of thyroxine on IBABP mRNA in this experiment, sucrase activity was measured in jejunum from these same animals to confirm that the thyroxine was active. In agreement with previous studies (28, 33) the enzyme activities obtained in the Dex group (5.36 ± 0.6 µmol · mg protein¹ · h¹) and the Dex plus thyroxine group (10.5 ± 0.6 µmol · mg protein¹ · h¹) showed evidence of synergy between the two hormones, indicating that the thyroxine administered was bioactive in these animals. Taken together, these results suggest that thyroxine does not have a role in IBABP mRNA expression in the suckling rat.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effect of thyroxine, Dex, and simultaneous Dex plus thyroxine on IBABP mRNA in day 14 rats. All groups received 6 days of treatment beginning on postnatal day 8. Graph shows quantitative data generated from all samples in study. V, saline; T, thyroxine; D, Dex; D/T, Dex plus thyroxine. IBABP mRNA is expressed as percentage of pooled adult standard. Values are means ± SE for 4 animals per treatment group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

IBABP mRNA is one of the most abundant transcripts of the ileal mucosa. Although expressed only in the villus epithelial cells (8, 14, 27), the mRNA shows a strong signal on Northern blots of total RNA prepared from the intact ileum (i.e., full thickness of muscle plus mucosa). Typical autoradiographs of such blots show that in adult rats the IBABP mRNA signal is detectable after less than 1 h of exposure. Thus IBABP mRNA can serve as a sensitive marker to study ileal maturation. In addition, because there is evidence to support IBABP being involved in the transport of bile acids across ileal enterocytes (13, 30, 31), understanding IBABP regulation may provide insight into that of other components of the ileal absorption of bile acids.

In the current study we first documented the postnatal development of IBABP mRNA in rat ileum. Although there is some variation in the time to reach peak mRNA levels, all prior studies of the ontogeny of IBABP mRNA expression in rodents show marked increases in mRNA levels during the third postnatal week (8, 14, 39), the time at which the protein becomes detectable (15, 43). Our data show that this aspect of intestinal maturation is precisely timed and is remarkably abrupt. The IBABP mRNA was first detectable at day 16 and by 6 days later had already reached adult levels. Admittedly a more sensitive measure might identify low levels of the transcript at earlier ages. Nevertheless, based on the limits of detection of our Northern blots, we estimate that the increase in the concentration of IBABP mRNA between day 16 and day 22 is at least 800-fold.

Glucocorticoids have been shown to have a role in many aspects of intestinal maturation (19, 22). A recurrent difficulty in interpreting previous data has been the variability in both the form and the dose of glucocorticoid administered. Hydrocortisone, corticosterone, Dex, and methylprednisolone are all examples of glucocorticoids of differing potencies that have been utilized in animal studies. Regardless of the form, it is important to distinguish between responses to pharmacological vs. physiological doses of glucocorticoids because the effects may be quite distinct (18). The question then lies in defining the physiological dose of glucocorticoids. For purposes of investigating the changes that occur during the suckling-weaning transition, an appropriate dose of exogenous glucocorticoids would be one that mimics the surge in serum corticosterone levels that occurs at the end of the second postnatal week. With different approaches (as described in METHODS) in previous studies (32, 34, 40), the conclusion was reached that such a dose would be ~10 µg corticosterone · g body wt¹ · day¹. Based on the relative potencies of the various forms of glucocorticoids (18), this dose of corticosterone is equivalent to ~0.1 µg Dex/g body wt. Prior studies using various glucocorticoids at doses up to 40-fold higher than this dose have reported the precocious induction of IBABP mRNA (14, 16). Our data show that glucocorticoids within the physiological dose range can elicit parallel increases in IBABP mRNA and protein and therefore support the possibility of a role for endogenous glucocorticoids in the normal developmental rise of IBABP expression during the third postnatal week.

The exact molecular mechanisms of the effect of glucocorticoids on intestinal maturation have not been delineated. In general, there have been three models proposed for the action of glucocorticoids on responsive genes: 1) a primary response in which the direct binding of the activated glucocorticoid receptor complex to the responsive target gene leads to a rapid increase in the respective mRNA, 2) a secondary response in which glucocorticoids induce a regulatory protein, which then binds to the transcription machinery of the target gene resulting in increased mRNA up to days later, and 3) a delayed primary response model in which there is direct binding of the receptor complex to the target gene but a delay dependent on the accumulation of a protein which is required in addition to the receptor complex to affect transcription (11). Our data indicate that glucocorticoids induce IBABP by either a secondary or delayed primary response mechanism because appreciable levels of IBABP mRNA were not observed until 24 h after the start of Dex administration. As a glucocorticoid response element has been identified in the mouse IBABP gene (8), a delayed primary response would appear to be the most likely mechanism. Further studies are needed to verify the functionality of the glucocorticoid response element and to identify the accessory proteins involved.

The slow time course observed for the precocious induction of IBABP mRNA by Dex is similar to that of other mRNAs (36, 37) elicited in the suckling intestine in response to glucocorticoid administration. For other intestinal genes that respond to glucocorticoids, hormone action has been shown to be mediated via the proliferating cells located in the intestinal crypts. For example, sucrase-isomaltase (which is the most studied intestinal gene) expression is first detected in the new population of cells at the villus base ~24 h after administration of glucocorticoid to suckling rats (17, 20, 23, 44, 45). With time, these cells migrate and eventually populate the entire villus (20, 23, 44). A similar phenomenon has been observed for IBABP in which the protein is also first detected in the upper crypt and then later appears in the enterocytes lining the villus (14). Taken together with our observation that 24 h are required to see an effect of glucocorticoid on levels of IBABP mRNA, this implies that the activated glucocorticoid receptor may engage the IBABP glucocorticoid response element in the proliferating zone of the crypt and then await onset of expression of an accessory protein at the time the affected cells move to the upper crypt. Thus the elucidation of the molecular mechanisms of glucocorticoid induction of IBABP mRNA will require careful analysis along the crypt-villus axis.

A study of IBABP expression along the longitudinal axis of the mouse ileum revealed a unique changing pattern of expression during normal development (8). On postnatal day 13 only the terminal 25% of the ileum has >95% of the villi staining positive for IBABP. There is increasing IBABP detected proximally with time until day 28 when the distal 75% of ileum has uniform IBABP staining on all villi, and it retains this pattern throughout adulthood. We were able to show that this distal-to-proximal expression also occurs at the mRNA level in the rat during normal development. Initially IBABP mRNA was detected only in the distal ileum (day 16). A proximal wave of IBABP mRNA expression started around day 19 with mRNA levels in the proximal ileum eventually (day 28) attaining ~50% of levels in the distal one-half. When IBABP mRNA was precociously induced by Dex, initially it was detected only in the distal ileum (after 2 days of treatment). Two days later, when levels in the distal ileum had increased markedly, the IBABP mRNA became detectable in the proximal ileum. These results with Dex are parallel to what is seen in early normal development and suggests that glucocorticoids may induce the normal regional pattern of IBABP mRNA expression in the ileum. Thus the glucocorticoid response seems to be influenced by preexisting positional control of IBABP mRNA expression.

Thyroxine was a logical candidate as an additional regulatory factor promoting the maturation of IBABP expression because of its increasing serum concentrations during the second postnatal week. Moreover, in studies of development of intestinal hydrolases, thyroxine has been shown previously to enhance both jejunal and ileal responses to glucocorticoid (29, 33, 46). However, our results show that there was no precocious induction of IBABP mRNA after administration of physiological doses of thyroxine and that there was no evidence of synergism between thyroxine and glucocorticoids. We conclude that in contrast to several other markers of intestinal maturation (29, 33), thyroxine does not seem to have a role in the developmental increase of IBABP mRNA expression. This lack of response to thyroxine is clearly a gene-specific issue rather than a regional one because Plateroti et al. (38) have documented marked influences of thyroid hormones on other aspects of the development of the rodent ileum.

Our results regarding the effects of glucocorticoids and thyroxine on IBABP are consistent with published data on the regulation of the overall process of bile acid uptake by the ileal mucosa. Using a villus incubation technique, Heubi and Gunn (25) showed that at postnatal day 14, transport of taurocholate was undetectable in control rat pups but was significant in corticosterone-treated pups. Interestingly, glucocorticoid-induced transport was only ~20% of adult levels and was nonsaturable. Both of these features could be explained by the IBABP induction we have observed with Dex. A subsequent study from the same laboratory indicated that pharmacological doses of thyroxine stimulated both saturable and nonsaturable transport of taurocholate in suckling rats (24). Inasmuch as these doses are known to elevate endogenous corticosterone (9), the most plausible interpretation in light of our IBABP data would be that the elevation of nonsaturable transport was due to glucocorticoid induction of IBABP, whereas the small amount of saturable transport may reflect an independent effect of thyroxine on the apical sodium-bile acid cotransporter.

Overall, our studies indicate that glucocorticoids can have a role in the regulation of the developmental expression of IBABP. The ability of Dex at doses in the physiological range to precociously induce IBABP mRNA and protein levels suggests that the endogenous surge in serum corticosterone levels during the third postnatal week may participate in the onset of rat IBABP expression. Interestingly, glucocorticoids induce IBABP to only about 20% of adult expression, which is not as dramatic compared with other studied intestinal genes (such as sucrase-isomaltase) in which 100% of adult expression can be attained (36). This indicates differences in glucocorticoid responsiveness between intestinal genes. Moreover, unlike other aspects of intestinal maturation, IBABP mRNA expression at the suckling-weaning transition does not seem to be influenced by thyroxine. Other factors must be involved in the developmental increase of IBABP expression because we have not shown full recapitulation of normal ontogeny with glucocorticoids alone. Further investigations of possible contributions from diet and bile acids may add more insight to the events involved in the dramatic increase of IBABP during the third postnatal week of rodent development.

Perspectives