Matúš Soták

Hepatic, Duodenal, and Colonic Circadian Clocks Differ in their Persistence under Conditions of Constant Light and in their Entrainment by Restricted Feeding

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. (Author correspondence: sumova@biomed.cas.cz)

Temporal Gradient in the Clock Gene and Cell-Cycle Checkpoint Kinase Wee1 Expression along the Gut

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-erbα, 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-erbα 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-erbα, 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.

Cross-talk between the circadian clock and the cell cycle in cancer

Abstract 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.

An association between clock genes and clock-controlled cell cycle genes in murine colorectal tumors

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.

Circadian regulation of electrolyte absorption in the rat colon

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, γEnac, 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 γEnac expression coincided with the rise in plasma aldosterone. Restricted feeding phase advanced the expression of Dra, Ae1, Nherf, and γEnac and decreased expression of Atp1a1. The genes Atp1b1, Cftr, αEnac, βEnac, Nkcc1, and Kcc1 did not show any diurnal variations in mRNA levels. A low-salt diet upregulated the expression of βEnac and γEnac during the subjective night but did not affect expression of αEnac. 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.

Heterogeneous expression of melatonin receptor MT1 mRNA in the rat intestine under control and fasting conditions

Abstract:  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.

Development and entrainment of the colonic circadian clock during ontogenesis

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.

Peripheral circadian clocks are diversely affected by adrenalectomy

ABSTRACT 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.

Differential impact of stress on hypothalamic–pituitary–adrenal axis: Gene expression changes in Lewis and Fisher rats

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.

Regulation of 11β-hydroxysteroid dehydrogenase type 1 and 7α-hydroxylase CYP7B1 during social stress.

11β-hydroxysteroid dehydrogenase type 1 (11HSD1) is an enzyme that amplifies intracellular glucocorticoid concentration by the conversion of inert glucocorticoids to active forms and is involved in the interconversion of 7-oxo- and 7-hydroxy-steroids, which can interfere with the activation of glucocorticoids. The presence of 11HSD1 in the structures of the hypothalamic-pituitary-adrenal (HPA) axis suggests that this enzyme might play a role in the regulation of HPA output. Here we show that the exposure of Fisher 344 rats to mild social stress based on the resident-intruder paradigm increased the expression of 11HSD1 and CYP7B1, an enzyme that catalyzes 7-hydroxylation of steroids. We found that social behavioral profile of intruders was significantly decreased whereas their plasma levels of corticosterone were increased more than in residents. The stress did not modulate 11HSD1 in the HPA axis (paraventricular nucleus, pituitary, adrenal cortex) but selectively upregulated 11HSD1 in some regions of the hippocampus, amygdala and prelimbic cortex. In contrast, CYP7B1 was upregulated not only in the hippocampus and amygdala but also in paraventricular nucleus and pituitary. Furthermore, the stress downregulated 11HSD1 in the thymus and upregulated it in the spleen and mesenteric lymphatic nodes whereas CYP7B1 was upregulated in all of these lymphoid organs. The response of 11HSD1 to stress was more obvious in intruders than in residents and the response of CYP7B1 to stress predominated in residents. We conclude that social stress induces changes in enzymes of local metabolism of glucocorticoids in lymphoid organs and in brain structures associated with the regulation of the HPA axis. In addition, the presented data clearly suggest a role of 11HSD1 in modulation of glucocorticoid feedback of the HPA axis during stress.