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Synergistic Effects of Thyroxine and Dexamethasone on Enzyme Ontogeny in Rat Small Intestine

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Synergistic Effects of Thyroxine and Dexamethasone on Enzyme Ontogeny in Rat Small Intestine 003 1-3998/92/3203-0306$03.00/0 PEDIATRIC RESEARCH Vol. 32, No. 3, 1992 Copyright O 1992 International Pediatric Research Foundation, Inc. Printed in U.S.A. Synergistic Effects of Thyroxine and Dexamethasone on Enzyme Ontogeny in Rat Small Intestine MEGHAN C. McDONALD AND SUSAN J. HENNING Department of Pediatrics, ~a~iorCollege of Medicine, Houston, Texas 77030 ABSTRACT. The synergistic effects of dexamethasone Postnatal maturation of the rodent small intestine is charac- (DEX) and thyroxine (T4) on the postnatal maturation of terized by significant modification in enzyme status during the the 13-d-old rodent small intestine has been studied. Pre- 3rd week of life. Enzymes such as sucrase and glucoamylase have vious studies have shown that hydrocortisone and T4 pro- almost undetectable activity at birth and increase rapidly in duced a synergistic response in enzyme maturation. How- activity around postnatal d 16, achieving adult levels between d ever, T4 elevates corticosteroid-binding globulin, which 2 1 and 24. Lactase and acid P-galactosidase have high activities reduces the clearance of hydrocortisone. Thus, the appar- at birth that decline during the 3rd postnatal week. These changes ent synergy between T4 and hydrocortisone may have been in enzyme status coincide with weaning and represent important due to increased glucocorticoid availability. DEX, which adaptations to the nutrient differences between milk and solid does not bind to corticosteroid-binding globulin, was given food (1, 2). (d 8-12) at 25 pmol (i.e. 0.01 pg)/g body wt/d as established Studies regarding enzyme development have suggested that by a dose-response study in which this dose of DEX modifications in enzyme activity are influenced by the hormonal induced one third the maximum response in sucrase activ- milieu of the rat pup (1, 2). Corticosterone, which increases in ity. In this way, synergy with T4 (130 pmol/g body wt/d, concentration toward the end of the 2nd postnatal week just i.e. 0.1 pglg body wt/d, d 5-12) could still be observed. before the time of weaning (3), is an important factor in intestinal Glucoamylase, lactase, acid @-galactosidase,alkaline phos- maturation, because GC have been shown to directly affect phatase, and sucrase activities were determined in two enzyme development in vitro (4, 5) and to induce precocious regions of the small intestine. Overall, the results for the maturation of enzyme activity in vivo (1, 6-8). In contrast, T4, two hormones administered alone showed intestinal matu- which increases in concentration early in the 2nd postnatal week ration to be not significantly affected in the T4 group and (9) and which has been studied extensively with regard to small partially stimulated in the DEX group. When combined, intestinal enzyme ontogeny (1, 2), has not been shown to affect DEX + T4 synergistically increased jejunal sucrase, ileal the intestinal epithelium directly (10). Assessment of the role of glmcoamylase, and duodenal alkaline phosphatase, and low- T4 in intestinal ontogeny is complicated by the fact that altera- ered ileal acid @-galactosidase.The striking exceptions to tions of -thyroid status have been shown to influence circulating the general pattern were two brush border enzymes that concentrations of both endogenous and exogenous GC (1 1, 12). normally decline during intestinal maturation, namely ileal Thus, for example, when the same doses of exogenous corticos- alkaline phosphatase and jejunal and ileal lactase. For terone were administered to hypothyroid and hyperthyroid rat these enzymes, DEX alone did not elicit precocious matu- pups, the circulating concentration of corticosterone was almost ration, and there was no evidence for a synergistic inter- 3-fold greater in the hyperthyroid pups (12). This influence of action of these two hormones. Serum corticosterone con- thyroid status on circulating concentrations of GC has been centrations also were measured. When corticosterone con- shown to be due to T4 causing increased production of CBG (1 1, centrations were compared with enzyme activity, no 13, 14). By increasing the circulating concentration of CBG (1 1) correlation was found. Thus, DEX + T4 act synergistically and the percent bound fraction of corticosterone (12, 15), T4 in the maturation of four enzymes in the rodent small decreases the volume of distribution and the metabolic clearance intestine. Lactase and ileal alkaline phosphatase are reg- rate of corticosterone (15). This results in an increased concen- ulated differently. Further studies are required to deter- tration of circulating corticosterone (12, 14). mine the mechanism through which T4 enhances the DEX Although studies in adrenalectomized animals have made a effect. (Pediatr Res 32: 306-311, 1992) convincing case that T4 in the absence of GC does not influence intestinal development (16), the fact remains that under normal circumstances both hormones are present. Thus, the possibility Abbreviations that T4 enhances the effects of GC is highly plausible. Previous studies showing synergism between the two hormones have been BW, body weight compromised by the interactions noted above. Specifically, all CBG, corticosteroid-binding globulin such studies have used hydrocortisone or cortisone as the exog- DEX, dexamethasone enous GC. Synergism has been deduced from the observation GC, glucocorticoid that combined administration of these GC and T4 has greater T4, thyroxine effects on intestinal enzymes than does the GC alone (8, 17, 18). However, in view of the recent findings regarding T4 and CBG (described above), these effects actually may have been simply a reflection of higher circulating hydrocortisone in the animals Received December 3, 1991; accepted April 20, 1992. receiving hydrocortisone plus T4. TO reexamine the question of Correspondence: Dr. Susan J. Henning, Department of Pediatrics, Baylor Col- lege of Medicine, One Baylor Plaza, Houston, TX 77030. synergy between T4 and GC, we performed a study using the Supported by Grant HD 14094 from the National Institutes of Health. synthetic steroid DEX, which does not bind to CBG and thus EFFECT OF DEX AND T4 ON ENZYME ONTOGENY 307 yields circulating concentrations unaffected by CBG status (14) were killed on d 13. Because there were no differences between and by thyroid status. the uninjected and the vehicle-treated groups, only the vehicle- Because previous studies indicated that conclusions regarding treated group was used in analysis, as shown in the graphs. intestinal development cannot be based on the behavior of a Collection of blood and quantitation of serum corticosterone. single enzyme (19, 20), our study encompassed five small intes- Animals were decapitated on postnatal d 13 between 0900 and tinal enzymes. The enzymes chosen for study included two 1100 h after an overnight isolation period during which the pups enzymes that normally increase in activity during postnatal and dams were not disturbed. Trunk blood was collected on ice, development, sucrase and glucoamylase (1, 6, 21), and two allowed to clot, and centrifuged at 1500 x g for 10 min at 4°C enzymes that show declining activity during postnatal develop- to separate the serum. The serum was then frozen at -10°C for ment, lactase and acid 0-galactosidase (1, 7). Each of these four later determination of corticosterone concentration using a com- enzymes was assayed in both jejunum and ileum because of petitive protein binding assay as described previously (23). recent evidence indicating that enzyme activity is controlled Collection of tissue. Immediately after collection of blood, the differently in these regions (19,22). The 5th enzyme studied was entire small intestine (from the pylorus to the ileocoecal valve) alkaline phosphatase, whose activity rises with development in was removed, flushed with ice-cold 150 mM NaCl, separated the proximal small intestine and declines in the distal small into duodenum, jejunum, and ileum, and stored at - 10°C. The intestine (20). It was therefore assayed in both the duodenum duodenum was defined as the segment proximal to the ligament and the ileum. of Treitz. The jejunum was defined as the proximal half of the segment distal to the ligament of Treitz, and the ileum was MATERIALS AND METHODS defined as the distal half of that segment. As explained earlier, jejunum and ileum were the regions used for assay of the four Chemicals. [1,2,6,7-3H]-corticosteronewas purchased from carbohydrase enzymes, whereas duodenum and ileum were used DuPont-NEN (Boston, MA). Dextran (molecular weight for assay of alkaline phosphatase. 500 OOO), L-thyroxine (T4), DEX, lactose, glycogen, glucose oxi- Enzyme assays. All tissues were thawed and the mucosa was dase reagent [glucose (Trinder) 1001, o-nitrophenyl-p-D-galacto- scraped with a glass microscope slide. The mucosa was homog- pyranoside (ONPG), p-nitrophenyl phosphate (PNP), p-nitro- enized in 9 volumes of 154 mM KC1 by 15 strokes of a Tri-R phenol, charcoal, and BSA were obtained from Sigma Chemical homogenizer (Tri-R Instruments, Rockville Centre, NY). Su- Co. (St. Louis, MO). Glucose (Trinder) 100 contains glucose crase, lactase, and acid p-galactosidase were assayed as previously oxidase together with peroxidase and a quinoneimine dye, which described (24, 25) using sucrose, lactose, and ONPG, respec- allows stoichiometric production of a colored compound from tively, as substrates. the reducing equivalents of the glucose oxidase reaction. Dex- Glucoamylase activity was determined by the method of Eg- tran-coated charcoal suspension was prepared by adding 37.5 germont (26) with minor modifications. The acid fraction of mg/mL activated charcoal to 20 pM (i.e. 10 mg/mL) dextran in adult and 13-d-old rat pup mucosal samples was found to ac- 40 mM sodium phosphate, pH 7.9. Ultrapure grade sucrose from count for 1.2-3.6% of the total glucoamylase activity. Because SchwartzIMann (Orangeburg, NJ) was used for sucrase assay. this was negligible, the acid fraction determination at pH 3.0 was DEX for injection was prepared in 550 mM ethanol in 150 mM omitted from our routine assay.
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