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.
Dietary sugars are linked to the development of non-alcoholic fatty liver disease (NAFLD) and dyslipidaemia, but it is unknown if NAFLD itself influences the effects of sugars on plasma lipoproteins. To study this further, men with NAFLD (n=11) and low liver fat ‘controls’ (n= 14) were fed two iso-energetic diets, high or low in sugars (26% or 6% total energy) for 12 weeks, in a randomised, cross-over design. Fasting plasma lipid and lipoprotein kinetics were measured after each diet by stable isotope trace-labelling. There were significant differences in the production and catabolic rates of VLDL subclasses between men with NAFLD and controls, in response to the high and low sugar diets. Men with NAFLD had higher plasma concentrations of VLDL1-triacylglycerol (TAG) after the high (P
Studying communities with different levels of urbanization may further the understanding of risk factors underlying metabolic diseases. The present study is unique by comprising detailed assessment of sleep and activity, biological rhythms, and metabolic factors of men from the same geographical location and place of birth that reside in different, rural vs. town, stages of urbanization. Sleep patterns, activity, and metabolic indicators in two groups (rural, n = 22 and town/urban, n = 20) of men residing in an Amazonian community (Xapuri, Acre, Brazil) were compared. Sociodemographic, anthropometric, and metabolic variables – fasting glucose, insulin resistance, triglycerides, total HDL cholesterol, LDL cholesterol, and VLDL cholesterol – were assessed. Sleep patterns, light exposure, and physical activity levels were additionally assessed by actigraphy, plus daily activities were recorded in diaries for 10 days. Town/urban dwellers were found to have significantly higher body weight, fasting glucose, insulin levels, and insulin resistance than rural dwellers, whereas triglycerides levels were similar. Town/Urban dwellers had shorter sleep duration (p < .01) and later sleep onset and offset times (p = .01). Our findings show an association between stage of urbanization and presence of risk factors for metabolic disorders, such as overweight, insulin resistance, increased glucose levels, short sleep duration, and less natural light exposure during work times.
Urbanization has contributed to extended wakefulness, which may in turn be associated with eating over a longer period. Here, we present a field study conducted in four groups with different work hours and places of living in order to investigate eating behavior (duration, content, and timing). Anthropometric measures were taken from the participants (rural (n = 22); town (n = 19); city-day workers (n = 11); city-night workers (n = 14)). In addition, a sociodemographic questionnaire was self-answered and 24-h food recalls were applied for three days. The 24-h food recalls revealed that fat intake varied according to the groups, with the highest consumption by the city-day workers. By contrast, city-day workers had the lowest intake of carbohydrate, whereas the rural group had the highest. In general, all groups had some degree of inadequacy in food consumption. Eating duration was negatively correlated with total energy intake, fat, and protein consumption in the rural and town groups. There was a positive correlation between body mass index and eating duration in both city groups. The rural group had the earliest start time of eating, and this was associated with a lower body mass index. This study suggested that food content and timing, as well as eating duration, differed according to place of living, which in turn may be linked to lifestyle.
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. It is widely accepted that obesity is the main risk factor for type 2 diabetes mellitus (T2DM) (1). The progression from obesity to T2DM is largely a result of comorbidities, such as systemic inflammation and insulin resistance. Metabolic profiling by using targeted metabolomics, which enables the quantification of more than 100 low-MW intermediates of metabolism, is increasingly used to characterize (pre)diabetic phenotypes and has identified differences in metabolite profiles between those individuals who are obese and those with T2DM (2–5). Recent work by our group and others has shown a 24-h variation in the human metabolome in healthy individuals, analyzed by using a range of analytical platforms (6–12), which has demonstrated that an estimated 15–20% of the metabolome is rhythmic in blood (6, 7). Transgenic mice that carry targeted genetic manipulation of circadian clock genes also exhibit a phenotype that involves defective metabolism, and associations between the circadian timing system and metabolic responses have been reported in humans (13). Reviews of these studies, including the higher incidence of obesity, T2DM, and related disorders in shift workers, have recently been published (14, 15). Existing metabolomics studies in T2DM have been restricted to the analysis of single-time-point, mostly fasting, samples, which cannot characterize the effect of increased body mass and T2DM on rhythmic metabolites. Characterizing 24-h metabolite rhythms in T2DM compared with age- and body mass–matched controls may therefore provide novel insights into the etiology and progression of T2DM. Identification of the optimal time of day for blood sampling—when metabolite levels show the biggest difference between T2DM and controls—would also provide more discriminating diagnostic biomarkers, rather than taking a single morning fasting sample. We thus assessed the effect of increased body mass [overweight/obese (OW/OB)] and T2DM on 24-h rhythms of circulating metabolites in men by using a quantitative targeted liquid chromatography/mass spectrometry (LC/MS) metabolomics approach. As T2DM is often accompanied by obesity, we set out to distinguish the effects of T2DM from those of increased body mass by incorporating both a lean and an OW/OB control group into the current study design.
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.
Intrinsically photosensitive retinal ganglion cells (ipRGCs), whose photopigment melanopsin has a peak of sensitivity in the short wavelength range of the spectrum, constitute a common light input pathway to the olivary pretectal nucleus (OPN), the pupillary light reflex (PLR) regulatory centre, and to the suprachiasmatic nuclei (SCN), the major pacemaker of the circadian system. Thus, evaluating PLR under short wavelength light (λmax 500 nm) and creating an integrated PLR parameter, as a possible tool to indirectly assess the status of the circadian system, becomes of interest. Nine monochromatic, photon-matched light stimuli (300 s), in 10 nm increments from λmax 420 to 500 nm were administered to 15 healthy young participants (8 females), analyzing: i) the PLR; ii) wrist temperature (WT) and motor activity rhythms (WA), iii) light exposure (L) pattern and iv) diurnal preference (Horne- Östberg), sleep quality (Pittsburgh) and daytime sleepiness (Epworth). Linear correlations between the different PLR parameters and circadian status index obtained from WT, WA and L recordings and scores from questionnaires were calculated. In summary, we found markers of robust circadian rhythms, namely high stability, reduced fragmentation, high amplitude, phase advance and low internal desynchronization, were correlated with a reduced PLR to 460–490 nm wavelengths. Integrated circadian (CSI) and PLR (cp-PLR) parameters are proposed, that also showed an inverse correlation. These results demonstrate, for the first time, the existence of a close relationship between the circadian system robustness and the pupillary reflex response, two non-visual functions primarily under melanopsin-ipRGC input.
The pupillary light reflex (PLR) is a neurological reflex driven by rods, cones, and melanopsin-containing retinal ganglion cells. Our aim was to achieve a more precise picture of the effects of 5-min duration monochromatic light stimuli, alone or in combination, on the human PLR, to determine its spectral sensitivity and to assess the importance of photon flux. Using pupillometry, the PLR was assessed in 13 participants (6 women) aged 27.2 ± 5.41 years (mean ± SD) during 5-min light stimuli of purple (437 nm), blue (479 nm), red (627 nm), and combinations of red+purple or red+blue light. In addition, nine 5-min, photon-matched light stimuli, ranging in 10 nm increments peaking between 420 and 500 nm were tested in 15 participants (8 women) aged 25.7 ± 8.90 years. Maximum pupil constriction, time to achieve this, constriction velocity, area under the curve (AUC) at short (0–60 s), and longer duration (240–300 s) light exposures, and 6-s post-illumination pupillary response (6-s PIPR) were assessed. Photoreceptor activation was estimated by mathematical modeling. The velocity of constriction was significantly faster with blue monochromatic light than with red or purple light. Within the blue light spectrum (between 420 and 500 nm), the velocity of constriction was significantly faster with the 480 nm light stimulus, while the slowest pupil constriction was observed with 430 nm light. Maximum pupil constriction was achieved with 470 nm light, and the greatest AUC0−60 and AUC240−300 was observed with 490 and 460 nm light, respectively. The 6-s PIPR was maximum after 490 nm light stimulus. Both the transient (AUC0−60) and sustained (AUC240−300) response was significantly correlated with melanopic activation. Higher photon fluxes for both purple and blue light produced greater amplitude sustained pupillary constriction. The findings confirm human PLR dependence on wavelength, monochromatic or bichromatic light and photon flux under 5-min duration light stimuli. Since the most rapid and high amplitude PLR occurred within the 460–490 nm light range (alone or combined), our results suggest that color discrimination should be studied under total or partial substitution of this blue light range (460–490 nm) by shorter wavelengths (~440 nm). Thus for nocturnal lighting, replacement of blue light with purple light might be a plausible solution to preserve color discrimination while minimizing melanopic activation.