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The gut microbiota is involved in a number of different metabolic processes of the host organism, including the metabolism of xenobiotics. In our study, we focused on liver cytochromes P450 (CYPs), which can metabolize a wide range of exo- and endogenous molecules. We studied changes in mRNA expression and CYP enzyme activities, as well as the mRNA expression of transcription factors that have an important role in CYP expression, all in stressed germ-free (GF) and stressed specific-pathogen-free (SPF) mice. Besides the presence of the gut microbiota, we looked at the difference between acute and chronic stress. Our results show that stress has an impact on CYP mRNA expression, but it is mainly chronic stress that has a significant effect on enzyme activities along with the gut microbiome. In acutely stressed mice, we observed significant changes at the mRNA level, however, the corresponding enzyme activities were not influenced. Our study suggests an important role of the gut microbiota along with chronic psychosocial stress in the expression and activity of CYPs, which can potentially lead to less effective drug metabolism and, as a result, a harmful impact on the organism.

Glucocorticoids (GCs) are hormones that are released in response to stressors and exhibit many activities, including immunomodulatory and anti-inflammatory activities. They are primarily synthesized in the adrenal gland but are also produced in peripheral tissues via regeneration of adrenal 11-oxo metabolites or by de novo synthesis from cholesterol. The present study investigated the influence of the microbiota on de novo steroidogenesis and regeneration of corticosterone in the intestine of germ-free (GF) and specific pathogen-free mice challenged with a physical stressor (anti-CD3 antibody i.p. injection). In the small intestine, acute immune stress resulted in increased mRNA levels of the proinflammatory cytokines IL1β, IL6 and Tnfα and genes involved in de novo steroidogenesis (Stard3 and Cyp11a1), as well as in regeneration of active GCs from their 11-oxo metabolites (Hsd11b1). GF mice showed a generally reduced transcriptional response to immune stress, which was accompanied by decreased intestinal corticosterone production and reduced expression of the GC-sensitive marker Fkbp5. In contrast, the interaction between stress and the microbiota was not detected at the level of plasma corticosterone or the transcriptional response of adrenal steroidogenic enzymes. The results indicate a differential immune stress-induced intestinal response to proinflammatory stimuli and local corticosterone production driven by the gut microbiota.

Introduction: Solute Carrier (SLC) and ATP-binding cassette (ABC) transporters expressed in the intestine, liver, and kidney determine the absorption, distribution, and excretion of drugs. In addit...

Aim We aimed to determine whether the sodium/glucose cotransporter family member SGLT3, a proposed glucose sensor, is expressed in the intestine and/or kidney, and if its expression is altered in mouse models of obesity and in humans before and after weight-loss surgery. Main methods We used in-situ hybridization and quantitative PCR to determine whether the Sglt3 isoforms 3a and 3b were expressed in the intestine and kidney of C57, leptin-deficient ob/ob, and diabetic BTBR ob/ob mice. Western blotting and immunohistochemistry were also used to assess SGLT3 protein levels in jejunal biopsies from obese patients before and after weight-loss Roux-en-Y gastric bypass surgery (RYGB), and in lean healthy controls. Key findings Sglt3a/3b mRNA was detected in the small intestine (duodenum, jejunum and ileum), but not in the large intestine or kidneys of mice. Both isoforms were detected in epithelial cells (confirmed using intestinal organoids). Expression of Sglt3a/3b mRNA in duodenum and jejunum was significantly lower in ob/ob and BTBR ob/ob mice than in normal-weight littermates. Jejunal SGLT3 protein levels in aged obese patients before RYGB were lower than in lean individuals, but substantially upregulated 6 months post-RYGB. Significance Our study shows that Sglt3a/3b is expressed primarily in epithelial cells of the small intestine in mice. Furthermore, we observed an association between intestinal mRNA Sglt3a/3b expression and obesity in mice, and between jejunal SGLT3 protein levels and obesity in humans. Further studies are required to determine the possible role of SGLT3 in obesity.

Colonic function is controlled by an endogenous clock that allows the colon to optimize its function on the daytime basis. For the first time, this study provided evidence that the clock is synchronized by rhythmic hormonal signals. In rat colon, adrenalectomy decreased and repeated applications of dexamethasone selectively rescued circadian rhythm in the expression of the clock gene Per1. Dexamethasone entrained the colonic clock in explants from mPer2Luc mice in vitro. In contrast, pinealectomy had no effect on the rat colonic clock, and repeated melatonin injections were not able to rescue the clock in animals maintained in constant light. Additionally, melatonin did not entrain the clock in colonic explants from mPer2Luc mice in vitro. However, melatonin affected rhythmic regulation of Nr1d1 gene expression in vivo. The findings provide novel insight into possible beneficial effects of glucocorticoids in the treatment of digestive tract-related diseases, greatly exceeding their anti-inflammato...

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.

Aging and tumorigenesis are associated with decline and disruption of circadian rhythms in many tissues and accumulating evidence indicates molecular link between circadian clock and cell cycle. The aim of this study was to investigate the effect of aging and tumorigenesis on coupling between cell cycle and circadian clock oscillators in colon, which undergoes regular rhythmicity of cell cycle and expresses peripheral circadian clock. Using healthy 14-week-old mice and 33-week-old mice with and without colorectal tumors, we showed that the 24-h expression profiles of clock genes and clock-controlled genes were mostly unaffected by aging, whereas the genes of cell cycle and cell proliferation were rhythmic in the young colons but were silenced during aging. On the other hand, tumorigenesis completely silenced or dampened the circadian rhythmicity of the clock genes but only a few genes associated with cell cycle progression and cell proliferation. These results suggest that aging impacts the colonic circadian clock moderately but markedly suppresses the rhythms of cell cycle genes and appears to uncouple the cell cycle machinery from circadian clock control. Conversely, tumorigenesis predominantly affects the rhythms of colonic circadian clocks but is not associated with uncoupling of circadian clock and cell cycle.

The commensal microbiota affects brain functioning, emotional behavior and ACTH and corticosterone responses to acute stress. However, little is known about the role of the microbiota in shaping the chronic stress response in the peripheral components of the hypothalamus-pituitary-adrenocortical (HPA) axis and in the colon. Here, we studied the effects of the chronic stress-microbiota interaction on HPA axis activity and on the expression of colonic corticotropin-releasing hormone (CRH) system, cytokines and 11β-hydroxysteroid dehydrogenase type 1 (11HSD1), an enzyme that determines locally produced glucocorticoids. Using specific pathogen-free (SPF) and germ-free (GF) BALB/c mice, we showed that the microbiota modulates emotional behavior in social conflicts and the response of the HPA axis, colon and mesenteric lymph nodes (MLN) to chronic psychosocial stress. In the pituitary gland, microbiota attenuated the expression of Fkbp5, a gene regulating glucocorticoid receptor sensitivity, while in the adrenal gland, it attenuated the expression of genes encoding steroidogenesis (MC2R, StaR, Cyp11a1) and catecholamine synthesis (TH, PNMT). The pituitary expression of CRH receptor type 1 (CRHR1) and of proopiomelanocortin was not influenced by microbiota. In the colon, the microbiota attenuated the expression of 11HSD1, CRH, urocortin UCN2 and its receptor, CRHR2, but potentiated the expression of cytokines TNFα, IFNγ, IL-4, IL-5, IL-6, IL-10, IL-13 and IL-17, with the exception of IL-1β. Compared to GF mice, chronic stress upregulated in SPF animals the expression of pituitary Fkbp5 and colonic CRH and UCN2 and downregulated the expression of colonic cytokines. Differences in the stress responses of both GF and SPF animals were also observed when immunophenotype of MLN cells and their secretion of cytokines were analyzed. The data suggest that the presence of microbiota/intestinal commensals plays an important role in shaping the response of peripheral tissues to stress and indicates possible pathways by which the environment can interact with glucocorticoid signaling.

The aim of the present work was to study the influence of variable stress on the expression of 11β-hydroxysteroid dehydrogenase type 1 (11HSD1) and the neuropeptides corticotropin-releasing hormone (CRH), urocortins 2 and 3(UCN2, UCN3), arginine vasopressin (AVP), oxytocin (OXT) and adenylate cyclase-activating polypeptide (PACAP) in two inbred rat strains: stress hypo-responsive Lewis (LEW) and hyper-responsive Fisher 344 (F344) rats. We found site-specific and strain-dependent differences in the basal and stress-stimulated expression of 11HSD1, CRH, UCN2, UCN3 and PACAP. In LEW rats, stress upregulated 11HSD1 in the prefrontal cortex and lateral amygdala, whereas in F344 rats 11HSD1 was upregulated in the central amygdala and hippocampal CA2 and ventral but not dorsal CA1 region; no effect was observed in the paraventricular nucleus, pituitary gland and adrenal cortex of both strains. The expression of glucocorticoid receptors did not parallel the upregulation of 11HSD1. Stress also stimulated the expression of paraventricular OXT, CRH, UCN3 and PACAP in both strains but amygdalar CRH only in LEW and UCN2/UCN3 in F344 rats, respectively. The upregulation of PACAP and CRH was paralleled only by increased expression of PACAP receptor PAC1 but not CRH receptor type 1. These observations provide evidence that inbred F344 and LEW rats exhibit not only the well-known phenotypic differences in the activity of the HPA axis but also strain- and stress-dependent differences in the expression of genes encoding 11HSD1 and neuropeptides associated with the HPA axis activity. Moreover, the differences in 11HSD1 expression suggest different local concentration of corticosterone and access to GR in canonical and noncanonical structures of the HPA axis.

The bioavailability of glucocorticoids is modulated by enzyme 11β-hydroxysteroid dehydrogenase type 1 (11HSD1), which catalyzes the conversion of inactive 11-oxo-glucocorticoids to active 11-hydroxy-glucocorticoids cortisol and corticosterone and is regulated by pro-inflammatory cytokines. Our aim was to assess the effect of colitis on the expression of 11HSD1 in specific microanatomical compartments of the mucosal immune system. Using qRT-PCR we quantified the expression of 11HSD1 and cytokines in the colon, mesenteric lymph nodes (MLN) and spleen of mice with colitis. Microsamples of the MLN cortex, paracortex and medulla, colonic crypt epithelium (CCE), lamina propria and isolated intestinal lymphoid follicles (ILF) were harvested by laser microdissection, whereas splenic and MLN lymphocytes by flow cytometry. Colitis increased 11HSD1 in the CCE, ILF, and MLN cortex but not in the lamina propria and the MLN paracortex and medulla. Expression of IL-4, IL-21 and TNFα was increased in both the cortex of MLN and ILF, whereas IL-1β and IL-10 were only increased in the follicles. No positive effect was observed in the case of IFNγ and TGFβ. 11HSD1 was positively correlated with TNFα and less strongly with IL-21, IL-1β, and IL-4. Colitis also upregulated the 11HSD1 expression of T cells in the spleen and MLN. The study demonstrates the stimulatory effect of inflammation on local glucocorticoid metabolism only in particular compartments of the mucosal immune system. The correlation between cytokines and 11HSD1 in the ILF and MLN cortex indicates that pro-inflammatory cytokines may amplify glucocorticoid signals in inductive compartments of the mucosal immune system.

Colonic morphology and function change significantly during ontogenesis. In mammals, many colonic physiological functions are temporally controlled by the circadian clock in the colon, which is entrained by the central circadian clock in the suprachiasmatic nuclei (SCN). The aim of this present study was to ascertain when and how the circadian clock in the colon develops during the perinatal period and whether maternal cues and/or the developing pup SCN may influence the ontogenesis of the colonic clock. Daily profiles of clock genes Per1, Per2, Cry1, Cry2, Rev-erbα, Bmal1, and Clock expression in the colon underwent significant modifications since embryonic day 20 (E20) through postnatal days (P) 2, 10, 20, and 30 via changes in the mutual phasing among the individual clock gene expression rhythms, their relative phasing to the light-dark regime, and their amplitudes. An adult-like state was achieved around P20. The foster study revealed that during the prenatal period, the maternal circadian phase may partially modulate development of the colonic clock. Postnatally, the absence and/or presence of rhythmic maternal care affected the phasing of the clock gene expression profiles in pups at P10 and P20. A reversal in the colonic clock phase between P10 and P20 occurred in the absence of rhythmic signals from the pup SCN. The data demonstrate ontogenetic maturation of the colonic clock and stress the importance of prenatal and postnatal maternal rhythmic signals for its development. These data may contribute to the understanding of colonic function-related diseases in newborn children.

The circadian clock system drives many physiological processes, including plasma concentration of glucocorticoids and epithelial transport of some ions and nutrients. As glucocorticoids entrain the circadian rhythms in various peripheral organs, we examined whether adrenalectomy affects the expression and circadian rhythmicity of intestinal transporters of the solute carrier (SLC) and ATP-binding cassette (ABC) families, which participate in intestinal barriers for absorption of nutrients, nonnutrients and oral drugs. The rat jejunum showed rhythmic circadian profiles of Sglt1, Pept1, Nhe3, Mdr1 and Mrp2 but not Mct1, Oct1, Octn1, Oatp1, Cnt1 and Bcrp. With the exception of Pept1 and Mct1, adrenalectomy decreased the expression of all rhythmic and arrhythmic transporters including the amplitude of Sglt1 and Nhe3 rhythms but minimally affected the phases of rhythmic transporters except of Nhe3. Similarly, adrenalectomy downregulated the expression of rhythmic (Pparα, Hlf, Pgc1α) and arrhythmic (Hnf1β, Hnf4α) transcription factors, which are known to regulate the expression of transporters. We conclude that endogenous corticosteroids have a profound effect on the expression of intestinal SLC and ABC transporters and their nuclear transcription factors. The circulating corticosteroids are necessary for maintaining upregulated expression of Sglt1, Oct1, Octn1, Oatp1, Cnt1, Nhe3, Mdr1, Bcrp, Mrp2, Pparα, Pgc1α, Hnf1β, Hnf4α and Hlf and for maintaining the high amplitude of Sglt1, Nhe3, Pparα, Pgc1α and Hlf circadian rhythms. The study demonstrates that signals from the adrenal gland are necessary for maintaining the expression of arrhythmic and rhythmic intestinal transporters and that changes in the secretion of corticosteroids associated with stress might reorganize intestinal transport barriers.

Many physiological functions exhibit a diurnal rhythmicity that is influenced by biological clocks and feeding rhythms. In this review, we discuss the growing evidence showing the important role of circadian rhythms in regulating intestinal mucosa. First, we introduce the molecular timing system and the interrelationship between the master biological clock in the suprachiasmatic nuclei of the brain and the peripheral intestinal clock and provide evidence that the intestinal clock is entrained with the external environment. Second, we review the circadian rhythmicity of enterocyte proliferation and the largely unknown regulatory mechanisms behind these rhythms. Finally, we focus on the circadian clock control of food processing that functions by regulating the expression of digestive enzymes and intestinal nutrient and salt transporters. The concepts to be discussed highlight the ability of the intestinal epithelium to utilize self-sustained clock signals together with signals associated with changes in the cellular environment and to use endogenous temporal control of the gastrointestinal functions to meet varying physiological and pathophysiological demands. The fact that internal de-synchronizations within the body, such as those that occur in shift workers or with changes in food intake behaviour, are often associated with malfunctions of the gastrointestinal tract indicates that more information about the connections between the circadian clock and intestinal mucosa/transporting enterocytes could provide clues for future therapies.

Disruption of circadian machinery appears to be associated with the acceleration of tumor development. To evaluate the function of the circadian clock during neoplastic transformation, the daily profiles of the core clock genes Per1, Per2, Rev-Erbα and Bmal1, the clock-controlled gene Dbp and the clock-controlled cell cycle genes Wee1, c-Myc and p21 were detected by real-time RT-PCR in chemically induced primary colorectal tumors, the surrounding normal tissue and in the liver. The circadian rhythmicity of Per1, Per2, Rev-Erbα and Dbp was significantly reduced in tumor compared with healthy colon and the rhythmicity of Bmal1 was completely abolished. Interestingly, the circadian expression of Per1, Per2, Rev-Erbα and Dbp persisted in the colonic tissue surrounding the tumor but the rhythmic expression of Bmal1 was also abolished. Daily profiles of Wee1, c-Myc and p21 did not exhibit any rhythmicity either in tumors or in the colon of healthy animals. The absence of diurnal rhythmicity of cell cycle genes was partially associated with ageing, because young healthy mice showed rhythmicity in the core clock genes as well as in the Wee1 and p21. In the liver of tumor-bearing mice the clock gene rhythms were temporally shifted. The data suggest that the circadian regulation is distorted in colonic neoplastic tissue and that the gene-specific disruption may be also observed in the non-neoplastic tissues. These findings reinforce the role of peripheral circadian clockwork disruption for carcinogenesis and tumor progression.

The intestinal transport of nutrients exhibits distinct diurnal rhythmicity, and the enterocytes harbor a circadian clock. However, temporal regulation of the genes involved in colonic ion transport, i.e., ion transporters and channels operating in absorption and secretion, remains poorly understood. To address this issue, we assessed the 24-h profiles of expression of genes encoding the sodium pump (subunits Atp1a1 and Atp1b1), channels (α-, β-, and γ-subunits of Enac and Cftr), transporters ( Dra, Ae1, Nkcc1, Kcc1, and Nhe3), and the Na+/H+ exchanger (NHE) regulatory factor ( Nherf1) in rat colonic mucosa. Furthermore, we investigated temporal changes in the spatial localization of the clock genes Per1, Per2, and Bmal1 and the genes encoding ion transporters and channels along the crypt axis. In rats fed ad libitum, the expression of Atp1a1, γ Enac, Dra, Ae1, Nhe3, and Nherf1 showed circadian variation with maximal expression at circadian time 12, i.e., at the beginning of the subjective night. The peak γ Enac expression coincided with the rise in plasma aldosterone. Restricted feeding phase advanced the expression of Dra, Ae1, Nherf, and γ Enac and decreased expression of Atp1a1. The genes Atp1b1, Cftr, α Enac, β Enac, Nkcc1, and Kcc1 did not show any diurnal variations in mRNA levels. A low-salt diet upregulated the expression of β Enac and γ Enac during the subjective night but did not affect expression of α Enac. Similarly, colonic electrogenic Na+ transport was much higher during the subjective night than the subjective day. These findings indicate that the transporters and channels operating in NaCl absorption undergo diurnal regulation and suggest a role of an intestinal clock in the coordination of colonic NaCl absorption.

Glucocorticoids are metabolized in vascular tissue by two types of 11β-hydroxysteroid dehydrogenases (11HSD1, 11HSD2) and thus these enzymes are considered to be important factors that modulate the diverse and complex effects of glucocorticoids on cardiovascular function. The present study evaluated the effect of peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone on 11HSD1 vascular smooth muscle cells (VSMC) and compared the effect with that of corticosterone. Using primary cultures of VSMC derived from rat aorta, we showed that pioglitazone significantly increases 11HSD1 activity and mRNA expression in a dose-dependent manner with EC50 243 nM and that this effect is not blocked by RU 486, an antagonist of the glucocorticoid receptor. In contrast, corticosterone had no effect on 11HSD1. Pioglitazone positively regulated transcription of two CCAAT/enhancer-binding proteins (C/EBPs), specifically C/EBPα a potent activator of 11HSD1 gene transcription in some cells types, and C/EBPζ, whereas C/EBPβ and C/EBPδ were not changed. In contrast, corticosterone stimulated the expression of C/EBPβ and C/EBPδ, but the levels of C/EBPα and C/EBPζ were not changed. In conclusion, activation of PPARγ in VSMC up-regulates vascular 11HSD1 and thus reactivates 11-oxo metabolites to biologically active glucocorticoids through a mechanism that seems to involve C/EBPα and C/EBPζ. Our data provide one of the possible explanations for PPARγ agonists’ effects on the cardiovascular system.

Elevated levels of survivin, telomerase catalytic subunit (TERT), integrin-linked kinase (ILK), cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS) and the regulatory factors c-MYB and Tcf-4 are often found in human cancers including colorectal cancer (CRC) and have been implicated in the development and progression of tumorigenesis. The aim of this study was to determine the expression of these genes in mouse models of sporadic and colitis-associated CRC. To address these issues, we used qRT-PCR approach to determine changes in gene expression patterns of neoplastic cells (high-grade dysplasia/intramucosal carcinoma) and surrounding normal epithelial cells in A/J and ICR mouse strains using laser microdissection. Both strains were injected with azoxymethane and ICR mice were also given drinking water that contained 2% dextran sodium sulphate. In both sporadic (A/J mice) and colitis-associated (ICR mice) models of CRC, the levels of TERT mRNA, COX-2 mRNA and Tcf-4 mRNA were higher in neoplastic cells than in surrounding normal epithelial cells. In contrast, survivin mRNA was upregulated only in neoplastic cells from A/J mice and ILK mRNA was upregulated only in neoplastic cells from ICR mice. However, the expression of iNOS mRNA was similar in normal and neoplastic cells in both models and c-MYB mRNA was actually downregulated in neoplastic cells compared with normal cells in both models. These findings suggest that the genetic background and/or the molecular mechanisms of tumorigenesis associated with genotoxic insults and colonic inflammation influence the gene expression of mTERT, COX-2, Tcf-4, c-MYB, ILK and survivin in colon epithelial neoplasia.

Objective. The objective of this study was to investigate the role of 11β-hydroxysteroid dehydrogenases (11HSD1 and 11HSD2) in determining the fetal concentration of glucocorticoids. Methods. The expression patterns for mRNA abundance, protein level, and enzyme activities of placental and fetal 11HSD1 and 11HSD2 were assessed from embryonic day 13 (E13) to day 21 (E21; term E22).The transplacental passage of maternal corticosterone and its contribution to fetal glucocorticoids was also studied. Results. Placental 11HSD1 mRNA decreased between days E13 and E14 and then remained at much lower values up to E21. Similarly, NADP+-dependent 11β-oxidation and 11-reduction were lower in late gestation. In contrast, placental 11HSD2 mRNA and protein decreased between E13 and E21. Dithiothreitol increased the activity of 11HSD2 and the output of 11-dehydrocorticosterone into fetal circulation.The fetal activity of 11HSD1 increased and 11HSD2 decreased between E16 and E21. Conclusions. The final third of gestation is accompanied by reciprocal changes in placental and fetal metabolism of corticosterone due to changes in 11HSD1 and 11HSD2 not only at the level of transcription but also at a posttranslational level.

NAD+-dependent 11β-hydroxysteroid dehydrogenase (11HSD2) converts glucocorticoids to 11-oxo derivatives and thus decreases their local concentration and prevents them from activating corticosteroid receptors. In this paper we report the partial cloning, characterization and tissue distribution of chicken 11HSD2. A cDNA of 991 bp was cloned from kidney mRNA by reverse transcription and polymerase chain reaction. At the amino acid level, the sequence of PCR product had 56–59% homology with mammalian and 46–48% with fish 11HSD2. The consensus sequences of the short-chain dehydrogenase/reductase superfamily such as the catalytic activity motif Tyr-X-X-X-Lys and cosubstrate-binding motif Gly-X-X-X-Gly-X-Gly, were found in the cloned cDNA. Analysis of the tissue expression of chicken 11HSD2 mRNA and NAD+-dependent 11β-oxidase activity showed a similar tissue distribution pattern in the majority of tissues. High levels of expression and activity were found in kidney, small intestine, colon and oviduct; low in ovary and almost zero in brain, liver and testis.

Melatonin is found in mammalian central nervous system and various peripheral tissues including gastrointestinal tract (GIT) where it participates in the regulation of intestinal motility, blood flow, immunomodulation, ion transport, cell proliferation and scavenging of free radicals. Some of these effects are achieved via melatonin binding to specific receptors, MT1 and MT2. As no thorough study on the expression of these receptors in the GIT has yet been done, the aim of this study was to determine the MT1 mRNA expression in the rat intestine under both control and fasting conditions. Our results suggest that MT1 mRNA is present in epithelial as well as subepithelial layer, with higher expression in the latter in all intestinal segments studied. The highest signal of the MT1 transcript along the rostro-caudal intestinal axis was found both in epithelial and subepithelial layers of the duodenum. Nevertheless, duodenal MT1 mRNA expression did not reach the level found in pituitary gland. In a 12:12-hr light:dark cycle a MT1 receptor expression in the subepithelial layer of rat distal colon did not manifest a significant diurnal rhythm. Short-term fasting increased the expression of MT1 transcript in the subepithelial layer of both the small and large intestine. During long-term fasting the increase persisted only in distal colon while a return to control levels was observed in small intestinal segments. In conclusion we demonstrated heterogeneous expression of MT1 receptor in the rat intestine and showed that its expression is up-regulated by nutritional deprivation.

Corticosteroids have been shown to play a role in cardiac remodeling, with the possibility of a direct effect of overexpression of 11β-hydroxysteroid dehydrogenase (11HSD) isoform 2 at the level of the cardiomyocytes. The aim of this study was to examine cardiac steroid metabolism in hypertensive rats with hearts that are hypertrophied and fibrotic and have structural alterations in the coronary circulation. To assess possible alterations of cardiac steroid metabolism the expression and activity of both isoforms of 11β-hydroxysteroid dehydrogenase (11HSD) were studied in spontaneously hypertensive rats (SHR), their normotensive controls Wistar–Kyoto (WKY), and in Dahl salt-sensitive (DS) and salt-resistant rats (DR) kept on a low- or high-salt diet. Using real-time quantitative RT-PCR and enzyme activity assay we found strain-dependent differences in cardiac metabolism of glucocorticoids. In Dahl rats expression of 11HSD1 and 11HSD2 mRNA was lower in DS than in DR rats and was not influenced by dietary salt intake; 11HSD1 mRNA was expressed at higher level than 11HSD2 mRNA. NADP+-dependent cardiac 11HSD activity showed similar distribution as 11HSD1 mRNA—lower activity in DS than in DR rats and no effect of salt intake. In SHR and WKY strains 11HSD2 mRNA expression was significantly higher in WKY than in SHR but no differences were observed in 11HSD1 mRNA abundance and NADP+-dependent 11HSD activity. These results show that the heart is able to metabolize glucocorticoids and that this metabolism is strain-dependent but do not support the notion of association between cardiac hypertrophy and changes of 11HSD1 and 11HSD2 expression.

The role of aldosterone in regulation of electrogenic Na+ transport is well established, though mineralocorticoid receptors bind glucocorticoids with similar binding affinity as aldosterone and plasma concentration of aldosterone is much lower than glucocorticoids. In mammals, the aldosterone specificity is conferred on the low-selective mineralocorticoid receptors by glucocorticoid inactivating enzyme 11beta-hydroxysteroid dehydrogenase (11HSD) that converts cortisol or corticosterone into metabolites (cortisone, 11-dehydrocorticosterone) with lower affinity for these receptors. The present study examined the chicken intestine, whether changes in 11HSD activity are able to modulate the effect of corticosterone on Na+ transport, and how the metabolism of this hormone is distributed within the intestinal wall. This study shows that not only aldosterone, but also corticosterone (B), was able to increase the electrogenic Na+ transport in chicken caecum in vitro. The effect of corticosterone was higher in the presence of carbenoxolone, an inhibitor of steroid dehydrogenases, and was comparable to the effect of aldosterone. The metabolism of B in the intestine was studied; results showed oxidation of this steroid to 11-dehydrocorticosterone (A) and reduction to 11-dehydro-20beta-dihydrocorticosterone (20diA) as the main metabolic products at low nanomolar concentration of the substrate. In contrast, 20beta-dihydrocorticosterone and 20diA were the major products at micromolar concentration of B. Progesterone was converted to 20beta-dihydroprogesterone. The metabolism of corticosterone was localized predominantly in the intestinal mucosa (enterocytes). In conclusion, the oxidation at position C11 and reduction at position C20 suggest that both 11HSD and 20beta-hydroxysteroid dehydrogenase (20HSD) operate in the chicken intestine and that the mucosa of avian intestine possesses a partly different system of modulation of corticosteroid signals than mammals. This system seems to protect the aldosterone target tissue against excessive concentration of corticosterone and progesterone.

11beta-Hydroxysteroid dehydrogenase (11betaHSD) converts endogenous glucocorticoids to their biologically inactive 11-dehydro derivatives and is therefore able to determine, at least in part, the biological action of glucocorticoids. Type 1 11betaHSD has both oxidase and reductase activities interconverting corticosterone and 11-dehydrocorticosterone, whereas type 2 11betaHSD has only oxidase activity converting corticosterone to 11-dehydrocorticosterone. Since 11betaHSD expression is regulated during development and by hormones in a tissue-specific manner and since glucocorticoids play an important role in postnatal intestinal maturation, we investigated the role of corticosteroids and cytodifferentiation in the regulation of intestinal 11betaHSD. Using rat intestinal organ cultures and epithelial cell lines derived from rat small intestine (IEC-6, IEC-18) and from human colon adenocarcinoma (Caco-2, HT-29), we analyzed the effect of corticosteroids and cytodifferentiation on 11betaHSD. Screening of the clonal cell lines showed that Caco-2 cells expressed by far the greatest 11betaHSD2 oxidase activity, lower activity was observed in HT-29 cells, and lowest activity was seen in IEC cells. Treatment with dexamethasone (50 nM) increased the activity of 11betaHSD2 in IEC-6 cells (+59%) and HT-29 cells (+31%), whereas aldosterone (50 nM) stimulated 11betaHSD2 in IEC-6 cells only (+31%). Caco-2 cells and IEC-18 cells did not respond to corticosteroids. Growth of IEC-6 cells on Matrigel, treatment of HT-29 cells with butyrate, and postconfluency of Caco-2 cells increased not only the markers of cytodifferentiation, such as alkaline phosphatase and sucrase, but also the activity of 11betaHSD2 in all of these cell lines (IEC-6, +96%; HT-29, +139%; Caco-2, +95%). Addition of corticosteroids to these more differentiated cell cultures did not enhance 11betaHSD2 activity. In intestinal organ cultures of suckling rat small intestine, dexamethasone and aldosterone stimulated 11betaHSD by more than 300%. We conclude that corticosteroids markedly and differentially regulate intestinal 11betaHSD2 and that cytodifferentiation of intestinal epithelial cells is associated with upregulation of 11betaHSD2 activity that is independent of corticosteroids.

The circadian clock is an endogenous timekeeper system that controls the daily rhythms of a variety of physiological processes. Accumulating evidence indicates that genetic changes or unhealthy lifestyle can lead to a disruption of circadian homeostasis, which is a risk factor for severe dysfunctions and pathologies including cancer. Cell cycle, proliferation, and cell death are closely intertwined with the circadian clock, and thus disruption of circadian rhythms appears to be linked to cancer development and progression. At the molecular level, the cell cycle machinery and the circadian clocks are controlled by similar mechanisms, including feedback loops of genes and protein products that display periodic activation and repression. Here, we review the circadian rhythmicity of genes associated with the cell cycle, proliferation, and apoptosis, and we highlight the potential connection between these processes, the circadian clock, and neoplastic transformations. Understanding these interconnections might have potential implications for the prevention and therapy of malignant diseases.

The regulation of intracellular pH (pH i ) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH i in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH i =7.24±0.04 and removal of extracellular Na + lowered pH i by 0.24 pH units. Acid-loaded colonocytes by an NH 3 /NH + 4 prepulse exhibited a spontaneous recovery that was partially Na + -dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na + -dependent recovery rate was enhanced by increasing the extracellular Na + concentration and was further stimulated by aldosterone. In an Na + - and K + -free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K + and this K + -dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH i exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO − 3 /CO 2 -containing solution, pH i of the proximal colonocytes was 7.20±0.04. Removal of external Cl − caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO − 3 /CO 2 from the medium was Cl − -dependent, Na + -independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na + -free HCO − 3 /CO 2 -containing solution was accelerated by addition of Na + . Removal of Cl − inhibited the effect of Na + . In summary, the freshly isolated proximal colonocytes of rats express Na + /H + exchanger, H + /K + exchanger ((H + -K + )-ATPase) and Na + -dependent Cl − /HCO − 3 exchanger that contribute to acid extrusion and Na + -independent Cl − /HCO − 3 exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH i in vivo. Proximal colonocytes are able to maintain a more stable pH i than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.

The role of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD) in hypertension remains unknown even if it appears that the inappropriately decreased 11betaHSD activity might be involved in a process that leads to high blood pressure. The possible changes of 11betaHSD were therefore investigated in rats with spontaneous or salt-induced hypertension. The adult male rats of the following genotypes were used: spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto rats (WKY), Dahl salt-sensitive rats fed either a high-salt diet containing 8% NaCl (DS-HS) or low-salt diet containing 0.2% NaCl (DS-LS), and Dahl salt-resistant rats fed the same diets (DR-HS, DR-LS). 11betaHSD was investigated in colon, aorta, renal cortex, and renal medulla and was assessed as percentage conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone in the presence of NAD or NADP. The results demonstrated that genotype exerts a significant effect on 11betaHSD. 11betaHSD activity was significantly increased in colon and renal medulla of SHR compared with WKY rats. No significant differences were observed in renal cortex and aorta. In Dahl rats kept on a low-salt diet, 11betaHSD activity was significantly higher in colon, renal medulla, and cortex of DS-LS than in DR-LS rats but no difference was observed in aorta. The differences disappeared in age-matched DS and DR rats fed the high-salt diet. Increased dietary sodium intake stimulated the activity of 11betaHSD in renal cortex and medulla of DR rats and decreased the activity in colon of DS rats. We conclude that the development of spontaneous and salt-induced hypertension is not associated with decreased activity of 11betaHSD. However, the results showed that salt intake is able to modulate the activity of 11betaHSD and that 11betaHSD in DS and DR rats responds to high dietary salt intake in a different manner.

The interrelationship between thyroid hormones and aldosterone has been examined in the regulation of rat colonic amiloride-sensitive Na+ transport which translocates Na+ through apical amiloride-sensitive Na+ channels and basolateral Na+, K+-ATPase. Electrogenic Na+ transport was measured in an Ussing chamber by the short-circuit current and identified by Na+ channel blocker amiloride. Na+-pumping activity of the basolateral Na+,K+-ATPase was investigated in nystatin-treated epithelium by measuring the equivalent short-circuit current after addition of mucosal Na+. The abundance of mRNA coding for alpha, beta and gamma subunits of the Na+ channel (rENaC) was estimated using Northern blot analysis. Hyperaldosteronism was induced by a low-salt diet and hypothyroidism by methimazole. The low-Na+ diet induced electrogenic Na+ transport in euthyroid rats but its effect was almost completely inhibited in hypothyroid animals even if the plasma concentration of aldosterone was high enough to stimulate this transport pathway both in euthyroid and hypothyroid rats. A kinetic study of the basolateral Na+,K+-ATPase revealed a decrease of Na+ transport capacity in hypothyroid rats kept on the low-Na+ diet in comparison with euthyroid animals fed the same diet. No significant differences in steady-state levels of alpha, beta and gamma rENaC mRNA were detected between euthyroid and hypothyroid rats. These data suggest that hypothyroidism decreases the efficacy of the basolateral Na+ pump but fails to inhibit it completely even though it inhibits the transepithelial electrogenic Na+ transport in response to aldosterone. We conclude that the permissive effect of thyroid hormones on the induction of electrogenic Na+ transport by aldosterone is localised beyond the transcriptional step of Na+ channel regulation.

The role of corticosteroids in the development of Na,K-ATPase and its distribution along the crypt base-colonic surface was investigated in suckling, weanling and adult rats using a biochemical and a histochemical approach. The cerium-based histochemical method for detection of ouabain-sensitive K+-dependent p-nitrophenylphosphatase (K-NPPase) component of the Na,K-ATPase complex was used to localize Na,K-ATPase in the epithelium. The activity of Na,K-ATPase was very low 2 days after birth and increased to a maximum in adulthood. Mature surface colonocytes and immature cells at the crypt base were similarly reactive and the reaction product was decreased by the addition of ouabain and inhibited by omission of K+. Adrenalectomy decreased colonic Na,K-ATPase activity in surface and deep crypt cells of suckling, weanling and adult animals. Mineralocorticoids (deoxycorticosterone acetate, DOCA) restored the Na,K-ATPase activity both in surface and crypt cells of adrenalectomized weanling rats and the effect of DOCA was inhibited by the mineralocorticoid receptor antagonist, spironolactone. Physiological doses of glucocorticoids (dexamethasone) stimulated Na,K-ATPase activity in surface colonocytes of adrenalectomized weanling rats; supraphysiological doses restored Na,K-ATPase probably via cross-over into mineralocorticoid receptors both in surface and crypt cells. High dietary Na+ intake during the weaning period reduced the reaction product to the level detected in adrenalectomized rats. The distribution of Na, K-ATPase activity in the epithelium of adrenalectomized rats with substitutional replacement hormone therapy was the same as in control animals or, in some animals, the surface absorptive epithelium exhibited a stronger reaction than the crypt cells. Similarly, the surface colonocytes of adult rats kept on a low-salt diet showed a stronger reaction than the crypt cells. These data indicate that postnatal development of Na,K-ATPase is regulated predominantly by aldosterone and that both surface and crypt cells are responsive to mineralocorticoids. Surface cells are also responsive to glucocorticoids.

We have previously demonstrated the developmental increase of the activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD) in the rat ileum which correlated with the developmental surge of plasma concentrations of corticosteroids, thyroid hormones and insulin. To ascertain whether these hormones directly stimulate 11betaHSD activity we used explant cultures of ileum and distal colon. The intestinal segments of young, 7-day-old rats, were cultured 48 hours in the presence of aldosterone (10[-7] M), dexamethasone (10[-7] M), triiodothyronine (10[-7] M) or insulin (10[-7] M) and 11betaHSD activity was evaluated by measuring the conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone. The activity of 11betaHSD was significantly increased following 48 h treatment with dexamethasone and aldosterone, whereas insulin and triiodothyronine were without any effect. Corticosterone oxidation was inhibited by carbenoxolone and progesterone. It is being concluded, that both glucocorticoids and mineralocorticoids but not insulin or triiodothyronine induce intestinal 11betaHSD activity.

Physiological functions of the gastrointestinal tract (GIT) are temporally controlled such that they exhibit circadian rhythms. The circadian rhythms are synchronized with the environmental light-dark cycle via signaling from the central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus, and by food intake. The aim of the study was to determine the extent to which disturbance in the SCN signaling via prolonged exposure to constant light affects circadian rhythms in the liver, duodenum, and colon, as well as to determine whether and to what extent food intake can restore rhythmicity in individual parts of the GIT. Adult male rats were maintained in constant light (LL) for 30 days and fed ad libitum throughout the entire interval or exposed to a restricted feeding (RF) regime for the last 14 days in LL. Locomotor and feeding behaviors were recorded throughout the experiment. On the 30th day, daily expression profiles of clock genes (Per1, Per2, Rev-erbα, and Bmal1) and of clock-controlled genes (Wee1 and Dbp) were measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in the duodenum, colon, and liver. By the end of the LL exposure, rats fed ad libitum had completely lost their circadian rhythms in activity and food intake. Daily expression profiles of clock genes and clock-controlled genes in the GIT were impaired to an extent depending on the tissue and gene studied, but not completely abolished. In the liver and colon, exposure to LL abolished circadian rhythms in expression of Per1, Per2, Bmal1, and Wee1, whereas it impaired, but preserved, rhythms in expression of Rev-erbα and Dbp. In the duodenum, all but Wee1 expression rhythms were preserved. Restricted feeding restored the rhythms to a degree that varied with the tissue and gene studied. Whereas in the liver and duodenum the profiles of all clock genes and clock-controlled genes became rhythmic, in the colon only Per1, Bmal1, and Rev-erbα-but not Per2, Wee1, and Dbp-were expressed rhythmically. The data demonstrate a greater persistence of the rhythmicity of the circadian clocks in the duodenum compared with that in the liver and colon under conditions when signaling from the SCN is disrupted. Moreover, disrupted rhythmicity may be restored more effectively by a feeding regime in the duodenum and liver compared to the colon.

Glucocorticoids exert anti-inflammatory and immunomodulatory effects that may be regulated in part by the activities of the glucocorticoid-activating and -inactivating enzymes, 11β-hydroxysteroid dehydrogenase type 1 (11HSD1) and type 2 (11HSD2), respectively. Previous studies have demonstrated that inflammatory bowel diseases in humans and experimental animals upregulate 11HSD1 and downregulate 11HSD2. We investigated whether proinflammatory cytokines modulate colonic 11HSDs as well as whether lymphoid organs exhibit any 11HSD response to inflammation. Colon tissue explants exposed to tumor necrosis factor α exhibited an upregulation of 11HSD1 mRNA whereas interleukin 1β downregulated 11HSD2 mRNA. Experimental colitis induced by the intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid stimulated 11HSD1 activity not only in the colon but also in mesenteric lymph nodes and the spleen. Analysis of mRNA for 11HSD1 in colon-draining lymph nodes and the spleen showed that inflammation upregulates the expression of this enzyme in mobile lymphoid cells similar to the intraepithelial and lamina propria leukocytes isolated from the colon. It is inferred that inflammation stimulates the reactivation of glucocorticoids in lymphoid organs and in gut-associated lymphoid tissue.

11β-Hydroxysteroid dehydrogenase type 1 (11HSD1) is a microsomal NADPH-dependent oxidoreductase which elevates intracellular concentrations of active glucocorticoids. Data obtained from mouse strains with genetically manipulated 11HSD1 showed that local metabolism of glucocorticoids plays an important role in the development of metabolic syndrome. Tissue specific dysregulation of 11HSD1 was also found in other models of metabolic syndrome as well as in a number of clinical studies. Here, we studied local glucocorticoid action in the liver, subcutaneous adipose tissue (SAT) and skeletal muscles of male and female Prague hereditary hypertriglyceridemic rats (HHTg) and their normotriglyceridemic counterpart, the Wistar rats. 11HSD1 bioactivity was measured as a conversion of [ 3 H]11-dehydrocorticosterone to [ 3 H]corticosterone or vice versa. Additionally to express level of active 11HSD1 protein, enzyme activity was measured in tissue homogenates. mRNA abundance of 11HSD1, hexoso-6-phosphate dehydrogenase (H6PDH) and the glucocorticoid receptor (GR) was measured by real-time PCR. In comparison with normotriglyceridemic animals, female HHTg rats showed enhanced regeneration of glucocorticoids in the liver and the absence of any changes in SAT and skeletal muscle. In contrast to females, the glucocorticoid regeneration in males of HHTg rats was unchanged in liver, but stimulated in SAT and downregulated in muscle. Furthermore, SAT and skeletal muscle exhibited not only 11-reductase but also 11-oxidase catalyzed by 11HSD1. In females of both strains, 11-oxidase activity largely exceeded 11-reductase activity. No dramatic changes were found in the mRNA expression of H6PDH and GR. Our data provide evidence that the relationship between hypertriglyceridemia and glucocorticoid action is complex and gender specific.

Background: Inflammatory bowel diseases including long-standing ulcerative colitis (UC) have an increased risk of evolving into colorectal cancer (CRC). The overexpression of some proproliferative and antiapoptotic genes, such as survivin, telomerase catalytic subunit (hTERT), integrin-linked kinase (ILK), and regulatory factors c-MYB and Tcf-4, has been implicated in the development and progression of several human malignancies including CRC. Methods: In this study we analyzed the expression alterations of these markers and proinflammatory enzymes cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) during the transition of colonic mucosa from chronic inflammation to epithelial neoplasia in biopsies of UC patients using quantitative real-time polymerase chain reaction and immunohistochemistry; additionally, we compared the expression profiles of this gene panel in samples of patients with CRC after tumor resection and in human tumor xenografts of SW620 malignant colonic cells. Results: The transcript levels of survivin, c-MYB, COX-2, iNOS, and Tcf-4 showed a statistically significant increase during neoplastic transformation of UC patient colonic mucosa, whereas hTERT and ILK were not elevated. In contrast, the specimens of CRC showed upregulated expression of not only survivin, c-MYB, Tcf-4, COX-2, and iNOS but also hTERT. A similar expression profile was observed in human tumor xenografts in which all transcripts with the exception of c-MYB were upregulated. Conclusions: These results suggest that telomerase and ILK activation occurs during the later stages of carcinoma progression, whereas upregulation of survivin, c-MYB, and Tcf-4 is a feature of the early stage of development of neoplasia, and thus, they might serve as early indicators for UC-associated colorectal carcinogenesis. (Inflamm Bowel Dis 2010)

Circadian clocks were recently discovered in the rat and mouse colon as well as mouse stomach and jejunum. The aim of this study was to determine whether clocks in the upper part of the gut are synchronized with those in the lower part, or whether there is a difference in their circadian phases. Moreover, the profiles of core clock-gene expression were compared with the profiles of the clock-driven Wee1 gene expression in the upper and lower parts of the gut. Adult rats were transferred to constant darkness on the day of sampling. 24 h expression profiles of the clock genes Per1, Per2, Rev-erbalpha, and Bmal1 and the cell-cycle regulator Wee1 were examined by a reverse transcriptase-polymerase chain reaction within the epithelium of the rat duodenum, ileum, jejunum, and colon. In contrast to the duodenum, the rhythms in expression of all genes but Rev-erbalpha and Bmal1 in the colon exhibited non-sinusoidal profiles. Therefore, a detailed analysis of the gene expression every 1 h within the 12 h interval corresponding to the previous lights-on was performed. The data demonstrate that rhythmic profiles of the clock gene Per1, Per2, Bmal1, Rev-erbalpha, and clock-driven Wee1 expression within the epithelium from different parts of the rat gut exhibited a difference in phasing, such that the upper part of the gut, as represented by the duodenum, was phase-advanced to the lower part, as represented by the distal colon. Our data demonstrate that the circadian clocks within each part of the gut are mutually synchronized with a phase delay in the cranio-caudal axis. Moreover, they support the view that the individual circadian clocks may control the timing of cell cycle within different regions of the gut.

11β-Hydroxysteroid dehydrogenase type 1 (11HSD1) is an enzyme that interconverts active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inactive 11-oxo derivatives (cortisone, 11-dehydrocorticosterone). Although bidirectional, it is considered to operate in vivo as an 11-reductase that regenerates active glucocorticoids and thus amplifies their local activity in mammals. Here we report the cloning, characterization and tissue distribution of chicken 11HSD1 (ch11HSD1). Its cDNA predicts a protein of 300 amino acids that share 51–56% sequence identity with known mammalian 11HSD1 proteins, while in contrast to most mammals, ch11HSD1 contains only one N-linked glycosylation site. Analysis of the tissue distribution pattern by RT-PCR revealed that ch11HSD1 is expressed in a large variety of tissues, with high expression in the liver, kidney and intestine, and weak in the gonads, brain and heart. 11-Reductase activity has been found in the liver, kidney, intestine and gonads with low or almost zero activity in the brain and heart. These results provide evidence for a role of 11HSD1 as a tissue-specific regulator of glucocorticoid action in non-mammalian vertebrates and may serve as a suitable model for further analysis of 11HSD1 evolution in vertebrates.

Although the gastrointestinal tract is a rich source of melatonin and possesses numerous melatonin-binding sites, the role of melatonin in this tissue has not yet been fully elucidated. In this work we focused on the role of melatonin in the modulation of ion transport in rat distal colon. Whereas melatonin had no effect on colonic secretion or caused only infrequent and small changes in the short circuit current (Isc) due to its solvent ethanol, this mediator significantly modulated the secretion elicited by some secretagogues. Out of the five substances tested (prostaglandin E(2); 5-hydroxytryptamine; bethanechol; histamine; sodium nitroprusside) melatonin inhibited the effect of prostaglandin E(2) (PGE(2)) and sodium nitroprusside (SNP). Melatonin concentration-dependently decreased PGE(2)-evoked Isc and this inhibitory effect was more obvious from the mucosal side. The basal level of cAMP in colonic mucosa was not influenced by melatonin, but this drug prevented a PGE(2)-induced increase in the level of cAMP. The neurotoxin tetrodotoxin blocked the inhibitory effect of melatonin on SNP-induced Isc. Our data suggests that melatonin takes part in the modulation of colonic ion transport. The modulatory effect of melatonin on PGE(2)-induced Isc occurs directly at the level of the epithelium, whereas the effect on SNP-induced Isc is indirect and located in tetrodotoxin-sensitive enteric neurons.

Background & Aims: The gastrointestinal tract exhibits diurnal rhythms in many physiologic functions. These rhythms are driven by food intake but are also preserved during food deprivation, suggesting the presence of endogenous circadian rhythmicity. The aim of the study was to provide insight into the circadian core clock mechanism within the rat colon. Moreover, the potency of a restricted feeding regime to shift the circadian clock in the colon was tested. The question of whether the colonic clock drives circadian expression in NHE3, an electroneutral Na+/H+ exchanger, was also addressed. Methods: Daily profiles in expression of clock genes Per1, Per2, Cry1, Bmal1, Clock, and Rev-erbα, and the NHE3 transporter were examined by reverse transcriptase–polymerase chain reaction and their mRNA levels, as well as PER1 and BMAL1 protein levels, were localized in the colonic epithelium by in situ hybridization and immunocytochemistry, respectively. Results: Expression of Per1, Per2, Cry1, Bmal1, Clock, Rev-erbα, and NHE3, as well as PER1 and BMAL1 protein levels, exhibited circadian rhythmicity in the colon. The rhythms were in phase with those in the liver but phase-delayed relative to the master clock in the suprachiasmatic nucleus. Restricted feeding entrained the clock in the colon, because rhythms in clock genes as well as in NHE3 expression were phase-advanced similarly to the clock in the liver. Conclusions: The rat colon harbors a circadian clock. The colonic clock is likely to drive rhythmic NHE3 expression. Restricted feeding resets the colonic clock similarly to the clock in the liver.

Intestinal secretory response is altered during colonic development. The aim of this report was to study the developmental changes of the Ca 2+ - and cAMP-induced regulatory pathways with special attention to the direct and indirect effect of secretagogues on the colonic epithelium. We investigated the effect of bethanechol, 5-hydroxytryptamine (5-HT), and histamine on Cl − secretion and stimulation of intracellular Ca 2+ ([Ca 2+ ] i ) and cAMP in the distal colon of suckling, weanling and adult rats. In the presence of tetrodotoxin, immature colon of suckling and weanling rats displayed higher potency (EC 50 ) of 5-HT to stimulate Cl − secretion, whereas the potency of histamine was not changed during development. The potency of bethanechol was reduced during weaning and partially restored in adulthood. 5-HT increased cAMP level similarly in both neonatal and adult colonic crypts, but the adults had higher basal level of cAMP than suckling rats. Also the effect of bethanechol on [Ca 2+ ] i was independent of colonic maturation. The results suggest that colonic Cl − secretion displays developmental changes of regulation depending on the non-neural secretagogue-signalling pathway and that these developmental changes seem to be localized somewhere outside colonocytes.

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.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.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.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.

A high-performance liquid chromatography–atmospheric pressure ionization–electrospray ionization mass spectrometry (HPLC–API– ESI–MS) method was developed for the analysis of steroids in a study of steroid-converting enzymes. Separations ware done on a Zorbax Eclipse XDB-C18 column (eluted with a linear methanol–water–acetic acid gradient) and identification of the steroids involved was done by API–ESI–MS using positive ion mode and extracted ion analysis. The applicability of the present method for studying steroid metabolism was proven in assaying two steroid-converting enzymes (20- hydroxysteroid dehydrogenase and 11-hydroxysteroid dehydrogenase) in various biological samples (rat and chicken intestine, chicken oviduct). © 2003 Elsevier B.V. All rights reserved.

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.

Glucocorticoid hormones have been reported to operate as regulators of cell proliferation and differentiation and to inhibit growth of several colon tumors and adenocarcinoma cell lines. The glucocorticoid action is regulated, in part, at the pre-receptor level through the expression of isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD1, 11betaHSD2) which are responsible for the interconversion of hormonally active cortisol to cortisone. Since both of these isoforms are expressed in the mammalian colon, we examined whether 11betaHSD1 and 11betaHSD2 are expressed in human colorectal cancer and whether their expression differs between neoplastic and autologous non-neoplastic tissue. We provide evidence that both isoforms of 11betaHSD are expressed in the colon adenocarcinoma, but their expression is not identical in neoplastic and non-neoplastic tissue. There is a significant decrease of 11betaHSD2 mRNA abundance and enzyme activity in neoplastic tissue. In contrast, 11betaHSD1 activity and mRNA abundance are increased in some but not all tumor samples. The results demonstrate that (1) neoplastic transformation is associated with decreasing steady-state levels of 11betaHSD2 mRNA and enzyme activity and in some cases also with increasing expression of 11betaHSD1, and (2) colorectal tumor cells have a decreased capability of autocrine inactivation of glucocorticoids.

The rat undergoes profound maturational changes in the intestinal structure and function during the third week of its life. To investigate the role of peripheral glucocorticoid metabolism in this process, we studied the postnatal maturation of intestinal structure and function. The peripheral metabolism of glucocorticoids depends on enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD), which is responsible for the interconversion of corticosterone to 11-dehydrocorticosterone and thus for the modulation of glucocorticoid access to corticosteroid receptors. The pups were treated with carbenoxolone (CBX), an inhibitor of 11betaHSD, for 10 d during the suckling (days 8-18) or weaning period (days 14-24 or days 20-30), and we determined the parameters of intestinal growth and activities of sucrase, alkaline phosphatase, and Na,K-ATPase. The CBX treatment increased plasma concentrations of corticosterone as a result of a significant reduction of peripheral degradation of corticosterone catalyzed by 11betaHSD. This also stimulated intestinal growth without changing somatic growth. The mucosal cell mass was significantly higher in CBX-treated suckling rats, whereas the effect of this treatment was less obvious in weanling animals. CBX increased the crypt depth and villus height in 18- and 24-d-old pups but not in 30-d-old animals. The small intestinal activities of sucrase, alkaline phosphatase, and Na,K-ATPase were not influenced by CBX. In contrast, colonic Na,K-ATPase was stimulated by CBX. We conclude that the administration of CBX results in acceleration of intestinal growth and structural maturation without any influence on the developmental pattern of brush-border hydrolases. The results indicate an important role of peripheral glucocorticoid metabolism in the regulation of intestinal growth during early postnatal life.

Active resorption of potassium ions in the colon is mediated by ouabain-sensitive and ouabain-insensitive H,K-ATPases localized in the apical membrane of colonic enterocytes (colonocytes). The present study was performed to investigate distribution patterns of apical ATPases using catalytic histochemistry and immunohistochemistry. Activity of the ATPases was localized both at the apical and basolateral regions of these cells. The basolateral activity was almost completely inhibited by ouabain, but apical ATPase activity was only partially inhibited by ouabain. Ultracytochemically, activity was localized at the cytoplasmic side of the apical and basolateral membrane and thus we assume that the activity represents isoforms of apical H,K-ATPases and basolateral Na,K-ATPase. With the use of a polyclonal antibody raised against H,K-ATPase alpha-subunit, we demonstrated immunostaining only in the apical region of colonic enterocytes whereas positive staining was not observed at the basolateral membrane. On the other hand, when antibodies against alpha1-subunit Na,K-ATPase were applied, immunostaining was localized only in the basolateral membrane domain. Therefore, we conclude that ATPases as demonstrated histochemically in the present study were identified immunohistochemically as colonic alpha-subunit H,K-ATPase (in the apical cell membrane of colonocytes along the entire length of crypts) and as alpha1-subunit Na,K-ATPase (in the basolateral membrane).