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Adipocytes are energy stores of the body which also play a role in physiological regulation and homeostasis through their endocrine activity. Adipocyte circadian clocks drive rhythms in gene expression, and dysregulation of these circadian rhythms associates with pathological conditions such as diabetes. However, although the role of circadian rhythms in adipose cells and related tissues has been studied from phsyiological and molecular perspectives, they have not yet been explored from an electrical perspective. Research into electro-chronobiology has revealed that electrical properties have important roles in peripheral clock regulation independently of transcription-translation feedback loops. We have used dielectrophoresis to study electrophysiological rhythms in pre-adipocytes - representing an adipocyte precursor and nucleated cell-based model, using serum shocking as the cellular method of clock entrainment. The results revealed significant electrophysiological rhythms, culminating in circadian (ca. 24 hourly) cycles in effective membrane capacitance and radius properties, whereas effective membrane conductance was observed to express ultradian (ca. 14 hourly) rhythms. These data shed new light into pre-adipocyte electrical behaviour and present a potential target for understanding and manipulation of metabolic physiology.

Context Daily variation in the thermic effect of food (TEF) is commonly reported and proposed as a contributing factor to weight gain with late eating. However, underlying circadian variability in resting metabolic rate (RMR) is an overlooked factor when calculating TEF associated with eating at different times of the day. Objective This work aimed to determine whether methodological approaches to calculating TEF contribute to the reported phenomena of daily variation in TEF. Methods Fourteen overweight to obese but otherwise healthy individuals had their resting and postprandial energy expenditure (EE) measured over 15.5 hours at a clinical research unit. TEF was calculated for breakfast, lunch, and dinner using standard methods (above a baseline and premeal RMR measure) and compared to a method incorporating a circadian RMR by which RMR was derived from a sinusoid curve model and TEF was calculated over and above the continuously changing RMR. Main outcome measures were TEF at breakfast, lunch, and dinner calculated by different methods. Results Standard methods of calculating TEF above a premeal measured RMR showed that morning TEF (60.8 kcal ± 5.6) (mean ± SEM) was 1.6 times greater than TEF at lunch (36.3 kcal ± 8.4) and 2.4 times greater than dinner TEF (25.2 kcal ± 9.6) (P = .022). However, adjusting for modeled circadian RMR nullified any differences between breakfast (54.1 kcal ± 30.8), lunch (49.5 kcal ± 29.4), and dinner (49.1 kcal ± 25.7) (P = .680). Conclusion Differences in TEF between morning and evening can be explained by the underlying circadian resting EE, which is independent of an acute effect of eating.

Whilst the treatment and prevention of overweight and obesity-related disease is managed by restricting daily energy intake, long-term adherence to dietary strategies appears unsustainable. Time-restricted eating (TRE) aims to position energy intake in an eating window under 12 h per day and offers an alternative behavioral intervention, which can aid weight management and improve cardiometabolic health. Adherence to previous TRE protocols is estimated at between 63 and 100%, although the accuracy of reporting is unclear. This study therefore aimed to provide an objective, subjective, and qualitative overview of adherence to a prescribed TRE protocol, and to identify any potential barriers affecting adherence. Adherence after 5 weeks of TRE was estimated at ~63% based on continuous glucose monitoring data when compared with time-stamped diet diaries. Subjective participant responses reported adherence at an average of ~61% per week. Barriers to adopting TRE, including work schedules, social events, and family life, were identified by participants during qualitative interviews. The findings of this study suggest that the development of personalized TRE protocols may help to navigate the barriers to adherence leading to improved health-related outcomes.

Gastric emptying (GE) is the process of food being processed by the stomach and delivered to the small intestine where nutrients such as lipids are absorbed into the blood circulation. The combination of an easy and inexpensive method to measure GE such as the CO

Background/Introduction Circadian rhythms, defined as biological oscillations with a period of circa 24h, regulate many physiological processes in the cardiovascular system, such as vascular function, vascular tone, blood pressure, heart rate and thrombus formation [1]. The vasculature responds to the main pacemaker located in the brain, but it also possesses its own clock. Indeed, a molecular clock has been identified in endothelial cells (EC) and smooth muscle cells (SMC). The disruption of the circadian clock profoundly affects cardiovascular functionality with adverse cardiovascular events such as myocardial infarction or stroke showing a 24h rhythmicity with a peak incidence in the early morning. Among several mechanisms affected by circadian dysregulation, angiogenesis plays a fundamental role in homeostasis and development of new blood vessels. EC and pericytes (PC) are the two main cell populations in the capillaries, and their physical and paracrine interaction drives and regulates the sprouting. However, the presence and the role of circadian rhythms in pericytes and whether the molecular clock affects the endothelial/pericyte interactions remain unexplored. Purpose The aim of this study is to identify a molecular clock in human vascular pericytes and elucidate the impact of the circadian clock on the formation of new blood vessels. Methods Human primary PC were synchronised and the rhythmicity of clock genes measured by luminescence, immunofluorescence, and qPCR. Synchronised PC were co-cultured with Bmal1::LUC human primary EC. The effect of PC synchronisation and circadian clock disruption by shRNA on EC clock genes and angiogenic potential were measured by luminescence and Matrigel assay, respectively. A macroporous polyurethane scaffold was developed for 3D co-cultures. Results PC presented rhythmic expression of the principal circadian genes with a circa 24h period but in our experimental setting, EC did not show circadian rhythmicity. Synchronised PC supported the rhythmic expression of the clock gene Bmal1 in EC in a contact co-culture system, suggesting a secondary form of EC molecular clock regulation. Non-contact co-cultures failed to synchronise EC. Furthermore, when the clock was disrupted in PC, their capacity to support EC's tube-forming capacity on Matrigel was impaired; clock disruption in EC did not affect angiogenesis, supporting the hypothesis that a disrupted clock in perivascular cells affects angiogenesis. In a 3D tissue engineering scaffold seeded with both EC and PC, the synchronisation of the clock led to the development of organised vascular-like structures around the scaffold's pores, as compared to the non-synchronised condition where cells appeared disorganised. Conclusion This study defines for the first time the existence of an endogenous molecular circadian clock in perivascular cells and suggests implications for circadian clock synchronisation in physiological and therapeutic angiogenesis. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Surrey Doctoral CollegeUniversity of Surrey Bioprocess and Biochemical Engineering (BioProChem) Group.

Morning loaded calorie intake in humans has been advocated as a dietary strategy to improve weight loss. This is also supported by animal studies suggesting time of eating can prevent weight gain. However, the underlying mechanisms through which timing of eating could promote weight loss in humans are unclear. In a randomized crossover trial (NCT03305237), 30 subjects with obesity/overweight underwent two 4-week calorie-restricted but isoenergetic weight loss diets, with morning loaded or evening loaded calories (45%:35%:20% versus 20%:35%:45% calories at breakfast, lunch, and dinner, respectively). We demonstrate no differences in total daily energy expenditure or resting metabolic rate related to the timing of calorie distribution, and no difference in weight loss. Participants consuming the morning loaded diet reported significantly lower hunger. Thus, morning loaded intake (big breakfast) may assist with compliance to weight loss regime through a greater suppression of appetite.

Constant routine and forced desynchrony protocols typically remove the effects of behavioral/environmental cues to examine endogenous circadian rhythms, yet this may not reflect rhythms of appetite regulation in the real world. It is therefore important to understand these rhythms within the same subjects under controlled diurnal conditions of light, sleep, and feeding. Ten healthy adults (9 M/1 F, means ±SD: age, 30 ± 10 yr; body mass index, 24.1 ± 2.7 kg·m-2) rested supine in the laboratory for 37 h. All data were collected during the final 24 h of this period (i.e., 0800-0800 h). Participants were fed hourly isocaloric liquid meal replacements alongside appetite assessments during waking before a sleep opportunity from 2200 to 0700 h. Hourly blood samples were collected throughout the 24-h period. Dim light melatonin onset occurred at 2318 ± 46 min. A diurnal rhythm in mean plasma unacylated ghrelin concentration was identified (P = 0.04), with the acrophase occurring shortly after waking (0819), falling to a nadir in the evening with a relative amplitude of 9%. Plasma leptin concentration also exhibited a diurnal rhythm (P < 0.01), with the acrophase occurring shortly after lights-out (0032 h) and the lowest concentrations at midday. The amplitude for this rhythm was 25%. Diurnal rhythms were established in all dimensions of appetite except for sweet preference (P = 0.29), with both hunger (2103 h) and prospective food consumption (1955 h) reaching their peak in the evening before falling to their nadir shortly after waking. Under controlled diurnal conditions, simultaneous measurement of leptin, unacylated ghrelin, and subjective appetite over a 24-h period revealed rhythmicity in appetite regulation in lean, healthy humans.NEW & NOTEWORTHY Simultaneous assessment of subjective appetite, unacylated ghrelin, and leptin was carried out over a continuous 37-h protocol for the first time under conditions of controlled light, sleep, and feeding in healthy, lean adults. Rhythms were observed in unacylated ghrelin, leptin, and components of subjective appetite, such as hunger, prospective consumption, and fullness. Concurrent measurement of rhythms in these variables is important to fully understand the temporal relationships between components of appetite as well as the influence of diurnal factors such as sleep, light, and feeding.

The circadian timing system governs daily biological rhythms, synchronising physiology and behaviour to the temporal world. External time cues, including the light-dark cycle and timing of food intake, provide daily signals for entrainment of the central, master circadian clock in the hypothalamic suprachiasmatic nuclei (SCN), and of metabolic rhythms in peripheral tissues, respectively. Chrono-nutrition is an emerging field building on the relationship between temporal eating patterns, circadian rhythms, and metabolic health. Evidence from both animal and human research demonstrates adverse metabolic consequences of circadian disruption. Conversely, a growing body of evidence indicates that aligning food intake to periods of the day when circadian rhythms in metabolic processes are optimised for nutrition may be effective for improving metabolic health. Circadian rhythms in glucose and lipid homeostasis, insulin responsiveness and sensitivity, energy expenditure, and postprandial metabolism, may favour eating patterns characterised by earlier temporal distribution of energy. This review details the molecular basis for metabolic clocks, the regulation of feeding behaviour, and the evidence for meal timing as an entraining signal for the circadian system in animal models. The epidemiology of temporal eating patterns in humans is examined, together with evidence from human intervention studies investigating the metabolic effects of morning compared to evening energy intake, and emerging chrono-nutrition interventions such as time-restricted feeding. Chrono-nutrition may have therapeutic application for individuals with and at-risk of metabolic disease and convey health benefits within the general population. (Figure presented.).

The interaction between time of day and energy intake, termed chrono-nutrition, has received considerable recent interest. One aspect of chrono-nutrition with potential to benefit long-term cardio-metabolic health is time-restricted feeding (TRF). Current support for TRF primarily derives from animal research, although recent small-scale human studies indicate possible translational benefit. Whether free-living humans, however, can incorporate TRF into their daily lives is poorly understood. This study reports data from participants (n = 608) who completed an online questionnaire to investigate daily routine, likelihood of TRF incorporation within work vs free-days, and key considerations influencing TRF uptake. The majority of participants reported a typical daily feeding window (time between first and last energy intake) of between 10 and 14 h on workdays and free days, 62.7 and 65.5% respectively. Likelihood of adherence to TRF declined with an increase in the proposed restriction of the feeding window by 0.5 to 4-h per day. We then examined data from participants with a typical daily feeding window of 12+ h on workdays (n = 221) and free-days (n = 223) to investigate the likelihood of using TRF, and the most important considerations in making this decision. Of these participants, (n = 132) on workdays and (n = 125) on free days would likely reduce their feeding window by 3-h. Multiple regression analysis revealed that key considerations determining the likelihood of adopting TRF were: cost, time availability, and perceived health benefits (on workdays); wake time, bed time, time availability, motivation to change and perceived health benefits (on free-days). These data provide novel information regarding public attitudes towards TRF and highlight important aspects to be considered when translating controlled laboratory studies to public dietary advice.

Studying circadian rhythms in most human tissues is hampered by difficulty in collecting serial samples. Here we reveal circadian rhythms in the transcriptome and metabolic pathways of human white adipose tissue. Subcutaneous adipose tissue was taken from seven healthy males under highly controlled ‘constant routine’ conditions. Five biopsies per participant were taken at six-hourly intervals for microarray analysis and in silico integrative metabolic modelling. We identified 837 transcripts exhibiting circadian expression profiles (2% of 41619 transcript targeting probes on the array), with clear separation of transcripts peaking in the morning (258 probes) and evening (579 probes). There was only partial overlap of our rhythmic transcripts with published animal adipose and human blood transcriptome data. Morning-peaking transcripts associated with regulation of gene expression, nitrogen compound metabolism, and nucleic acid biology; evening-peaking transcripts associated with organic acid metabolism, cofactor metabolism and redox activity. In silico pathway analysis further indicated circadian regulation of lipid and nucleic acid metabolism; it also predicted circadian variation in key metabolic pathways such as the citric acid cycle and branched chain amino acid degradation. In summary, in vivo circadian rhythms exist in multiple adipose metabolic pathways, including those involved in lipid metabolism, and core aspects of cellular biochemistry.

Chrononutrition is an emerging discipline that builds on the intimate relation between endogenous circadian (24-h) rhythms and metabolism. Circadian regulation of metabolic function can be observed from the level of intracellular biochemistry to whole-organism physiology and even postprandial responses. Recent work has elucidated the metabolic roles of circadian clocks in key metabolic tissues, including liver, pancreas, white adipose, and skeletal muscle. For example, tissue-specific clock disruption in a single peripheral organ can cause obesity or disruption of whole-organism glucose homeostasis. This review explains mechanistic insights gained from transgenic animal studies and how these data are being translated into the study of human genetics and physiology. The principles of chrononutrition have already been demonstrated to improve human weight loss and are likely to benefit the health of individuals with metabolic disease, as well as of the general population.

A growing body of evidence highlights the importance of the biological clock as a modulator of energy balance and metabolism. Recent studies in humans have shown that ingested calories are apparently utilised more efficiently in the morning than in the evening and this is manifest through improved weight loss, even under iso‐energetic calorie intake. The mechanisms behind this enhanced morning energy metabolism are not yet clear, although it may result from behavioural adaptations or circadian driven variations in physiology and energy metabolism. A major objective of the newly funded Big Breakfast Study therefore is to investigate the mechanistic basis of this amplified morning thermogenesis leading to enhanced weight loss, by exploring behavioural and physiological adaptations in energy expenditure alongside the underlying circadian biology. This report briefly discusses the current research linking meal timing, circadian rhythms and metabolism; highlights the research gaps; and provides an overview of the studies being undertaken as part of the Medical Research Council‐funded Big Breakfast Study.

Circadian regulation of transcriptional processes has a broad impact on cell metabolism. Here, we compared the diurnal transcriptome of human skeletal muscle conducted on serial muscle biopsies in vivo with profiles of human skeletal myotubes synchronized in vitro. More extensive rhythmic transcription was observed in human skeletal muscle compared to in vitro cell culture as a large part of the in vivo mRNA rhythmicity was lost in vitro. siRNA-mediated clock disruption in primary myotubes significantly affected the expression of ~8% of all genes, with impact on glucose homeostasis and lipid metabolism. Genes involved in GLUT4 expression, translocation and recycling were negatively affected, whereas lipid metabolic genes were altered to promote activation of lipid utilization. Moreover, basal and insulin-stimulated glucose uptake were significantly reduced upon CLOCK depletion. Our findings suggest an essential role for the circadian coordination of skeletal muscle glucose homeostasis and lipid metabolism in humans.

The isolation of melatonin was first reported in 1958. Since the demonstration that pineal melatonin synthesis reflects both daily and seasonal time, melatonin has become a key element of chronobiology research. In mammals, pineal melatonin is essential for transducing day-length information into seasonal physiological responses. Due to its lipophilic nature, melatonin is able to cross the placenta and is believed to regulate multiple aspects of perinatal physiology. The endogenous daily melatonin rhythm is also likely to play a role in the maintenance of synchrony between circadian clocks throughout the adult body. Pharmacological doses of melatonin are effective in resetting circadian rhythms if taken at an appropriate time of day, and can acutely regulate factors such as body temperature and alertness, especially when taken during the day. Despite the extensive literature on melatonin physiology, some key questions remain unanswered. In particular, the amplitude of melatonin rhythms has been recently associated with diseases such as type 2 diabetes mellitus but understanding of the physiological significance of melatonin rhythm amplitude remains poorly understood.

The power of the application of bioinformatics across multiple publicly available transcriptomic data sets was explored. Using 19 human and mouse circadian transcriptomic data sets, we found that NR1D1 and NR1D2 which encode heme-responsive nuclear receptors are the most rhythmic transcripts across sleep conditions and tissues suggesting that they are at the core of circadian rhythm generation. Analyzes of human transcriptomic data show that a core set of transcripts related to processes including immune function, glucocorticoid signalling, and lipid metabolism is rhythmically expressed independently of the sleep-wake cycle. We also identify key transcripts associated with transcription and translation that are disrupted by sleep manipulations, and through network analysis identify putative mechanisms underlying the adverse health outcomes associated with sleep disruption, such as diabetes and cancer. Comparative bioinformatics applied to existing and future data sets will be a powerful tool for the identification of core circadian- and sleep-dependent molecules.

This pilot study explored the feasibility of a moderate time-restricted feeding (TRF) intervention and its effects on adiposity and metabolism. For 10 weeks, a free-living TRF group delayed breakfast and advanced dinner by 1·5 h each. Changes in dietary intake, adiposity and fasting biochemistry (glucose, insulin, lipids) were compared with controls who maintained habitual feeding patterns. Thirteen participants (29 (sem2) kg/m2) completed the study. The average daily feeding interval was successfully reduced in the TRF group (743 (sem32) to 517 (sem22) min/d;P n7), although questionnaire responses indicated that social eating/drinking opportunities were negatively impacted. TRF participants reduced total daily energy intake (P = 0·019) despitead libitumfood access, with accompanying reductions in adiposity (P = 0·047). There were significant between-group differences in fasting glucose (P = 0·008), albeit driven primarily by an increase among controls. Larger studies can now be designed/powered, based on these novel preliminary qualitative and quantitative data, to ascertain and maximise the long-term sustainability of TRF.

International audience; Circadian clocks play an important role in lipid homeostasis, with impact on various metabolic diseases. Due to the central role of skeletal muscle in whole-body metabolism, we aimed at studying muscle lipid profiles in a temporal manner. Moreover, it has not been shown whether lipid oscillations in peripheral tissues are driven by diurnal cycles of rest-activity and food intake or are able to persist in vitro in a cell-autonomous manner. To address this, we investigated lipid profiles over 24 h in human skeletal muscle in vivo and in primary human myotubes cultured in vitro. Glycerolipids, glycerophos-pholipids, and sphingolipids exhibited diurnal oscillations, suggesting a widespread circadian impact on muscle lipid metabolism. Notably, peak levels of lipid accumulation were in phase coherence with core clock gene expression in vivo and in vitro. The percentage of oscillating lipid metabolites was comparable between muscle tissue and cultured myotubes, and temporal lipid profiles correlatedwith transcript profiles of genes implicated in their biosynthesis. Lipids enriched in the outer leaflet of the plasma membrane oscillated in a highly coordinated manner in vivo and in vitro. Lipid metabolite oscillations were strongly attenuated upon siRNA-mediated clock disruption in human primary myotubes. Taken together, our data suggest an essential role for endogenous cell-autonomous human skeletal muscle oscillators in regulating lipid metabolism independent of external synchronizers, such as physical activity or food intake.

Metabolic profiling of individuals with type 2 diabetes mellitus (T2DM) has previously been limited to single-time-point samples, ignoring time-of-day variation. Here, we tested our hypothesis that body mass and T2DM affect daily rhythmicity and concentrations of circulating metabolites across a 24-h day in 3 age-matched, male groups—lean, overweight/obese (OW/OB), and OW/OB with T2DM—in controlled laboratory conditions, which were not confounded by large meals. By using targeted liquid chromatography/mass spectrometry metabolomics, we quantified 130 plasma metabolites every 2 h over 24 h, and we show that average metabolite concentrations were significantly altered by increased body mass (90 of 130) and T2DM (56 of 130). Thirty-eight percent of metabolites exhibited daily rhythms in at least 1 study group, and where a metabolite was rhythmic in >1 group, its peak time was comparable. The optimal time of day was assessed to provide discriminating biomarkers. This differed between metabolite classes and study groups—for example, phospholipids showed maximal difference at 5:00 AM (lean vs. OW/OB) and at 5:00 PM (OW/OB vs. T2DM). Metabolites that were identified with both robust 24-h rhythms and significant concentration differences between study groups emphasize the importance of controlling the time of day for diagnosis and biomarker discovery, offering a significant improvement over current single sampling.—Isherwood, C. M., Van der Veen, D. R., Johnston, J. D., Skene, D. J. Twenty-four-hour rhythmicity of circulating metabolites: effect of body mass and type 2 diabetes.

Circadian rhythms, metabolism, and nutrition are intimately linked [1, 2], although effects of meal timing on the human circadian system are poorly understood. We investigated the effect of a 5-hr delay in meals on markers of the human master clock and multiple peripheral circadian rhythms. Ten healthy young men undertook a 13-day laboratory protocol. Three meals (breakfast, lunch, dinner) were given at 5-hr intervals, beginning either 0.5 (early) or 5.5 (late) hr after wake. Participants were acclimated to early meals and then switched to late meals for 6 days. After each meal schedule, participants' circadian rhythms were measured in a 37-hr constant routine that removes sleep and environmental rhythms while replacing meals with hourly isocaloric snacks. Meal timing did not alter actigraphic sleep parameters before circadian rhythm measurement. In constant routines, meal timing did not affect rhythms of subjective hunger and sleepiness, master clock markers (plasma melatonin and cortisol), plasma triglycerides, or clock gene expression in whole blood. Following late meals, however, plasma glucose rhythms were delayed by 5.69 ± 1.29 hr (p 0.001), and average glucose concentration decreased by 0.27 ± 0.05 mM (p 0.001). In adipose tissue, PER2 mRNA rhythms were delayed by 0.97 ± 0.29 hr (p 0.01), indicating that human molecular clocks may be regulated by feeding time and could underpin plasma glucose changes. Timed meals therefore play a role in synchronizing peripheral circadian rhythms in humans and may have particular relevance for patients with circadian rhythm disorders, shift workers, and transmeridian travelers.

Circadian rhythms, metabolism and nutrition are closely interlinked. A great deal of recent research has investigated not only how aspects of metabolic physiology are driven by circadian clocks, but also how these circadian clocks are themselves sensitive to metabolic change. At the cellular level, novel feedback loops have been identified that couple circadian 'clock genes' and their proteins to expression of nuclear receptors, regulation of redox state and other major pathways. Using targeted disruption of circadian clocks, mouse models are providing novel insight into the role of tissue-specific clocks in glucose homeostasis and body weight regulation. The relationship between circadian rhythms and obesity appears complex, with variable alteration of rhythms in obese individuals. However, it is clear from animal studies that the timing and nutritional composition of meals can regulate circadian rhythms, particularly in peripheral tissues. Translation of these findings to human physiology now represents an important goal.

Circadian rhythms act to optimise many aspects of our biology and thereby ensure that physiological processes are occurring at the most appropriate time. The importance of this temporal control is demonstrated by the strong associations between circadian disruption, morbidity and disease pathology. There is now a wealth of evidence linking the circadian timing system to metabolic physiology and nutrition. Relationships between these processes are often reciprocal, such that the circadian system drives temporal changes in metabolic pathways and changes in metabolic/nutritional status alter core molecular components of circadian rhythms. Examples of metabolic rhythms include daily changes in glucose homeostasis, insulin sensitivity and postprandial response. Time of day alters lipid and glucose profiles following individual meals whereas, over a longer time scale, meal timing regulates adiposity and body weight; these changes may occur via the ability of timed feeding to synchronise local circadian rhythms in metabolically active tissues. Much of the work in this research field has utilised animal and cellular model systems. Although these studies are highly informative and persuasive, there is a largely unmet need to translate basic biological data to humans. The results of such translational studies may open up possibilities for using timed dietary manipulations to help restore circadian synchrony and downstream physiology. Given the large number of individuals with disrupted rhythms due to, for example, shift work, jet-lag, sleep disorders and blindness, such dietary manipulations could provide widespread improvements in health and also economic performance.

Sleep complaints and irregular sleep patterns, such as curtailed sleep during workdays and longer and later sleep during weekends, are common. It is often implied that differences in circadian period and in entrained phase contribute to these patterns, but few data are available. We assessed parameters of the circadian rhythm of melatonin at baseline and in a forced desynchrony protocol in 35 participants (18 women) with no sleep disorders. Circadian period varied between 23 h 50 min and 24 h 31 min, and correlated positively (n = 31, rs = 0.43, P = 0.017) with the timing of the melatonin rhythm relative to habitual bedtime. The phase of the melatonin rhythm correlated with the Insomnia Severity Index (n = 35, rs = 0.47, P = 0.004). Self-reported time in bed during free days also correlated with the timing of the melatonin rhythm (n = 35, rs = 0.43, P = 0.01) as well as with the circadian period (n = 31, rs = 0.47, P = 0.007), such that individuals with a more delayed melatonin rhythm or a longer circadian period reported longer sleep during the weekend. The increase in time in bed during the free days correlated positively with circadian period (n = 31, rs = 0.54, P = 0.002). Polysomnographically assessed latency to persistent sleep (n = 34, rs = 0.48, P = 0.004) correlated with the timing of the melatonin rhythm when participants were sleeping at their habitual bedtimes in the laboratory. This correlation was significantly stronger in women than in men (Z = 2.38, P = 0.017). The findings show that individual differences in circadian period and phase of the melatonin rhythm associate with differences in sleep, and suggest that individuals with a long circadian period may be at risk of developing sleep problems.

Although daily rhythms regulate multiple aspects of human physiology, rhythmic control of the metabolome remains poorly understood. The primary objective of this proof-of-concept study was identification of metabolites in human plasma that exhibit significant 24-h variation. This was assessed via an untargeted metabolomic approach using liquid chromatography-mass spectrometry (LC-MS). Eight lean, healthy, and unmedicated men, mean age 53.6 (SD ± 6.0) yrs, maintained a fixed sleep/wake schedule and dietary regime for 1 wk at home prior to an adaptation night and followed by a 25-h experimental session in the laboratory where the light/dark cycle, sleep/wake, posture, and calorific intake were strictly controlled. Plasma samples from each individual at selected time points were prepared using liquid-phase extraction followed by reverse-phase LC coupled to quadrupole time-of-flight MS analysis in positive ionization mode. Time-of-day variation in the metabolites was screened for using orthogonal partial least square discrimination between selected time points of 10:00 vs. 22:00 h, 16:00 vs. 04:00 h, and 07:00 (d 1) vs. 16:00 h, as well as repeated-measures analysis of variance with time as an independent variable. Subsequently, cosinor analysis was performed on all the sampled time points across the 24-h day to assess for significant daily variation. In this study, analytical variability, assessed using known internal standards, was low with coefficients of variation

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

Seasonal photoperiodic responses in mammals depend on the pineal hormone melatonin. The pars tuberalis (PT) region of the anterior pituitary has emerged as a principal melatonin target tissue, controlling endocrine responses. Rising melatonin levels acutely influence the expression of a small cluster of genes either positively (exemplified by cryptochrome-1, cry1) or negatively (exemplified by the type 1 melatonin receptor, mt1). The purpose of this study was to characterize the pathways through which these evening actions of melatonin are mediated. In vitro experiments showed that cAMP signaling in the PT directly influences mt1 but not cry1 expression. Analysis of nuclear extracts from sheep PT tissue collected 90 min after melatonin or saline control injections highlighted the response element for the immediate early gene egr1 (EGR1-RE) as a candidate for acute melatonin-dependent transcriptional regulation. We identified putative EGR1-RE’s in the proximal promoter regions of the ovine cry1 and mt1 genes, and confirmed their functionality in luciferase reporter assays. Egr1 expression is suppressed by melatonin in PT cell cultures, and is rhythmic in the ovine PT with a nadir in the early night. We propose that melatonin-dependent effects on EGR1-RE’s contribute to evening gene expression profiles in this pituitary melatonin target tissue.—Fustin J. M., Dardente H., Wagner G. C., Carter D. A., Johnston J. D., Lincoln G. A., Hazlerigg D. G. Egr1 involvement in evening gene regulation by melatonin.

Pineal secretion of melatonin provides a neuroendocrine representation of the light–dark cycle, which is used to synchronise daily and annual rhythms of physiology and behaviour. In mammals, melatonin primarily acts through MT 1 melatonin receptors that exhibit a highly restricted tissue distribution. Expression of MT 1 receptors is subject to developmental and circadian control, which likely modulates the physiological actions of melatonin. To investigate the mechanisms controlling MT 1 expression we cloned the proximal 1.5 kb region of the ovine MT 1 promoter. Sequence analysis revealed putative cis -elements for transcription factors involved in pituitary development, namely Pitx-1 and Egr-1, and multiple putative E-boxes, which are involved in both circadian and developmental gene regulation. Nuclear protein from ovine pars tuberalis (PT) cells, a site of high endogenous MT 1 expression, stimulated gene expression from a MT 1 expression construct, indicating the presence of a functional promoter. Pitx-1 was strongly expressed in the ovine PT and stimulated MT 1 promoter activity in transfection assays. Co-transfection with Egr-1 induced promoter-specific effects: Pitx-1-stimulated MT 1 activity was inhibited, whereas βLH promoter activity was enhanced. In addition to Pitx-1 the circadian clock genes Clock and Bmal1 were also expressed in the PT. However, despite multiple putative E-boxes in the MT 1 promoter, transfected Clock and Bmal1 were unable to regulate either basal or Pitx-1-stimulated MT 1 promoter activity. The current data, in conjunction with our previous study of the rat MT 1 promoter, suggests a general model in which melatonin receptor expression in the mammalian pituitary is determined by the developmentally changing balance between stimulatory and inhibitory transcription factors. Furthermore, our data suggest that circadian variation in MT 1 gene expression does not depend upon the direct action of circadian clock genes on E-box cis -elements.

In mammals, circadian and photoperiodic information is encoded in the pineal melatonin signal. The pars tuberalis (PT) of the pituitary is a melatonin target tissue, which transduces photoperiodic changes and drives seasonal changes in prolactin secretion from distal lactotroph cells. Measurement of photoperiodic time in the PT is believed to occur through melatonin dependent changes in clock gene expression, although it is unclear whether the PT should be considered a melatonin sensitive peripheral oscillator. We tested this hypothesis in the Siberian hamster (Phodopus sungorus) firstly by investigating the effects of melatonin injection, and secondly by determining whether temporal variation in gene expression within the PT persists in the absence of a rhythmic melatonin signal. Hamsters preconditioned to long days were treated with melatonin during the late light phase, to advance the timing of the nocturnal melatonin peak, or placed in constant light for one 24 h cycle, thereby suppressing endogenous melatonin secretion. Gene expression in the PT was measured by in situ hybridization. We show that melatonin rapidly induces cry1 mRNA expression without the need for a prolonged melatonin-free interval, acutely inhibits mt1 expression, advances the timing of peak rev-erb alpha expression and modulates per1 expression. With the exception of cry1, these genes continue to show temporal changes in expression over a first cycle in the absence of a melatonin signal. Our data are consistent with the hypothesis that the hamster PT contains a damped endogenous circadian oscillator, which requires a rhythmic melatonin signal for long-term synchronization.

In mammals, changing day length modulates endocrine rhythms via nocturnal melatonin secretion. Studies of the pituitary pars tuberalis (PT) suggest that melatonin-regulated clock gene expression is critical to this process. Here, we considered whether clock gene rhythms continue in the PT in the absence of melatonin and whether the effects of melatonin on the expression of these genes are temporally gated. Soay sheep acclimated to long photoperiod (LP) were transferred to constant light for 24 h, suppressing endogenous melatonin secretion. Animals were infused with melatonin at 4-h intervals across the final 24 h, and killed 3 h after infusion. The expression of five clock genes (Per1, Per2, Cry1, Rev-erbα, and Bmal1) was measured by in situ hybridization. In sham-treated animals, PT expression of Per1, Per2, and Rev-erbα showed pronounced temporal variation despite the absence of melatonin, with peak times occurring earlier than predicted under LP. The time of peak Bmal1 expression remained LP-like, whereas Cry1 expression was continually low. Melatonin infusion induced Cry1 expression at all times and suppressed other genes, but only when they showed high expression in sham-treated animals. Hence, 3 h after melatonin treatment, clock gene profiles were driven to a similar state, irrespective of infusion time. In contrast to the PT, melatonin infusions had no clear effect on clock gene expression in the suprachiasmatic nuclei. Our results provide the first example of acute sensitivity of multiple clock genes to one endocrine stimulus and suggest that rising melatonin levels may reset circadian rhythms in the PT, independently of previous phase.

During development, melatonin receptors are transiently expressed in multiple neuroendocrine tissues, suggesting a novel role for melatonin in developmental physiology. The best characterised model of melatonin signalling during development is the pars distalis of the rat pituitary. However, although many studies have characterised the postnatal decline of melatonin receptors in the rat pars distalis, the mechanism(s) that time the developmental onset of receptor expression during embryogenesis are unknown. Analysis of these mechanisms may yield important information regarding the putative role of melatonin in neuroendocrine development. Here, we report the expression of MT(1) melatonin receptor mRNA in the rat pituitary from embryonic day 15.5 (e15.5). Prior to e15.5, the homeodomain transcription factor Msx-1, an inhibitor of cellular differentiation, is widely expressed throughout the pituitary. In transient transfection experiments, Msx-1 potently inhibited pituitary homeobox-1 (Pitx-1)-induced MT(1) promoter activity and therefore may represent a key inhibitor of MT(1) expression in early pituitary development. During late embryogenesis, MT(1) mRNA was expressed in both the ventral and dorsal pituitary. Analysis of a 1.5-kb fragment of the rat MT(1) promoter revealed four putative cis-elements for the POU domain factor Pit-1, which is associated with mid-dorsal cell lineages. Although Pit-1 induced a strong, dose-dependent stimulation of MT(1) promoter activity in vitro, dual-labelled in situ hybridisation revealed no colocalisation of MT(1) and Pit-1 mRNAs in vivo at e19.5. By contrast, all MT(1) positive cells colocalised with alphaGSU and most with betaTSH mRNA. Our data therefore implicate the decline of Msx-1 expression as a key event that times the onset of melatonin receptor expression to the differentiation of endocrine cells types in the developing pituitary gland, and suggest that the melatonin-sensitive cells in the embryonic pituitary are primarily Pit-1-independent thyrotrophs in the rostral pituitary, with a secondary population of pars distalis gonadotrophs.

Day-length (photoperiod) is the primary environmental signal used to synchronise endogenous rhythms of physiology and behaviour. In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus house the master circadian clock. The SCN incorporate photoperiodic information and therefore measure both daily and seasonal time. Over the past decade, there have been significant advances in the understanding of the molecular basis of circadian clocks. It is now becoming apparent that the core molecular clock mechanism is itself regulated by photoperiod, although there is currently debate as to how this occurs. One recent model proposes that distinct groups of core 'clock genes' are associated with either morning or evening phases of the daily light/dark cycle. However, the validity of associating particular genes to morning and evening has been questioned. This article reviews the evidence for photoperiodic regulation of circadian clock function and then discusses alternative models that may explain the available data.

Photoperiod regulates the seasonal physiology of many mammals living in temperate latitudes. Photoperiodic information is decoded by the master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus and then transduced via pineal melatonin secretion. This neurochemical signal is interpreted by tissues expressing melatonin receptors (e.g. the pituitary pars tuberalis, PT) to drive physiological changes. In this study we analysed the photoperiodic regulation of the circadian clockwork in the SCN and PT of the Siberian hamster. Female hamsters were exposed to either long or short photoperiod for 8 weeks and sampled at 2-h intervals across the 24-h cycle. In the SCN, rhythmic expression of the clock genes Per1, Per2, Cry1, Rev-erbalpha, and the clock-controlled genes arginine vasopressin (AVP) and d-element binding protein (DBP) was modulated by photoperiod. All of these E-box-containing genes tracked dawn, with earlier peak mRNA expression in long, compared to short, photoperiod. This response occurred irrespective of the presence of additional regulatory cis-elements, suggesting photoperiodic regulation of SCN gene expression through a common E-box-related mechanism. In long photoperiod, expression of Cry1 and Per1 in the PT tracked the onset and offset of melatonin secretion, respectively. However, whereas Cry1 tracked melatonin onset in short period, Per1 expression was not detectably rhythmic. We therefore propose that, in the SCN, photoperiodic regulation of clock gene expression primarily occurs via E-boxes, whereas melatonin-driven signal transduction drives the phasing of a subset of clock genes in the PT, independently of the E-box.

Most mammals use changing annual day-length cycles to regulate pineal melatonin secretion and thereby drive many physiological rhythms including reproduction, metabolism, immune function, and pelage. Prolonged exposure to short winter day lengths results in refractoriness, a spontaneous reversion to long-day physiological status. Despite its critical role in the timing of seasonal rhythms, refractoriness remains poorly understood. The aim of this study was therefore to describe cellular and molecular mechanisms driving the seasonal secretion of a key hormone, prolactin, in refractory Syrian hamsters. We used recently developed single cell hybridization and reporter assays to show that this process is initiated by timed reactivation of endocrine signaling from the pars tuberalis (PT) region of the pituitary gland, a well-defined melatonin target site, causing renewed activation of prolactin gene expression. This timed signaling is independent of per1 clock gene expression in the suprachiasmatic nuclei and PT and of melatonin secretion, which continue to track day length. Within the PT, there is also a continued short day-like profile of ICER expression, suggesting that the change in hormone secretion is independent of cAMP signaling. Our data thus identify the PT as a key anatomical structure involved in endogenous seasonal timing mechanisms, which breaks from prevailing day length-induced gene expression.

Melatonin inhibits the gonadotropin-releasing hormone (GnRH)-stimulated secretion of luteinizing hormone and follicle-stimulating hormone from the pars distalis region of the neonatal rat pituitary gland. Over the initial weeks of postnatal life, this response to melatonin declines in parallel with a loss of iodo-melatonin binding sites. Although neonatal gonadotrophs have since been extensively used to study melatonin receptor signalling pathways, the mechanisms driving this phenomenon, together with its physiological significance, remain unknown. Melatonin receptors are expressed in the foetal pars distalis before activation of the GnRH system. Furthermore, the MT1 melatonin receptor promoter contains response elements for transcription factors involved in both pituitary differentiation and gonadotroph regulation. These data, coupled with the known ability of melatonin to regulate rhythmical gene expression in adult pars tuberalis cells, leads us to propose that melatonin acts in the developing animal as a regulator of internal synchrony between tissues.

As inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, statins are an important first-line treatment for hypercholesterolemia. However, a recognized side-effect of statin therapy is myopathy, which in severe cases can present as potentially fatal rhabdomyolysis. This represents an important impediment to successful statin therapy, and despite decades of research the molecular mechanisms underlying this side-effect remain unclear. Current evidence supports a role for reduced levels of mevalonate pathway intermediates, with the most accepted hypothesis being a reduction in isoprenoids formation, leading to faulty post-translational modifications of membrane-associated proteins. We have undertaken a comprehensive analysis of the impact of nine statins on two human cell lines; Huh7 hepatoma and RD rhabdomyosarcoma. In both cell lines, concentration-dependent inhibition of prenylation was observed for cerivastatin and simvastatin, which could be rescued with the pathway intermediate mevalonate; in general, muscle cells were more sensitive to this effect, as measured by the levels of unprenylated Rap1A, a marker for prenylation by geranylgeranyl transferase I. Concentration-dependent toxicity was observed in both cell lines, with muscle cells again being more sensitive. Importantly, there was no correlation between inhibition of prenylation and cell toxicity, suggesting they are not causally linked. The lack of a causal relationship was confirmed by the absence of cytotoxicity in all cell lines following exposure to specific inhibitors of geranylgeranyl transferases I and II, and farnesyl transferase. As such, we provide strong evidence against the commonly accepted hypothesis linking inhibition of prenylation and statin-mediated toxicity, with the two processes likely to be simultaneous but independent.

Circadian rhythms, metabolism and nutrition are closely interlinked. A great deal of recent research has investigated not only how aspects of metabolic physiology are driven by circadian clocks, but also how these circadian clocks are themselves sensitive to metabolic change. At the cellular level, novel feedback loops have been identified that couple circadian 'clock genes' and their proteins to expression of nuclear receptors, regulation of redox state and other major pathways. Using targeted disruption of circadian clocks, mouse models are providing novel insight into the role of tissue-specific clocks in glucose homeostasis and body weight regulation. The relationship between circadian rhythms and obesity appears complex, with variable alteration of rhythms in obese individuals. However, it is clear from animal studies that the timing and nutritional composition of meals can regulate circadian rhythms, particularly in peripheral tissues. Translation of these findings to human physiology now represents an important goal.

Syrian hamsters exhibit a marked seasonal variation in prolactin secretion. The aim of this study was to analyse the nature of the photoperiodic regulation of prolactin gene expression, and to define the role of melatonin and the pars tuberalis of the anterior pituitary in this process. Pituitary prolactin gene expression, restricted to the pars distalis, was increased in hamsters maintained in long daylengths (16 h : 8 h, light : dark) compared to hamsters exposed to short daylengths (8 h : 16 h, light : dark) for 8–12 weeks. Analysis of single cells by in situ hybridization showed that photoperiod had no effect on the percentage of pars distalis cells expressing prolactin mRNA, but shifted the frequency distribution of prolactin mRNA expression per cell, such that in long photoperiods a greater proportion of cells were recruited to a higher expressing population. In vitro coculture of hamster pars tuberalis fragments increased prolactin promoter-driven luciferase activity in stably transfected GH3 cells in a dose- and duration-dependent manner. Conditioned medium from hamster and ovine pars tuberalis also activated the prolactin promoter. Furthermore, basal and forskolin-stimulated conditioned medium from hamster pars tuberalis increased prolactin mRNA expression in primary cultures of pars distalis cells. Melatonin attenuated the activity of pars tuberalis-conditioned medium but had no direct effect on either prolactin mRNA expression or secretion in pars distalis cell cultures. Finally, pars tuberalis fragments from long photoperiod hamsters stimulated prolactin gene promoter activity to a greater extent than those from short photoperiod hamsters. In conclusion, this study provides the first evidence in a seasonal mammal that the synthesis of prolactin depends on photoperiodic modulation of a pars tuberalis-derived factor. Our data support further the hypothesis that seasonal modulation of prolactin gene expression depends upon a melatonin-dependent paracrine action of the pars tuberalis on pars distalis lactotrophic cells.

The effect of benzodiazepine agonists of varying efficacy on γ -aminobutyric acid A receptor α 1 subunit protein expression was determined in primary cultured cerebellar granule cells. After 48 h exposure to 1 μ M drug concentrations, flunitrazepam, diazepam, and the partial agonists Ro 19-8022 and bretazenil, but not the partial agonists Ro 42-8773, Ro 41-7812 or imidazenil, decreased α 1 subunit protein expression. The grading of effect of the benzodiazepine partial agonists on α 1 subunit protein expression is consistent with their agonist efficacies. This model, therefore, appears to act as a sensitive indicator of benzodiazepine agonist efficacy with the ability to differentiate between partial agonists.

Emerging links between circadian rhythms and metabolism promise much for the understanding of metabolic physiology and pathophysiology, in which white adipose tissue (WAT) plays a prominent role. Many WAT endocrine molecules, termed adipokines, display rhythmic plasma concentration. Moreover, similar to most other tissues, WAT exhibits widespread 24-h variation in gene expression, with approximately 20% of the murine adipose transcriptome estimated to undergo daily variation. A major limitation to human chronobiology research is the availability of physiologically defined peripheral tissues. To date most analyses of in vivo human peripheral clocks has been limited to blood leucocytes. However, subcutaneous adipose tissue represents a novel opportunity to study peripheral molecular rhythms that are of clearly defined metabolic relevance. This review summarises basic concepts of circadian and metabolic physiology before then comparing alternative protocols used to analyse the rhythmic properties of human adipose tissue.