Peripheral circadian clocks are diversely affected by adrenalectomy.

Glucocorticoids are considered to synchronize the rhythmicity of clock genes in peripheral tissues; however, the role of circadian variations of endogenous glucocorticoids is not well defined. In the present study, we examined whether peripheral circadian clocks were impaired by adrenalectomy. To achieve this, we tested the circadian rhythmicity of core clock genes (Bmal1, Per1-3, Cry1, RevErbα, Rora), clock-output genes (Dbp, E4bp4) and a glucocorticoid- and clock-controlled gene (Gilz) in liver, jejunum, kidney cortex, splenocytes and visceral adipose tissue (VAT). Adrenalectomy did not affect the phase of clock gene rhythms but distinctly modulated clock gene mRNA levels, and this effect was partially tissue-dependent. Adrenalectomy had a significant inhibitory effect on the level of Per1 mRNA in VAT, liver and jejunum, but not in kidney and splenocytes. Similarly, adrenalectomy down-regulated mRNA levels of Per2 in splenocytes and VAT, Per3 in jejunum, RevErbα in VAT and Dbp in VAT, kidney and splenocytes, whereas the mRNA amounts of Per1 and Per2 in kidney and Per3 in VAT and splenocytes were up-regulated. On the other hand, adrenalectomy had minimal effects on Rora and E4bp4 mRNAs. Adrenalectomy also resulted in decreased level of Gilz mRNA but did not alter the phase of its diurnal rhythm. Collectively, these findings suggest that adrenalectomy alters the mRNA levels of core clock genes and clock-output genes in peripheral organs and may cause tissue-specific modulations of their circadian profiles, which are reflected in changes of the amplitudes but not phases. Thus, the circulating corticosteroids are necessary for maintaining the high-amplitude rhythmicity of the peripheral clocks in a tissue-specific manner.

Glucocorticoid availability in colonic inflammation of rat.

Recent in vitro studies have shown the involvement of pro-inflammatory cytokines in the regulation of the local metabolism of glucocorticoids via 11beta-hydroxysteroid dehydrogenase type 1 and type 2 (11HSD1 and 11HSD2). However, direct in vivo evidence for a relationship among the local metabolism of glucocorticoids, inflammation and steroid enzymes is still lacking. We have therefore examined the changes in the local metabolism of glucocorticoids during colonic inflammation induced by TNBS and the consequences of corticosterone metabolism inhibition by carbenoxolone on 11HSD1, 11HSD2, cyclooxygenase 2 (COX-2), mucin 2 (MUC-2), tumor necrosis factor alpha (TNF-alpha), and interleukin 1beta (IL-1beta). The metabolism of glucocorticoids was measured in tissue slices in vitro and their 11HSD1, 11HSD2, COX-2, MUC-2, TNF-alpha, and IL-1beta mRNA abundances by quantitative reverse transcription-polymerase chain reaction. Colitis produced an up-regulation of colonic 11HSD1 and down-regulation of 11HSD2 in a dose-dependent manner, and these changes resulted in a decreased capacity of the inflamed tissue to inactivate tissue corticosterone. Similarly, 11HSD1 transcript was increased in colonic intraepithelial lymphocytes of TNBS-treated rats. Topical intracolonic application of carbenoxolone stimulated 11HSD1 mRNA and partially inhibited 11HSD2 mRNA and tissue corticosterone inactivation and these changes were blocked by RU-486. The administration of budesonide mimicked the effect of carbenoxolone. In contrast to the local metabolism of glucocorticoids, carbenoxolone neither potentiates nor diminishes gene expression for COX-2, TNF-alpha, and IL-1beta, despite the fact that budesonide down-regulated all of them. These data indicate that inflammation is associated with the down-regulation of tissue glucocorticoid catabolism. However, these changes in the local metabolism of glucocorticoids do not modulate the expression of COX-2, TNF-alpha, and IL-1beta in inflamed tissue.

Cloning and expression of chicken 20-hydroxysteroid dehydrogenase.

The ligand specificity and activation of steroid receptors depend considerably on the enzymatic activities involved in local pre-receptor synthesis and the metabolism of the steroids. Several enzymes in particular, steroid dehydrogenases have been shown to participate in this process. Here we report the isolation of 20-hydroxysteroid dehydrogenase (ch20HSD) cDNA from chicken intestine and the distribution of ch20HSD mRNA and 20-reductase activity in various avian tissues. Using a reverse transcription PCR and comparison with the known sequences of mammalian 20betaHSDs, we have isolated a new ch20HSD cDNA. This cDNA predicted 276 amino acid residues that shared about 75% homology with mammalian 20betaHSD. Sequences specific to the short-chain dehydrogenase/reductase superfamily (SDR) were found, the Gly-X-X-X-Gly-X-Gly cofactor-binding motif (residues 11-17) and the catalytic activity motif Tyr-X-X-X-Lys (residues 193-197). The cDNA coding for ch20HSD was expressed in Escherichia coli by placing it under isopropylthiogalactoside (IPTG) inducible control. Both the IPTG cells of E. coli and the isolated recombinant protein reduced progesterone to 20-dihydroprogesterone, corticosterone to 20-dihydrocorticosterone and 5alpha-dihydrotestosterone to its 3-ol derivative. The 20-reductase and 3-reductase activities of ch20HSD catalyzed both 3alpha/beta- and 20alpha/20beta-epimers. The mRNA transcripts of ch20HSD were found in the kidney, colon, and testes; weaker expression was also found in the heart, ovaries, oviduct, brain, liver, and ileum. 20-Reductase activity has been proven in tissue slices of kidney, colon, ileum, liver, oviduct, testis, and ovary; whereas the activity was nearly absent in the heart and brain. A similar distribution of 20-reductase activity was found in tissue homogenates measured under V(max) conditions. These results suggest that chicken 20HSD is the latest member of the SDR superfamily to be found, is expressed in many avian tissues and whose precise role remains to be determined.

Colitis up-regulates local glucocorticoid activation and down-regulates inactivation in colonic tissue.

BACKGROUND: Pro-inflammatory processes are counteracted by anti-inflammatory factors such as glucocorticoids. The response of target cells to glucocorticoids depends on several factors including prereceptor modulation of glucocorticoid signals via local glucocorticoid metabolism. This is determined by two isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD); 11betaHSD1 operates in vivo as a reductase converting inactive 11-oxo glucocorticoids to active glucocorticoids cortisol or corticosterone, whereas 11betaHSD2 catalyses oxidation of active glucocorticoids to their inactive 11-oxo derivatives. The aim of this study was to investigate the changes in local metabolism of glucocorticoids and in the expression of 11betaHSD1 and 11betaHSD2 mRNA during colonic inflammation. METHODS: Acute colitis was induced by intracolonic administration of 2,4,6-trinitrobenzenesulphonic acid (TNBS) or by drinking a dextran sodium sulphate (DSS) solution. Metabolism of glucocorticoids was measured in tissue fragments in vitro and 11betaHSD1 and 11betaHSD2 mRNA abundance was quantified using real-time RT-PCR one week after administration of TNBS and 10 days after drinking the DSS solution. RESULTS: In both models of inflammatory bowel disease we observed down-regulation of corticosterone oxidation to 11-dehydrocorticosterone by 64% (TNBS) and 53% (DSS) and reciprocal stimulation of reduction of 11-dehydrocorticosterone to corticosterone by 83% and 54%, respectively. A similar pattern was observed at the level of mRNA; 11betaHSD1 mRNA was significantly higher (TNBS: increase by 660%; DSS: increase by 760%) and 11betaHSD2 mRNA lower (TNBS: decrease by 85%; DSS: decrease by 60%) during inflammation. CONCLUSIONS: Colitis induces local glucocorticoid activation from 11-oxo steroids and decreases glucocorticoid inactivation; i.e. inflammation increases local tissue ratio of active and inactive glucocorticoids. The results indicate that the changes in local metabolism of glucocorticoids could contribute to the control of an overshoot of inflammation processes in the colon.

Corticosteroid regulation of colonic ion transport during postnatal development: methods for corticosteroid analysis.

Many mammalian species including human are immature at birth and undergo major developmental changes during suckling and weaning period. This problem is also conspicuous for the gastrointestinal tract that undergoes abrupt transitions coinciding with birth and weaning. This review deals with the maturation of ion transport functions in colon, the intestinal segment that plays an important role in sodium and potassium absorption and secretion. The purpose of the present review is to summarize the mechanism of sodium and potassium transport pathways and show how these transport processes change postnatally and how hormones, particularly corticosteroids, modify the pattern of development. Finally we describe some of the ways, how to analyze corticosteroid metabolism in target tissue.

Corticosteroid effect on Caco-2 cell lipids depends on cell differentiation.

Previous studies from our laboratory have indicated that secondary hyperaldosteronism affects phospholipids of rat colonic enterocytes. To assess whether this represents a direct effect of mineralocorticoids on enterocytes, the role of aldosterone and dexamethasone in the regulation of lipid metabolism was examined in Caco-2 cells during development of their enterocyte phenotype. Differentiation of Caco-2 cells was associated with increased levels of triglycerides (TG) and cholesteryl esters (CE), a decreased content of cholesterol and phospholipids and changes in individual phospholipid classes. The phospholipids of differentiated cells had a higher content of n-6 polyunsaturated fatty acids (PUFA) and lower amounts of monounsaturated (MUFA) and saturated fatty acids than subconfluent undifferentiated cells. Differentiated cells exhibited a higher ability to incorporate [3H]arachidonic acid (AA) into cellular phospholipids and a lower ability for incorporation into TG and CE. Incubation of subconfluent undifferentiated cells with aldosterone or dexamethasone was without effect on the content of lipids, their fatty acids and [3H]AA incorporation. In contrast, aldosterone treatment of differentiated cells diminished the content of TG, increased the content of phospholipids and modulated their fatty acid composition. The percentage of n-6 and n-3 PUFA in phospholipids was increased and that of MUFA decreased, whereas no changes in TG were observed. The incorporation of [3H]AA into phospholipids was increased and into TG decreased and these changes were blocked by spironolactone. Treatment of differentiated cells with dexamethasone increased their CE content but no effect was identified upon other lipids, their fatty acid composition and on the incorporation of [3H]AA. As expected for the involvement of corticosteroid hormones the mineralocorticoid and glucocorticoid receptors were identified in Caco-2 cells by RT-PCR. The results suggest that aldosterone had a profound influence on lipid metabolism in enterocytes and that its effect depends on the stage of differentiation. The aldosterone-dependent changes occurring in phospholipids and their fatty acid composition may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.