Insight Into the Circadian Clock Within Rat Colonic Epithelial Cells

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. 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. See editorial on page 1373. See editorial on page 1373. Diurnal rhythms in physiologic functions are observed in various organs and peripheral tissues, including the gastrointestinal tract (GIT).1Scheving L.A. Biological clocks and the digestive system.Gastroenterology. 2000; 119: 536-549Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar The GIT exhibits daily rhythms in gut motility,2Furukawa Y. Cook I.J. Panagopoulos V. McEvoy R.D. Sharp D.J. Simula M. 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However, when access to food is limited only to the light part of a light–dark cycle (ie, during the rest period of nocturnal rodents), clock gene expression in the liver, pancreas, heart, and other tissues becomes phase-advanced relative to that in animals fed ad libitum.18Damiola F. Le Minh N. Preitner N. Kornmann B. Fleury-Olela F. Schibler U. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus.Genes Dev. 2000; 14: 2950-2961Crossref PubMed Scopus (1777) Google Scholar, 19Stokkan K.A. Yamazaki S. Tei H. Sakaki Y. Menaker M. Entrainment of the circadian clock in the liver by feeding.Science. 2001; 291: 490-493Crossref PubMed Scopus (1390) Google Scholar The rate of this advance is tissue specific (eg, lung responds slower than the liver). The reversed FR, however, does not affect the phase of clock gene expression within the SCN.18Damiola F. Le Minh N. Preitner N. Kornmann B. Fleury-Olela F. Schibler U. 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Daily profiles in expression of selected clock genes, namely of Per1, Per2, Cry1, Bmal1, Clock, and Rev-erbα, were determined by reverse transcriptase–polymerase chain reaction (RT-PCR), and their expression was localized within intestinal sections by in situ hybridization (ISH). Moreover, the levels of clock gene products PER1 and BMAL1 were detected within the intestinal sections by immunocytochemistry. The phases of oscillations in clock gene expression in the colon were compared with those in a well-characterized peripheral clock, the rat liver, and also with those in the SCN master clock. Moreover, the daily expression profile of the colonic electroneutral Na+/H+ exchanger (NHE3) was studied. NHE3 was chosen because it represents one of the major transporters in the colonic epithelium and because its promoter contains the E-box sequence,37Saifur Rohman M. Emoto N. Nonaka H. Okura R. Nishimura M. Yagita K. van der Horst G.T. Matsuo M. Okamura H. Yokoyama M. Circadian clock genes directly regulate expression of the Na(+)/H(+) exchanger NHE3 in the kidney.Kidney Int. 2005; 67: 1410-1419Crossref PubMed Scopus (90) Google Scholar through which CLOCK-BMAL1 heterodimers may control circadian expression of the NHE3 gene. To elucidate whether the food intake may entrain the intestinal clock independently of the SCN, the profiles of several core clock genes and NHE3 expression were studied in the rat colon under conditions of a reversed, restricted FR. Two-month-old male Wistar rats (Bio Test, Konarovice, Czech Republic) were maintained for at least 4 weeks in a temperature of 23°C ± 2°C under light–dark cycle with 12 hours of light and 12 hours of darkness per day. Light was provided by overhead 40-W fluorescent tubes, and illumination was between 50 and 300 lux, depending on cage position in the animal room. Animals had free access to food and water. On the day of the experiment, animals were divided into 2 groups. The control group was fed ad libitum as previously, the light was not turned on at the time of usual dark-to-light transition, designated as circadian time 0 (CT0), and the animals were released into constant darkness. Starting from the CT0 (or from CT4), 3 animals per each time point were sampled every 4 (occasionally every 2) hours throughout the whole circadian cycle. The experimental group was subjected to a restricted FR, and access to food was permitted only for 6 hours during the light period (ie, between CT3 and CT9). Access to drinking water was not limited. The food restriction began with the removal of food pellets at CT9 and continued for the next 14 days. Thereafter, the animals were released into constant darkness and sampled as described in the control group. Food was not provided on the day of sampling. Rats of both groups were killed after deep anesthesia (thiopental 50 mg/kg intraperitoneally) by decapitation. All experiments were conducted under license no. A5228-01 with the US National Institutes of Health and in accordance with Animal Protection Law of the Czech Republic (license no. 42084/2003-1020). For in situ hybridization, the brains and transversal sections of the distal colon were removed, colons were rinsed in phosphate buffer, and both tissues were immediately frozen on dry ice and stored at −80°C. For RNA isolation, roughly 4-mm–thick pieces of liver tissue were dissected and immediately placed into RNAlater stabilization reagent (Qiagen, Valencia, CA). Samples of the distal part of the colon, just above the pelvic brim, were rinsed with phosphate-buffered saline and cut longitudinally. Gentle scraping of the mucosal layer yielded material rich in epithelial cells (denoted as epithelial tissue), which was immersed into RNAlater. Samples in RNAlater were stored at 4°C (for not longer than 1 week) before isolation of total RNA and subsequent real-time RT-PCR. Each brain was cut in 12-μm–thick sections throughout the whole rostro-caudal extent of the SCN and processed for ISH to determine levels of Per1, Per2 and Bmal1 gene mRNAs. Similarly, 12-μm–thick transversal slices of the colon were processed for ISH to determine levels of Per1, Per2, Cry1, and Bmal1 mRNAs. The method of ISH as well as the cDNA fragments of rat Per1, Per2, and Bmal1 and mouse Cry1 were described previously.38Shearman L.P. Jin X. Lee C. Reppert S.M. Weaver D.R. Targeted disruption of the mPer3 gene: subtle effects on circadian clock function.Mol Cell Biol. 2000; 20: 6269-6625Crossref PubMed Scopus (257) Google Scholar, 39Sumova A. Jac M. Sladek M. Sauman I. Illnerova H. Clock gene daily profiles and their phase relationship in the rat suprachiasmatic nucleus are affected by photoperiod.J Biol Rhythms. 2003; 18: 134-144Crossref PubMed Scopus (95) Google Scholar All sections hybridized with the same probe were processed simultaneously under identical conditions. Relative optical density (OD) of the specific hybridization signal was analyzed with the use of an image analysis system (Image Pro; Olympus, New Hyde Park, NY). The SCN slides were counterstained with cresyl violet, and the mRNA was quantified at the midcaudal SCN section. Each measurement was corrected for nonspecific background by subtracting the OD values from the same adjacent area in the hypothalamus. In the colon, alternating sections were counterstained with methylene blue, and the signal was measured selectively in the area corresponding to the colonic epithelium. The surrounding background staining in submucosa served as an internal standard value and was subtracted from the OD value of the epithelium. Coronal sections (12-μm thick) of rat colon were cut, mounted on slides, fixed in 4% paraformaldehyde in phosphate-buffered saline, and processed for immunohistochemistry by using the standard avidin-biotin method with diaminobenzidine as the chromogen (Vector Laboratories, Peterborough, United Kingdom) as described elsewhere.40Sumova A. Sladek M. Jac M. Illnerova H. The circadian rhythm of Per1 gene product in the rat suprachiasmatic nucleus and its modulation by seasonal changes in daylength.Brain Res. 2002; 947: 260-270Crossref PubMed Scopus (47) Google Scholar The PER1 polyclonal primary antiserum was synthesized at the Massachusetts General Hospital Biopolymer Core Facility. It was generated against the amino acids 6–21 of the peptide sequence of mPER1 and characterized elsewhere.41Hastings M.H. Field M.D. Maywood E.S. Weaver D.R. Reppert S.M. Differential regulation of mPER1 and mTIM proteins in the mouse suprachiasmatic nuclei: new insights into a core clock mechanism.J Neurosci. 1999; 19: RC11PubMed Google Scholar The BMAL1 antibody was raised against the C-terminal 15 residues of mBMAL1 (GLGGPVDFSDLPWPL) using the Sigma-Aldrich (St Louis, MO) custom peptide antibody service and was characterized previously.29Reddy A.B. Karp N.A. Maywood E.S. Sage E.A. Deery M. O’Neill J.S. Wong G.K. Chesham J. Odell M. Lilley K.S. Kyriacou C.P. Hastings M.H. Circadian orchestration of the hepatic proteome.Curr Biol. 2006; 16: 1107-1115Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar As controls for background staining, parallel sections were treated simultaneously through the immunohistochemical procedure without incubation with the specific primary antibody. Total RNA was isolated from 20 to 50 mg homogenized liver tissue and from colon epithelial tissue by means of Trizol-based reagent (RNABlue; Top-Bio, Prague, Czech Republic) according to the manufacturer’s instructions. RNA concentrations were determined by spectrophotometry at 260 nm, and RNA quality was assessed by electrophoresis on 1.5% agarose gel. Moreover, the integrity of randomly selected samples of total RNA was tested by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The RT-PCR method used to detect the clock genes as well as the sequences of the primers for Per1, Per2, Rev-erbα, Bmal1, Clock, Cry1, and β2-microglobulin were described previously.42Sladek M. Jindrakova Z. Bendova Z. Sumova A. Postnatal ontogenesis of the circadian clock within the rat liver.Am J Physiol Regul Integr Comp Physiol. 2007; 292: R1224-R1229Crossref PubMed Scopus (85) Google Scholar Total RNA (0.5 μg) was reverse transcribed to cDNA using the Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) with oligo(dT) 12–18 primers (Roche Diagnostic, Lewes, United Kingdom) according to the manufacturer’s instructions. Then, 20 μL of the cDNA reaction was diluted 10 times in diethylpyrocarbonate-treated water to eliminate inhibition of Taq polymerase. Diluted cDNA (1 μL) was amplified in 10-μL PCR reaction containing commercial SYBR Green and Hot Start Taq polymerase master mix (LC-Fast Start DNA Master SYBR Green I; Roche Diagnostics) and specific primers with the following sequences: β-actin (NM_031144) forward, 5′-TACAACCTCCTTGCAGCTCC-3′, and reverse, 5′-TTCTGACCCATGCCCACCA-3′; NHE3 (M.85300.1) forward, 5′-TGCGCTACACTA TGAAGATGC-3′, and reverse, 5′-AGGCCCTGAAAGATGACTGTGA-3′. Both primers were designed in our laboratory and were intron-spanning. The concentration of primers was 0.5 μmol/L, whereas the MgCl2 concentration was 3 mmol/L for NHE3, and 4 mmol/L for β-actin. Real-time PCR reactions were performed on a LightCycler system (Roche Diagnostic). The protocol for β-actin was as follows: 10-minute activation of polymerase followed by 30 cycles amplification of target cDNA (95°C for 15 seconds, 55°C for 10 seconds, and 72°C for 8 seconds). Amplification of NHE3 was performed using a different protocol: 10-minute activation of polymerase followed by 38 cycles amplification of target cDNA (95°C for 15 seconds, 55°C for 10 seconds, and 72°C for 14 seconds). At the end of each run, melting curve analysis was performed to ascertain the presence of a single amplicon. Standard curves were generated for each PCR run from single time-point cDNAs (CT 24) by 3-fold serial dilution. Quantification was achieved by using the LightCycler software version 3.5 and performing second derivative maximum analysis. The expression of NHE3 was normalized to the expression of β-actin. Levels of β-actin did not vary significantly as a function of time. Non-template (without cDNA) and no-RT (30 ng of total RNA instead of cDNA) control reactions were performed in each PCR run, and the identity of all PCR products was verified by sequencing. Daily profiles of mRNAs in the colon, liver, and SCN were analyzed by one-way analysis of variance (ANOVA) for time differences. The effect of restricted feeding on the mRNA profiles in the colon and the SCN was analyzed by 2-way ANOVA for FR and time differences. Subsequently, the Student-Newman-Keuls multiple range test was used, with P < .05 being required for significance. Data are expressed as means of 3 animals ± standard error of the mean (SEM) per time point. Rhythms were considered to be present when the one-way ANOVA revealed a significant effect of time and at the same time the maximum and minimum values were clustered into 2 separate time intervals. In the rat colon (Figure 1, colon), the one-way ANOVA revealed a significant effect of time on expression of Per1, Per2, Cry1 (P < .05), Rev-erbα, and Bmal1 (P < .01) but not on expression of Clock. Per1 mRNA level at CT12 was significantly higher than those at CT8, CT20, and CT24 (P < .05); the levels thus rose between CT8 and CT12 and declined between CT12 and CT20. Per2 mRNA level at CT12 and CT16