Caffeine alters circadian rhythms and expression of disease and metabolic markers

The circadian clock regulates many aspects of physiology, energy metabolism, and sleep. Restricted feeding (RF), a regimen that restricts the duration of food availability entrains the circadian clock. Caffeine has been shown to affect both metabolism and sleep. However, its effect on clock gene and clock-controlled gene expression has not been studied. Here, we tested the effect of caffeine on circadian rhythms and the expression of disease and metabolic markers in the serum, liver, and jejunum of mice supplemented with caffeine under ad libitum (AL) feeding or RF for 16 weeks. Caffeine significantly affected circadian oscillation and the daily levels of disease and metabolic markers. Under AL, caffeine reduced the average daily mRNA levels of certain disease and inflammatory markers, such as liver alpha fetoprotein (Afp), C-reactive protein (Crp), jejunum alanine aminotransferase (Alt), growth arrest and DNA damage 45β (Gadd45β), Interleukin 1α (Il-1α), Il-1β mRNA and serum plasminogen activator inhibitor 1 (PAI-1). Under RF, caffeine reduced the average daily levels of Alt, Gadd45β, Il-1α and Il-1β mRNA in the jejunum, but not in the liver. In addition, caffeine supplementation led to decreased expression of catabolic factors under RF. In conclusion, caffeine affects circadian gene expression and metabolism possibly leading to beneficial effects mainly under AL feeding.     

All-trans retinoic acid modifies the expression of clock and disease marker genes

Restricted feeding (RF), a regimen that restricts the duration of food availability with no calorie restriction, entrains the circadian clock in peripheral tissues. Restricted feeding leads to high-amplitude circadian rhythms, which have been shown to promote wellness and reduce disease and inflammatory markers. Retinoids, such as all-trans retinoic acid (ATRA), act as anti-inflammatory agents. Thus far, the effect of ATRA combined with RF on the ability to delay the occurrence of age-associated changes, such as cancer and inflammation, is not known. We measured circadian expression of clock genes, disease marker genes and inflammatory markers in the serum, liver and jejunum in mice fed ad libitum (AL) or RF supplemented with 15 or 250 μg/kg body/day ATRA for 16 weeks. Our results show that ATRA supplementation led to phase shifts and reduced amplitudes in clock genes. Under AL, ATRA reduced the average daily messenger RNA (mRNA) levels of some disease markers, such as liver Afp and jejunum Afp, Alt and Gadd45β and aspartate transaminase (AST) protein in the serum, but increased the expression level of liver Crp mRNA. Under RF, ATRA reduced the average daily levels of jejunum Alt and Gadd45β and AST protein in the serum, but increased liver Afp, Alt, Gadd45β and Arginase mRNA. Altogether, our findings suggest that ATRA strongly affects circadian oscillation and disease marker levels. Moreover, its impact is different depending on the feeding regimen (AL or RF).     

Peripheral circadian clocks are diversely affected by adrenalectomy

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

The Tilapia collagen peptide mixture TY001 protects against LPS-induced inflammation,disruption of glucose metabolism,and aberrant expression of circadian clock genes in mice

The Tilapia collagen peptide mixture TY001 has been shown to accelerate wound healing in streptozotocin-induced diabetic mice and to protect against streptozotocin-induced inflammation and elevation in blood glucose. The goals of the present study are to further study TY001 effects on lipopolysaccharide (LPS)-induced inflammation and metabolic syndrome. LPS is known to disrupt circadian clock to produce toxic effects, the effects of TY001 on rhythmic alterations of serum cytokines and hepatic clock gene expressions were examined. Mice were given TY001 (30 g/L, ≈ 40 g/kg) through the drinking water for 30 days, and on the 21^st day of TY001 supplementation, LPS (0.25 mg/kg, ip, daily) was given for 9 days to establish the inflammation model. Repeated LPS injections produced inflammation, impaired glucose metabolism, and suppressed the expression of circadian clock core genes Bmal1 and Clock; clock feedback gene Cry1, Cry2, Per1, and Per2; clock target gene Rev-erbα and RORα. TY001 prevented LPS-induced elevations of TNFα, IL-1β, IL-6, and IL-10 in the liver, along with improved histopathology. TY001 reduced LPS-elevated fasting blood glucose and increased LPS-reduced serum insulin levels, probably via increased glucose transporter GLUT2, enhanced insulin signaling p-Akt and p-IRS-1^Try612. Importantly, LPS-induced circadian elevations of serum TNFα and IL-1β and aberrant expression of circadian clock genes in the liver were ameliorated by TY001. Immunohistochemistry revealed that the LPS decreased Bmal1 and Clock protein in the liver, which was recovered by TY001. Taken together, TY001 is effective against LPS-induced inflammation, disruption of glucose metabolism and disruption of circadian clock gene expressions.

Abbreviations: TY001: Tilapia collagen peptide mixture; LPS: Lipopolysaccharide; TNFα: Tumor necrosis factor-α; IL-1β: Interleukin-1β; GLUT2: Glucose transporter 2     

Influence of light and food on the circadian clock in liver of rainbow trout,Oncorhynchus mykiss

Several reports support the existence of multiple peripheral oscillators in fish, which may be able to modulate the rhythmic functions developed by those tissues hosting them. Thus, a circadian oscillator has been proposed to be located within fish liver. In this vertebrate group, the role played by the circadian system in regulating metabolic processes in liver is mostly unknown. We, therefore investigated the liver of rainbow trout (Oncorhynchus mykiss) as a potential element participating in the regulation of circadian rhythms in fish by hosting a functional circadian oscillator. The presence and expression pattern of main components of the circadian molecular machinery (clock1a, bmal1, per1 and rev-erbβ-like) were assessed. Furthermore, the role of environmental cues such as light and food, and their interaction in order to modulate the circadian oscillator was also assessed by exposing animals to constant conditions (absence of light for 48 h, and/or a 4 days fasting period). Our results demonstrate the existence of a functional circadian oscillator within trout liver, as demonstrated by significant rhythms of all clock genes assessed, independently of the environmental conditions studied. In addition, the daily profile of mRNA abundance of clock genes is influenced by both light (mainly clock1a and per1) and food (rev-erbβ-like), which is indicative of an interaction between both synchronizers. Our results point to rev-erbβ-like as possible mediator between the influence of light and food on the circadian oscillator within trout liver, since its daily profile is influenced by both light and food, thus affecting that of bmal1.     

Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV‐2 Microglial Cells

Naegleria fowleri, a free‐living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV‐2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV‐2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL‐1α and TNF‐α. NfESP‐induced IL‐1α and TNF‐α expression in BV‐2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP‐induced IL‐1α and TNF‐α production in BV‐2 cells were effectively downregulated by inhibition of NF‐kB and AP‐1. These results collectively suggest that NfESP stimulates BV‐2 cells to release IL‐1α and TNF‐α via NF‐kB‐ and AP‐1‐dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection.     

Effect of peroxisome proliferator-activated receptor-alpha ligands in the interaction between adipocytes and macrophages in obese adipose tissue

Objective: This study was designed to examine the effect of peroxisome proliferator‐activated receptor‐α (PPAR‐α) ligands on the inflammatory changes induced by the interaction between adipocytes and macrophages in obese adipose tissue. Methods and Procedures: PPAR‐α ligands (Wy‐14,643 and fenofibrate) were added to 3T3‐L1 adipocytes, RAW264 macrophages, or co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages in vitro, and monocyte chemoattractant protein‐1 (MCP‐1) and tumor necrosis factor‐α (TNF‐α) mRNA expression and secretion were examined. PPAR‐α ligands were administered to genetically obese ob/ob mice for 2 weeks. Moreover, the effect of PPAR‐α ligands was also evaluated in the adipose tissue explants and peritoneal macrophages obtained from PPAR‐α‐deficient mice. Results: In the co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages, PPAR‐α ligands reduced MCP‐1 and TNF‐α mRNA expression and secretion in vitro relative to vehicle‐treated group. The anti‐inflammatory effect of Wy‐14,643 was observed in adipocytes treated with macrophage‐conditioned media or mouse recombinant TNF‐α and in macrophages treated with adipocyte‐conditioned media or palmitate. Systemic administration of PPAR‐α ligands inhibited the inflammatory changes in adipose tissue from ob/ob mice. Wy‐14,643 also exerted an anti‐inflammatory effect in the adipose tissue explants but not in peritoneal macrophages obtained from PPAR‐α‐deficient mice. Discussion: This study provides evidence for the anti‐inflammatory effect of PPAR‐α ligands in the interaction between adipocytes and macrophages in obese adipose tissue, thereby improving the dysregulation of adipocytokine production and obesity‐related metabolic syndrome.     

Testing the potency of anti‐TNF‐α and anti‐IL‐1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor‐matched chondrogenically differentiated mesenchymal stem cells

Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor‐alpha (TNF‐α) and interleukin 1β (IL‐1β). New in vitro testing systems are needed to evaluate efficacies of new anti‐inflammatory biological drugs, ideally in a patient‐specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti‐inflammatory drugs, spheroids were exposed to TNF‐α, IL‐1β, or to supernatant containing secretome from activated macrophages (MCM). The anti‐inflammatory efficacies of anti‐TNF‐α biologicals adalimumab, infliximab, and etanercept, and the anti‐IL‐1β agent anakinra were assessed in short‐term microspheroid and long‐term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF‐α or IL‐1β. The differences in potency of anti‐TNF‐α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short‐term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti‐TNF‐α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045–1058, 2018     

Monocyte chemoattractant protein-1 in obesity and type 2 diabetes. Insulin sensitivity study

Objective: Our goal was to test any association between human plasma circulating levels of monocyte chemoattractant protein‐1 (cMCP‐1) and insulin resistance and to compare monocyte chemoattractant protein‐1 (MCP‐1) adipose tissue gene expression and cMCP‐1 in relation with inflammatory markers. Research Methods and Procedures: cMCP‐1 was measured in n = 116 consecutive control male subjects to whom an insulin sensitivity (S_i) test was performed. Circulating levels of soluble CD14, soluble tumor necrosis factor receptor type 2 (sTNFR2), soluble interleukin‐6 (sIL‐6), and adiponectin also were measured. Subcutaneous adipose tissue samples were obtained from n = 107 non‐diabetic and type 2 diabetic subjects with different degrees of obesity. Real‐time polymerase chain reaction was used to measure gene expression of MCP‐1, CD68, tumor necrosis factor‐α (TNF‐α), and its receptor TNFR2. Results: In the S_i study, no independent effect of cMCP‐1 levels on insulin sensitivity was observed. In the expression study, in non‐diabetic subjects, MCP‐1 mRNA had a positive correlation with BMI (r = 0.407, p = 0.003), TNF‐α mRNA (r = 0.419, p = 0.002), and TNFR2 mRNA (r = 0.410, p = 0.003). In these subjects, cMCP‐1 was found to correlate with waist‐to‐hip ratio (r = 0.322, p = 0.048). In patients with type 2 diabetes, MCP‐1 mRNA was up‐regulated compared with non‐diabetic subjects. TNF‐α mRNA was found to independently contribute to MCP‐1 mRNA expression. In this group, CD68 mRNA was found to correlate with BMI (r = 0.455, p = 0.001). Discussion: cMCP‐1 is not associated with insulin sensitivity in apparently healthy men. TNF‐α is the inflammatory cytokine associated with MCP‐1 expression in subcutaneous adipose tissue.     

Interaction of MAGED1 with nuclear receptors affects circadian clock function

The circadian clock has a central role in physiological adaption and anticipation of day/night changes. In a genetic screen for novel regulators of circadian rhythms, we found that mice lacking MAGED1 (Melanoma Antigen Family D1) exhibit a shortened period and altered rest–activity bouts. These circadian phenotypes are proposed to be caused by a direct effect on the core molecular clock network that reduces the robustness of the circadian clock. We provide in vitro and in vivo evidence indicating that MAGED1 binds to RORα to bring about positive and negative effects on core clock genes of Bmal1, Rev‐erbα and E4bp4 expression through the Rev‐Erbα/ROR responsive elements (RORE). Maged1 is a non‐rhythmic gene that, by binding RORα in non‐circadian way, enhances rhythmic input and buffers the circadian system from irrelevant, perturbing stimuli or noise. We have thus identified and defined a novel circadian regulator, Maged1, which is indispensable for the robustness of the circadian clock to better serve the organism.     

Effect of insoluble fibre on intestinal morphology and mRNA expression pattern of inflammatory,cell cycle and growth marker genes in a piglet model

The effects of insoluble dietary fibre differing in lignin content on intestinal morphology and mRNA expression was tested in an animal model of 48 weaned piglets. Engaged fibre sources were wheat bran (rich in cellulose and hemicellulose) and pollen from Chinese Masson pine (Pinus massoniana) (rich in lignin), respectively. The fibre sources were added to a basal diet as follows: no addition (control), 3.0% wheat bran, 1.27% pine pollen, and 2.55% pine pollen. The 12 animals of each feeding group were fed four experimental diets ad libitum for 37 days and were then slaughtered for retrieving tissue samples from stomach, jejunum, ileum, colon and mesenterial lymph nodes. Both fibre sources increased villus height of mucosa in jejunum (+10% on average) and ileum (+16% on average). Results of mRNA expression rates of inflammatory, cell cycle and growth marker genes (NFκB, TNFα, TGFβ, Caspase3, CDK4, IGF1) were specific to fibre source and tissue: wheat bran induced an up-regulation of NFκB in stomach and jejunum, as well as TNFα and TGFβ, and Caspase3 in jejunum. Pine pollen induced down regulation of NFκB, TNFα, TGFβ, Caspase3, CDK4 and IGF1 in the colon as well as up-regulation of NFκB and TGFβ in mesenterial lymph nodes. Finally, an overall data comparison based on a hierarchical cluster analysis showed a close relation between gene regulation in different gut sections and organs, as well as between small intestine morphology and zootechnical performance.     

Time-imposed daily restricted feeding induces rhythmic expression of Fgf21 in white adipose tissue of mice

Fibroblast growth factor 21 (FGF21) is a key metabolic regulator that is induced by fasting and starvation, and its expression is thought to be regulated by the circadian clock in the liver. To evaluate the functional role of FGF21 in the circadian regulation of physiology and behavior, we examined the temporal expression profiles of Fgf21 and circadian clock genes in addition to behavioral activity rhythms under adlibitum feeding (ALF) and time-imposed restricted feeding (RF) in mice. Four hours of daily restricted feeding during the daytime induced over an 80-fold increase in feeding-dependent rhythmic Fgf21 mRNA expression in epididymal white adipose tissue (eWAT), although the expression levels were continuously increased 10-fold in the liver of wild-type (WT) mice. Refeeding subsequent to transient fasting revealed that refeeding but not fasting remarkably induces Fgf21 expression in eWAT, although fasting-induced hepatic Fgf21 expression is completely reversed by refeeding. The free-running period of locomotor activity rhythm under ALF and the food anticipatory activity (FAA) under RF remained intact in Fgf21 knockout (KO) mice, suggesting that FGF21 is dispensable for both the central clock in the suprachiasmatic nucleus (SCN) and the food-entrainable oscillator that governs the FAA. Temporal expression profiles of circadian genes such as mPer2 and BMAL1 were essentially identical in both tissues between WT and Fgf21 KO mice under RF. The physiological role of the refeeding-induced adipose Fgf21 expression remains to be elucidated.     

Upregulation of uncoupling protein 2 mRNA in genetic obesity: lack of an essential role for leptin,hyperphagia, increased tissue lipid content,and TNF-α

Uncoupling protein 2 (UCP2) has been proposed to play a prominent role in the regulation of energy balance. UCP2 mRNA expression is upregulated in white adipose tissue (WAT) and liver, but is not altered in skeletal muscle in genetically obese ob/ob mice. The mechanisms involved in the upregulation of UCP2 in obesity have not been investigated. We have now examined the potential role of leptin, hyperphagia, increased tissue lipid content, and overexpression of tumor necrosis factor (TNF)-α in the upregulation of UCP2 mRNA expression in the liver and WAT in ob/ob mice. Treatment of ob/ob mice with leptin for 3 days significantly reduced their food intake but had no effect on the upregulation of UCP2 mRNA levels in the liver or WAT. To investigate the effect of feeding and higher tissue lipid content on the upregulation of UCP2 in liver and WAT, we compared UCP2 mRNA levels in ad-libitum fed and 72-h fasted control and ob/ob mice. In controls, fasting had no effect on UCP2 mRNA levels in liver, but increased UCP2 mRNA in WAT suggesting that the effects of fasting on UCP2 mRNA levels are tissue-specific. In ob/ob mice, fasting did not lower UCP2 mRNA levels in liver or WAT suggesting that the upregulation of UCP2 in ob/ob mice is not merely a direct consequence of increased food intake. 72-h fasting lowered hepatic total lipid content by 34% and 36% in control and ob/ob mice, respectively, without any corresponding decrease in hepatic UCP2 mRNA levels, suggesting that the enhanced UCP2 expression in the liver of ob/ob mice is not secondary to lipid accumulation in their livers. Although TNF-α has been shown to acutely increase UCP2 mRNA levels in liver and WAT, and is overexpressed in adipose tissue in obesity, deletion of the genes for both TNF receptors in ob/ob mice produces a further increase in UCP2 mRNA expression in liver and adipose tissue indicating a paradoxical inhibitory role. Taken together, these results suggest that the upregulation of UCP2 mRNA levels in the liver and WAT of ob/ob mice is not due to the lack of leptin, hyperphagia, increased tissue lipid content, or over-expression of TNF-α.     

Expression of Clock Genes in Human Peripheral Blood Mononuclear Cells throughout the Sleep/Wake and Circadian Cycles

The rhythmic expression of circadian clock genes in the neurons of the suprachiasmatic nucleus (SCN) underlies the manifestation of endogenous circadian rhythmicity in behavior and physiology. Recent evidence demonstrating rhythmic clock gene expression in non‐SCN tissues suggests that functional clocks exist outside the central circadian pacemaker of the brain. In this investigation, the nature of an oscillator in peripheral blood mononuclear cells (PBMCs) is evaluated by assessing clock gene expression throughout both a typical sleep/wake cycle (LD) and during a constant routine (CR). Six healthy men and women aged (mean±SEM) 23.7±1.6 yrs participated in this five‐day investigation in temporal isolation. Core body temperature and plasma melatonin concentration were measured as markers of the central circadian pacemaker. The expression of HPER1, HPER2, and HBMAL1 was quantified in PBMCs sampled throughout an uninterrupted 72 h period. The core body temperature minimum and the midpoint of melatonin concentration measured during the CR occurred 2:17±0:20 and 3:24 ±0:09 h before habitual awakening, respectively, and were well aligned to the sleep/wake cycle. HPER1 and HPER2 expression in PBMCs demonstrated significant circadian rhythmicity that peaked early after wake‐time and was comparable under LD and CR conditions. HBMAL1 expression was more variable, and peaked in the middle of the wake period under LD conditions and during the habitual sleep period under CR conditions. For the first time, bi‐hourly sampling over three consecutive days is used to compare clock gene expression in a human peripheral oscillator under different sleep/wake conditions.     

Relationship between FGF21 and UCP1 levels under time-restricted feeding and high-fat diet

Fibroblast growth factor 21 (FGF21) exhibits a circadian oscillation, and its induction is critical during fasting. When secreted by liver and skeletal muscle, FGF21 enhances thermogenic activity in brown adipose tissue (BAT) by utilizing uncoupling protein 1 (UCP1) to dissipate energy as heat. Recently, it has been reported that UCP1 is not required for FGF21-mediated reduction in body weight or improvements in glucose homeostasis. As the relationship between FGF21 and UCP1 induction in tissues other than BAT is less clear, we tested the effect of restricted feeding (RF) and high dietary fat on FGF21 circadian expression and its correlation with UCP1 expression in liver and white adipose tissue (WAT). High dietary fat disrupted Fgf21 mRNA circadian oscillation but increased its levels in WAT. RF led to increased liver FGF21 protein levels, whereas those of UCP1 decreased. In contrast, WAT FGF21 protein levels increased under high-fat diet, whereas those of UCP1 decreased under RF. In summary, FGF21 exhibits circadian oscillation, which is disrupted with increased dietary fat. The relationship between FGF21 and UCP1 levels depends on the tissue and the cellular energy status.     

Human Intestinal Circadian Clock: Expression of Clock Genes in Colonocytes Lining the Crypt

Biological clock components have been detected in many epithelial tissues of the digestive tract of mammals (oral mucosa, pancreas, and liver), suggesting the existence of peripheral circadian clocks that may be entrainable by food. Our aim was to investigate the expression of main peripheral clock genes in colonocytes of healthy humans and in human colon carcinoma cell lines. The presence of clock components was investigated in single intact colonic crypts isolated by chelation from the biopsies of 25 patients (free of any sign of colonic lesions) undergoing routine colonoscopy and in cell lines of human colon carcinoma (Caco2 and HT29 clone 19A). Per‐1, per‐2, and clock mRNA were detected by real‐time RT‐PCR. The three‐dimensional distributions of PER‐1, PER‐2, CLOCK, and BMAL1 proteins were recorded along colonic crypts by immunofluorescent confocal imaging. We demonstrate the presence of per‐1, per‐2, and clock mRNA in samples prepared from colonic crypts of 5 patients and in all cell lines. We also demonstrate the presence of two circadian clock proteins, PER‐1 and CLOCK, in human colonocytes on crypts isolated from 20 patients (15 patients for PER‐1 and 6 for CLOCK) and in colon carcinoma cells. Establishing the presence of clock proteins in human colonic crypts is the first step toward the study of the regulation of the intestinal circadian clock by nutrients and feeding rhythms.