Jonathan David Johnston

Experimental inversion of circadian and behavioural rhythms by 12-hours adversely affects markers of metabolic health. We investigated the effects of a more modest 5-hour delay in behavioural cycles. Fourteen participants completed an 8-day in-patient laboratory protocol, with controlled sleep-wake opportunities, light-dark cycles, and diet. The 5-hour delay in behavioural cycles was induced by delaying sleep opportunity. We measured: melatonin to confirm central circadian phase; fasting markers and postprandial metabolism; energy expenditure; subjective sleepiness; and appetite, throughout the waking period. After the phase delay, there was slower gastric emptying at breakfast, lower fasting plasma glucose, higher postprandial plasma glucose and triglycerides, and lower thermic effect of feeding. Any changes were abolished or attenuated within 48-72 hours. These data extend our previous findings, which showed no time-of-day effect in healthy adults on daytime energy expenditure or thermic effect of feeding when accounting for circadian variation in resting metabolic rate.

Circadian rhythms, metabolism, and nutrition are closely linked.¹ Timing of a 3-meal daily feeding pattern synchronises some human circadian rhythms.² Despite animal data showing anticipation of food availability, linked to a Food Entrainable Oscillator³, it is unknown whether human physiology predicts mealtimes and restricted food availability. In a controlled laboratory protocol, we tested the hypothesis that the human circadian system anticipates large meals. Twenty-four male participants undertook an 8-day laboratory study, with strict sleep-wake schedules, light-dark schedules, and food intake. For six days, participants consumed either hourly small meals throughout the waking period, or two large daily meals (7.5 and 14.5-h after wake-up). All participants then undertook a 37-hour constant routine. Interstitial glucose was measured every 15 minutes throughout the protocol. Hunger was assessed hourly during waking periods. Saliva melatonin was measured in the constant routine. During the 6-day feeding pattern, both groups exhibited increasing glucose concentration early each morning. In the small meal group, glucose concentrations continued to increase across the day. However, in the large meal group, glucose concentrations decreased from 2-h after waking until the first meal. Average 24-h glucose concentration did not differ between groups. In the constant routine, there was no difference in melatonin onset between groups, but antiphasic glucose rhythms were observed, with low glucose at the time of previous meals in the large meal group. Moreover, in the large meal group, constant routine hunger scores increased before the predicted meal times. These data support the existence of human food anticipation.

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.

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.

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.

This pilot study explored the feasibility of a moderate time-restricted feeding (TRF) intervention and its effects on adiposity and metabolism. For ten weeks, a free-living TRF group (n=9) delayed breakfast and advanced dinner by 1.5-hours each. Changes in dietary intake, adiposity and fasting biochemistry (glucose, insulin, lipids) were compared to controls (n=7) who maintained habitual feeding patterns. Thirteen participants (29±2kg/m2) completed. The average daily feeding interval was successfully reduced in the TRF group (743±32 to 517±22 mins/day (p<0.001); n=7), although questionnaire responses indicated that social eating/drinking opportunities were negatively impacted. TRF participants reduced total daily energy intake (p=0.019) despite ad libitum food 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 maximize the long-term sustainability of TRF.

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.

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, glycerophospholipids 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 correlated with 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.

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-hour 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-hour intervals, beginning either 0.5 (early) or 5.5 (late) hours 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-hour 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 hours (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 hours (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 synchronising peripheral circadian rhythms in humans, and may have particular relevance for patients with circadian rhythm disorders, shift workers, and transmeridian travellers.