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Introduction Bronchial asthma is a chronic respiratory disease characterized by airway inflammation, allergen-induced hypersensitivity and dyspnea. Most asthmatic patients demonstrate oscillations of disease symptoms within 24 hours regulated by circadian clock genes. We hypothesized that these genes may be regulators of childhood asthma risk. Objectives The aim was to investigate whether single-nucleotide polymorphisms (SNPs) in the circadian clock genes are associated with childhood asthma risk. We also aimed to analyze the mRNA level of clock genes in the blood of asthmatic children and NHBE cells stimulated with IL-13. Materials and methods Peripheral blood was collected from 165 asthmatic and 138 healthy Polish children. NHBE cells were culture at the air-liquid interface (ALI) with IL-13 as an in vitro model of allergic inflammation. Using TaqMan probes, we genotyped 32 SNPs in: CLOCK, BMAL1, PER3 and TIMELESS. Expression analysis for TIMELESS was performed using real-time PCR with SYBR Green. For haplotype and genotype statistical analysis we used Haploview 4.2 and STATISTICA version 12, respectively. Gene expression analysis was performed in DataAssist v3.01. Results We found that three polymorphisms in TIMELESS (rs2291739, rs10876890, rs11171856) and two haplotypes (TTTT and CTAC) were associated with asthma risk. We also found significantly decreased expression of TIMELESS in the blood of asthmatic children as compared to the healthy children (P = 0.0289) and in NHBE cells stimulated with IL-13 (P = 0.0302). Conclusions In our study, we showed for the first time that TIMELESS variants and expression may be associated with childhood asthma.

Occupational stress is associated with sleep quality among workers and the human variable number tandem repeat (VNTR) polymorphism of the period circadian regulator 3 (PER3) gene relates to sleep-wake regulation. The main aims of the present study were to examine the effects of PER3 VNTR genotypes, occupational stress, and their interactions on sleep quality. A cross-sectional study was conducted and 729 workers were recruited in Sichuan. Sleep quality were assessed using the Pittsburgh Sleep Quality Index. Occupational stress was measured using the Generic Job Stress Questionnaire. PER3 genotypes were determined with polymerase chain reaction. High and medium occupational stress were linked to a higher risk of poor sleep quality than low levels. Unconditional logistic regression indicated that PER3 genotype was significantly associated with sleep quality, and an increased risk of poor sleep of >1.5-times was observed in those with the allele 5 compared to allele 4. The 5/5 genotype was associated with both sleep latency and sleep duration. Crossover analysis showed an occupational stress × PER3 interaction. Compared to subjects with both low and medium occupational stress and 4/4 + 4/5 genotype, those with both high occupational stress and 5/5 genotype had a higher risk of poor sleep quality. Stratified logistic analyses found that compared with low and medium occupational stress, high occupational stress increased the risk of poor sleep by more than five-times in 5/5 genotype carriers. Occupational stress and PER3 genotype had both separate and combined effects on poor sleep quality of workers. The results suggest that occupational stress may increase the risk of poor sleep quality through interaction with the PER3 gene polymorphism.

Previous studies have shown that mouse Period3 (mPer3) is dispensable for the generation of autonomous oscillations in the circadian clock. However, human studies have suggested that human Period3 (hPer3) may have more important roles in the core clock machinery than mPer3. To investigate the role of hPer3 protein in the cell-autonomous circadian oscillator, we conducted gene knockout of the hPer3 gene in human bone osteosarcoma epithelial cells using genome-editing technology. We examined the circadian transcription of endogenous clock genes in hPer3-deficient cell clones and found that hPer3-deficient cells showed a phase advance in circadian transcription compared to wild-type cells. We subsequently transfected wild-type and mutant cells with an adenovirus carrying a luciferase gene whose expression was driven by a clock gene promotor, and monitored bioluminescence in real time. Cosinor analysis showed that the circadian period length in all hPer3-deficient cells was significantly shorter than that in wild-type cells, demonstrating that the phase advance in endogenous clock gene expression in hPer3-deficient cell clones was attributable to a shortened circadian period length rather than a phase shift. Together these findings are consistent with previous studies in mice lacking functional mPer3, indicating that the Per3 protein functions similarly in both mice and humans.

It is well known that circadian clocks are mainly regulated by light targeting signaling pathways in the hypothalamic suprachiasmatic nucleus. However, an entrainment mediated by non-photic sensory stimuli was also suggested for peripheral clocks. Exposure to extremely low frequency (ELF) electromagnetic fields might affect circadian rhythmicity. The goal of this research was to investigate effects of ELF magnetic fields (ELF-MF) on circadian clock genes in a human fibroblast cell line. We found that an ELF-MF (0.1 mT, 50 Hz) exposure was capable of entraining expression of clock genes BMAL1, PER2, PER3, CRY1, and CRY2. Moreover, ELF-MF treatment induced an alteration in circadian clock gene expression previously entrained by serum shock stimulation. These results support the hypothesis that ELF-MF may be able to drive circadian physiologic processes by modulating peripheral clock gene expression. Bioelectromagnetics. 36:294–301, 2015. © 2015 Wiley Periodicals, Inc.

New Findings What is the central question of this study? Disrupting circadian rhythms in rodents perturbs glucose metabolism and increases adiposity. In this study, we asked whether circadian rhythm disruption, induced by exposure of sheep to rapidly alternating photoperiods (RAPs), also disrupts metabolic homeostasis in a large diurnal animal model. What is the main finding and its importance? Exposure to RAPs disrupted central (melatonin and core body temperature) and peripheral rhythmicity (skeletal muscle clock gene expression). This led to reduced nocturnal plasma glucose concentrations, but did not affect glucose tolerance and glucose-stimulated insulin secretion. These results suggest that RAP-induced circadian rhythm disruption has minimal effect on glucose homeostasis in the sheep. Disrupting circadian rhythms in rodents perturbs glucose metabolism and increases adiposity. To determine whether these effects occur in a large diurnal animal, we assessed the impact of circadian rhythm disruption upon metabolic function in sheep. Adult ewes (n = 7) underwent 3 weeks of a control 12 h light–12 h dark photoperiod, followed by 4 weeks of rapidly alternating photoperiods (RAPs) whereby the time of light exposure was reversed twice each week. Measures of central (melatonin secretion and core body temperature) and peripheral rhythmicity (clock and metabolic gene expression in skeletal muscle) were obtained over 24 h in both conditions. Metabolic homeostasis was assessed by glucose tolerance tests and 24 h glucose and insulin profiles. Melatonin and core body temperature rhythms resynchronized within 2 days of the last photoperiod shift. High-amplitude Bmal1, Clock, Nr1d1, Cry2 and Per3 mRNA rhythms were apparent in skeletal muscle, which were phase advanced by up to 3.5 h at 2 days after the last phase shift, whereas Per1 expression was downregulated at this time. Pparα, Pgc1α and Nampt mRNA were constitutively expressed in both conditions. Nocturnal glucose concentrations were reduced following chronic phase shifts (zeitgeber time 0, −5.5%; zeitgeber time 12, −2.9%; and zeitgeber time 16, −5.7%), whereas plasma insulin, glucose tolerance and glucose-stimulated insulin secretion were not altered. These results demonstrate that clock gene expression within ovine skeletal muscle oscillates over 24 h and responds to changing photoperiods. However, metabolic genes which link circadian and metabolic clocks in rodents were arrhythmic in sheep. Differences may be due to the ruminant versus monogastric digestive organization in each species. Together, these results demonstrate that despite disruptions to central and peripheral rhythmicity following exposure to rapidly alternating photoperiods, there was minimal impact on glucose homeostasis in the sheep.

Objectives The therapeutic action of lithium in bipolar mood disorder may be connected with its effect on biological rhythms. In the present study, an attempt was made to investigate an association between multiple single nucleotide polymorphisms (SNPs) and their haplotypes pertaining to four genes involved in regulation of biological rhythms [circadian locomotor output cycle kaput (CLOCK), aryl hydrocarbon receptor nuclear translocator-like (ARNTL), timeless circadian clock (TIMELESS), period circadian clock 3 (PER 3)], and the efficacy of lithium prophylaxis. Methods The study was performed on 115 patients with bipolar mood disorder (45 males, 70 females) with a mean age of 52 ± 12 years, with lithium prophylaxis for 22 ± 8 years, recruited from the outpatients in the Department of Psychiatry, Poznan University of Medical Sciences. The assessment of the lithium prophylactic response was made retrospectively using the Alda scale. Genotyping was done for nine SNPs of the CLOCK gene, 18 SNPs of the ARNTL gene, six SNPs of the timeless circadian clock (TIM) gene, and nine SNPs of the PER3 gene. Results An association with the degree of lithium prophylaxis was found for six SNPs and three haplotype blocks of the ARNTL gene, and two SNPs and one haplotype block of the TIM gene. No association with SNPs or haplotypes of the CLOCK and PER3 genes was observed. Conclusions The results suggest that the ARNTL and TIM genes may be associated with the lithium prophylactic response in bipolar illness. This association may be related to the role of these genes in the predisposition to bipolar mood disorder. Of special interest may be polymorphisms of these genes involved both in the predisposition to bipolar mood disorder and the lithium response.

We compared the period of the rhythm of plasma melatonin, driven by the hypothalamic circadian pacemaker, to in vitro periodicity in cultured peripheral fibroblasts to assess the effects on these rhythms of a polymorphism of PER3 (rs57875989), which is associated with sleep timing. In vitro circadian period was determined using luminometry of cultured fibroblasts, in which the expression of firefly luciferase was driven by the promoter of the circadian gene Arntl (Bmal1). The period of the melatonin rhythm was assessed in a 9-d forced desynchrony protocol, minimizing confounding effects of sleep-wake and light-dark cycles on circadian rhythmicity. In vitro periods (32 participants, 24.61±0.33 h, mean±sd) were longer than in vivo periods (31 participants, 24.16±0.17 h; P0.4). Analyses of replicate in vitro assessments demonstrated that circadian period was reproducible within individuals (intraclass correlation=0.62), but in vivo and in vitro period assessments did not correlate (P>0.9). In accordance with circadian entrainment theory, in vivo period correlated with the timing of melatonin (P

In mammals, the retina contains a clock system that oscillates independently of the master clock in the suprachiasmatic nucleus and allows the retina to anticipate and to adapt to the sustained daily changes in ambient illumination. Using a combination of laser capture micro-dissection and quantitative PCR in the present study, the clockwork of mammalian photoreceptors has been recorded. The transcript amounts of the core clock genes Clock, Bmal1, Period1 (Per1), Per3, Cryptochrome2, and Casein kinase Ie in photoreceptors of rat retina have been found to undergo daily changes. Clock and Bmal1 peak with Per1 and Per3 around dark onset, whereas Casein kinase Ie and Cryptochrome2 peak at night. As shown for Clock, Per1, and Casein kinase Ie, the oscillation of transcript amounts results in daily changes of the protein products. The in-phase oscillation of Clock/Bmal1 with Pers and the rhythmic expression of Casein kinase Ie do not occur in molecular clocks of other tissues including the suprachiasmatic nucleus. Therefore, the findings presented suggest that the photoreceptor clock is unique not only in its position outside the clock hierarchy mastered by the suprachiasmatic nucleus, but also with regard to the intrinsic rhythmic properties of its molecular components.

There are clinical reports uncovering a circadian rhythmicity of alcohol intoxication and overdose (Raymond et al., 1992). There also appears to be a relationship between alcohol drinking behavior and altered body circadian functions. In line with these views, alcohol intake has been shown to alter circadian blood pressure (Nakashita et al., 2009; Ohkubo et al., 2009), core body temperature (Devaney et al., 2003), hormone release in humans (Rojdmark et al., 2001), circadian phase shifting and free running period in mice (Seggio et al., 2009), circadian phase resetting and nocturnal activity patters in the hamster (Ruby, 2009), and rhythmicity in hypothalamic proopiomelanocortin (POMC) neurons in rats (Chen et al., 2006). Maternal alcohol drinking profoundly and permanently affects circadian function in the offspring. It is known that prenatal ethanol exposure alters core body temperature, corticosterone rhythms (Handa et al., 2007), NK cell function rhythms (Arjona et al., 2006a), phase shifting ability (Sakata-Haga et. al, 2006), and rhythmic pituitary-adrenal function in rats (Taylor et al., 1982), as well as circadian blood pressure and the heart rate of adolescent humans (Alvarez et al., 1995). Why is the body’s circadian system so vulnerable to alcohol toxicity? One logical explanation for the circadian system vulnerability, may point to a possible alcohol specific target for the gene(s) that regulate circadian functions. The circadian clock-work involves the interaction of the so-called clock genes such as the Period (Per1, Per2, Per3), Clock, Bmal1 and Cryptochrome (Cry1, Cry2) genes into two tightly coupled transcriptional and translational feedback loops, thereby self-sustaining a circadian period of activity of the cell and modulating the expression of clock-controlled genes (Ko and Takahashi, 2006). Of these genes, the Per2 gene appears to be a putative target for alcohol. Support for this view comes from evidence showing that chronic alcohol exposure alters the circadian expression of Period genes in the hypothalamus of adult rats (Chen et al., 2004). Similarly, prenatal alcohol feeding alters circadian expression of Period genes in the hypothalamus and peripheral tissues (Chen et al., 2006; Arjona et al, 2006b; Farnell et al, 2008). Additionally, Per2 gene mutant (Per2Brdm1) mice display enhanced alcohol consumption and preference (Spanagel et al., 2005a), whereas Per1Brdm1 mutant mice do not show such an enhancement in alcohol drinking behavior (Zghoul et al., 2007). Evidence has shown, alcoholics with a specific set of polymorphisms in the Per2 gene consume less alcohol than alcoholics without the polymorphisms (Spanagel et al., 2005a, b). Hence, Per2 appears to be a targeted gene where alcohol may act on to alter circadian functions. In order to more closely elucidate the role of the circadian clock in ethanol’s effects on the brain, we examined the effect of the Per2 mutation on hypothalamic β-endorphin neuronal sensitivity to ethanol.

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long-term diseases including hepatocellular carcinoma (HCC). Melatonin has been reported to alleviate promotion and progression of HCC, but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight ip) once a week for 8 weeks. Melatonin was given at 5 or 10 mg kg-1 d-1 ip beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK1e, Rev-erbα, and Rev-erbβ decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT1 receptor and reduced levels of the hypoxia-inducible factors Hif-1α and Hif-2α. An increased expression of p21, p53, and PARP1/2, a higher Bax/Bcl-2 ratio, and a lower expression of Cyclin D1, CDK6, HSP70, HSP90, and GRP78 proteins were also observed in melatonin-treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV-ERB agonist SR9009 in human Hep3B cells, and BMAL1 knocking down attenuated the pro-apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.

The premammillary nucleus (PMM) has been shown to contain a daily endogenous dual-oscillation in dopamine (DA)/melatonin (MEL) as well as c-fos mRNA expression that is associated with the daily photo-inducible phase of gonad growth in turkeys. In the present study, the expression of clock genes (Bmal1, Clock, Cry1, Cry2, Per2 and Per3) in the PMM was determined under short (8 : 16 h light/dark cycle) and long (16 : 8 h light/dark cycle) photoperiods relative to changes associated with the diurnal rhythm of DA and MEL. Constant darkness (0 : 24 h light/dark cycle) was used to assess the endogenous response of clock genes. In addition, light pulses were given at zeitgeber time (ZT) 8, 14 and 20 to ascertain whether clock gene expression is modulated by light pulse stimulation and therefore has a daily phase-related response. In the PMM, the temporal clock gene expression profiles were similar under short and long photoperiods, except that Per3 gene was phase-delayed by approximately 16 h under long photoperiod. In addition, Cry1 and Per3 genes were light-induced at ZT 14, the photosensitive phase for gonad recrudescence, whereas the Clock gene was repressed. Gene expression in established circadian pacemakers, the visual suprachiasmatic nucleus (vSCN) and the pineal, was also determined. Clock genes in the pineal gland were rhythmic under both photoperiods, and were not altered after light pulses at ZT 14, which suggests that pineal clock genes may not be associated with the photosensitive phase and reproductive activities. In the vSCN, clock gene expression was phase-shifted depending on the photoperiod, with apexes at night under short day length and during the day under long day length. Furthermore, light pulses at ZT 14 induced the Per2 gene, whereas it repressed the Bmal1 gene. Taken together, the changes in clock gene expression observed within the PMM were unique compared to the pineal and vSCN, and were induced by long photoperiod and light during the daily photosensitive phase; stimuli that are also documented to promote reproductive activity. These results show that Cry1 and Per3 are involved in the photic response associated with the PMM neuronal activation and are coincident with an essential circadian mechanism (photosensitive phase) controlling the reproductive neuroendocrine system.

Objective: Genes encoding the circadian transcriptional apparatus exhibit robust oscillatory expression in murine adipose tissues. This study tests the hypothesis that human subcutaneous adipose-derived stem cells (ASCs) provide an in vitro model in which to monitor the activity of the core circadian transcriptional apparatus. Research Methods and Procedures: Primary cultures of undifferentiated or adipocyte-differentiated ASCs were treated with dexamethasone, rosiglitazone, or 30% fetal bovine serum. The response of undifferentiated ASCs to dexamethasone was further evaluated in the presence of lithium chloride. Lithium inhibits glycogen synthase kinase 3, a key component of the circadian apparatus. Total RNA was harvested at 4-hour intervals over 48 hours and examined by real-time reverse transcription polymerase chain reaction (RT-PCR). Results: Adipocyte-differentiated cells responded more rapidly to treatments than their donor-matched undifferentiated controls; however, the period of the circadian gene oscillation was longer in the adipocyte-differentiated cells. Dexamethasone generated circadian gene expression patterns with mean periods of 25.4 and 26.7 hours in undifferentiated and adipocyte-differentiated ASCs, respectively. Both rosiglitazone and serum shock generated a significantly longer period in adipocyte-differentiated ASCs relative to undifferentiated ASCs. The Bmal1 profile was phase-shifted by ∼8 to 12 hours relative to Per1, Per3, and Cry2, consistent with their expression in vivo. Lithium chloride inhibited adipogenesis and significantly lengthened the period of Per3 and Rev-erbα gene expression profiles by >5 hours in dexamethasone-activated undifferentiated ASCs. Discussion: These results support the initial hypothesis and validate ASCs as an in vitro model for the analysis of circadian biology in human adipose tissue.

Several studies have demonstrated that the mammalian retina contains an autonomous circadian clock. Dopaminergic and other inner retinal neurons express many of the clock genes, whereas some of these genes seem to be absent from the photoreceptors. This observation has led to the suggestion that in mammalian retina the circadian pacemaker driving retinal rhythms is located in the inner nuclear layer. However, other evidence points to the photoreceptor layer as the site of the mammalian retinal clock. The goal of the present study was to demonstrate the presence of a functional circadian clock in photoreceptors. First, using laser capture microdissection and reverse transcriptase-polymerase chain reaction, we investigated which of the clock genes are expressed in rat photoreceptors. We then prepared photoreceptor layer cultures from the retina to test whether these isolated cultures were viable and could drive circadian rhythms. Our data indicated that Per1, Per3, Cry1, Cry2, Clock, Bmal1, Rev-erb alpha, and Rora RNAs were present in the photoreceptors, whereas we were unable to amplify mRNA for Per2 and Npas2. Photoreceptor layers obtained from Period1-luciferase rats expressed a robust circadian rhythm in bioluminescence and melatonin synthesis. These results demonstrate that mammalian photoreceptors contain the circadian pacemaker driving rhythmic melatonin synthesis.

We hypothesize that circadian dysfunction could underlie, at least partially, the liability for bipolar 1 disorder (BD1). Our hypothesis motivated tests for the association between the polymorphisms of genes that mediate circadian function and liability for BD1. The US Caucasian patients with BD1 (DSM-IV criteria) and available parents were recruited from Pittsburgh and surrounding areas (n = 138 cases, 196 parents) and also selected from the NIMH Genetics Collaborative Initiative (n = 96 cases, 192 parents). We assayed 44 informative single-nucleotide polymorphisms (SNPs) from eight circadian genes in the BD1 samples. A population-based sample, specifically cord blood samples from local live births, served as community-based controls (n = 180). It was used as a contrast for genotype and haplotype distributions with those of patients. US patients with schizophrenia/schizoaffective disorder (SZ/SZA, n = 331) and available parents from Pittsburgh (n = 344) were assayed for a smaller set of SNPs based on the results from the BD1 samples. Modest associations with SNPs at ARNTL (BmaL1) and TIMELESS genes were observed in the BD1 samples. The associations were detected using family-based and case-control analyses, albeit with different SNPs. Associations with TIMELESS and PERIOD3 were also detected in the Pittsburgh SZ/SZA group. Thus far, evidence for association between specific SNPs at the circadian gene loci and BD1 is tentative. Additional studies using larger samples are required to evaluate the associations reported here.

The influence of seasonal lighting conditions on expression of clock genes and the circadian pacemaker was investigated in the rat retina. For this purpose, the 24-h profiles of nine clock genes (bmal1, clock, per1, per2, per3, dec1, dec2, cry1 and cry 2) and the arylalkylamine N-acetyltransferase gene as an indicator of the circadian pacemaker output were compared between light-dark periods of 8 : 16 and 16 : 8 h. The photoperiod influenced the daily patterns of the amount of transcript for per1, per3, dec2 and arylalkylamine N-acetyltransferase. This indicates that photoperiodic information modulates clock gene expression in addition to the circadian pacemaker of the retina. Under constant darkness, photoperiod-dependent changes in the daily profile of the level of transcript persisted for the arylalkylamine N-acetyltransferase gene but not for any of the clock genes. Hence, quantitative expression of each clock gene is influenced by the photoperiod only under the acute light-dark cycle, whereas the pacemaker is capable of storing photoperiodic information from past cycles.

All free‐living organisms are regulated by the 24‐h light and dark cycle produced by Earth’s rotation. In mammals, circadian rhythms are indispensble for the regulation of physiological and biochemical processes in the body. In 1972, the suprachiasmatic nucleus was shown to be required for daily rhythms in animal behavior1. Light signals through the optic nerves give the cue to set the clock and to synchronize the various behavioral outputs. Interstingly, both the central nervous system and almost all peripheral tissues and cells express the circadian oscillator transcriptional factors CLOCK and BMAL1, which regulate cellular functions. CLOCK and BMAL1 heterodimerize and activate downstream target genes rhythmically, including the period (Per1, Per2 and Per3) and cryptochrome (Cry1 and Cry2) genes2. All cells in the body are regulated by this clock gene system according to the day–night cycle in an organized way. Several findings in mammals have suggested that clocks outside the brain paticipate in metabolic regulation. For example, expression of the receptors, transporters and enzymes that regulate key steps of cellular metabolism are under circadian regulation. In addition, circadian clocks outside the suprachiasmatic nucleus rely on feeding cues rather than light signals. Disruption of this molecular clock system has been shown to induce deleterious metabolic outcomes, diseases and tumors. Because disruption of clock genes affects both locomotor activity and feeding behavior, and also might indirectly change metabolism3, it has been considered that clocks outside the brain have a local function and that an effect on metabolism is slight. Recent work on organ‐specific inactivation of genes, such as BMAL1, have shown this for liver circadian clocks in glucose metabolism; however, many questions regarding circadian activity outside the brain remain to be answered. Recently, Marcheva et al.4 reported that disruption of the clock componets, CLOCK and BMAL1, leads to diabetes (Figure 1). They showed evidence that the mouse pancreas has a functional circadian clock; pancreatic islets also show robust rhythmical activity, even if they are excluded from the tissue environment. This mammalian islet clock regulates the expression of genes involved in glucose sensing, insulin secretion, and islet cell growth and development. Figure 1 Pancreatic clock machinery and glucose metabolism. Regulatory mechanisms of CLOCK/BMAL1 in pancreatic insulin secretion in day and night through the step of insulin granule exocytosis. Disruption of this mechanism leads to diabetes as a result ... An insulin secretory defect should result from β‐cell dysfunction or reduction in β‐cell mass5. Morphometric studies indicate that type 2 diabetes patients have lost approximately 50% of their β‐cells. As a 50%β‐cell loss in experimental animals does not produce the fasting hyperglycemia regulary seen in type 2 diabetes, additional factors, such as dysfunction of the remaining β‐cells or concomittent insulin resistance, might contribute6. It has long been recognized that normal glucose‐induced insulin secretion in humans varies across the day–night cycle, and that disruption of the circadian oscillation of glucose metabolism is a feature of type 2 diabetes7. Marcheva et al.4 described the mechanism of this pathophysiological state. The authors showed that mice in which the circadian clock is disrupted produce less insulin in the resting state and secrete less insulin in response to glucose. In addition, morphometric analyses show that the pancreatic islets are smaller and that production of insulin is reduced. These results suggest direct involvement of the islet clock in the production of insulin. Although an impaired acute insulin response to intravenous glucose challenge is typical in human type 2 diabetes, non‐glucose stimulated insulin secretion is also reduced when plasma glucose levels are matched by either a glucose infusion in normal sujects or an insulin infusion in type 2 diabetic subjects. Animals with conditional pancreatic clock ablation represent an interesting form of diabetes – the islets show not only an impaired insulin response to glucose, but to KCl and the adenylate cyclase activators, froskolin and exendin‐4, which suggests a defect in insulin exocytosis, but not in glucose metabolism. One of the major issues in the circadian clock’s role in diabetes is whether disruption of CLOCK or BMAL 1 leads to metabolic defects by affecting circadian rhythms or by mechanisms not related to their clock function. To answer this question is essential – if disruption of clock genes is involved in development of diabetes by affecting circadian rhythms directly, industrialized, modern lifestyle itself has a pathological impact. Unusual rhythms of eating, fasting, sleeping, wakefulness and activity are more or less forced on more and more individuals by requirements of living, especially by shift work. From a clincal point of view, an epidemiological study of circadian disruption by shift work or other lifestyle disruptions of the day–night cycle in the development of diabetes and related disorders in the developed and developing world is required. Several reports have shown that CLOCK and BMAL1 are associated with features of metabolic syndrome. Polymorphisms in the clock gene have been correlated with predisposition to obesity, and two BMAL1 haplotypes are associated with type 2 diabetes and hypertension2. Shift work involves 20–25% of the employees in the manufacturing industry worldwide, and is becoming increasingly prevalent in contemporary life. Although a precise definition of shift work is lacking, most studies classify shift workers as those who work regularly outside the usual daytime shift hours. Shift work is thought to be associated with several health problems, such as metabolic syndrome, diabetes mellitus and cardiovascular disease, that are possibly the result of impaired biological circadian rhythms. Indeed, a higher prevalence of metabolic syndrome has recently been shown among shift workers8. Some Japanese reports have shown shift work to be an independent risk factor for impaired glucose metabolism and higher prevalence of diabetes mellitus. Other problems of 24‐h contemporary life have been investigated, such as night eating and breakfast skipping, which are suggested to have an association with obesity and visceral adiposity in overweight youth9,10. Sleep disorders (short duration and/or poor quality sleep) also have a profound impact on the development of type 2 diabetes and obesity2. Thus, the mechanism by which disruption of CLOCK‐related genes affects circadian rhythms directly might underlie the recent rapid increase of diabetes worldwide. Technical developments that allow modification of specific and selective controls of clock function are required, in addition to continued study of the physiological and pathphysiological significance of circadian rhythms in the development of diabetes mellitus.