Characterising 24-h skeletal muscle gene expression alongside metabolic & endocrine responses under diurnal conditions.

CONTEXT: Skeletal muscle plays a central role in the storage, synthesis, and breakdown of nutrients, yet little research has explored temporal responses of this human tissue, especially with concurrent measures of systemic biomarkers of metabolism. OBJECTIVE: To characterise temporal profiles in skeletal muscle expression of genes involved in carbohydrate metabolism, lipid metabolism, circadian clocks, and autophagy and descriptively relate them to systemic metabolites and hormones during a controlled laboratory protocol. METHODS: Ten healthy adults (9M/1F, mean ± SD: age: 30 ± 10 y; BMI: 24.1 ± 2.7 kg·m-2) rested in the laboratory for 37 hours with all data collected during the final 24 hours of this period (i.e., 0800-0800 h). Participants ingested hourly isocaloric liquid meal replacements alongside appetite assessments during waking before a sleep opportunity from 2200-0700 h. Blood samples were collected hourly for endocrine and metabolite analyses, with muscle biopsies occurring every 4 h from 1200 h to 0800 h the following day to quantify gene expression. RESULTS: Plasma insulin displayed diurnal rhythmicity peaking at 1804 h. Expression of skeletal muscle genes involved in carbohydrate metabolism (Name - Acrophase; GLUT4 - 1440 h; PPARGC1A -1613 h; HK2 - 1824 h) and lipid metabolism (FABP3 - 1237 h; PDK4 - 0530 h; CPT1B - 1258 h) displayed 24 h rhythmicity that reflected the temporal rhythm of insulin. Equally, circulating glucose (0019 h), NEFA (0456 h), glycerol (0432 h), triglyceride (2314 h), urea (0046 h), CTX (0507 h) and cortisol concentrations (2250 h) also all displayed diurnal rhythmicity. CONCLUSION: Diurnal rhythms were present in human skeletal muscle gene expression as well systemic metabolites and hormones under controlled diurnal conditions. The temporal patterns of genes relating to carbohydrate and lipid metabolism alongside circulating insulin are consistent with diurnal rhythms being driven in part by the diurnal influence of cyclic feeding and fasting.

Daily rhythms of diabetogenic factors in men: role of type 2 diabetes and body weight.

Obesity is a major cause of type 2 diabetes. Transition from obesity to type 2 diabetes manifests in the dysregulation of hormones controlling glucose homeostasis and inflammation. As metabolism is a dynamic process that changes across 24 h, we assessed diurnal rhythmicity in a panel of 10 diabetes-related hormones. Plasma hormones were analysed every 2 h over 24 h in a controlled laboratory study with hourly isocaloric drinks during wake. To separate effects of body mass from type 2 diabetes, we recruited three groups of middle-aged men: an overweight (OW) group with type 2 diabetes and two control groups (lean and OW). Average daily concentrations of glucose, triacylglycerol and all the hormones except visfatin were significantly higher in the OW group compared to the lean group (P < 0.001). In type 2 diabetes, glucose, insulin, C-peptide, glucose-dependent insulinotropic peptide and glucagon-like peptide-1 increased further (P < 0.05), whereas triacylglycerol, ghrelin and plasminogen activator inhibitor-1 concentrations were significantly lower compared to the OW group (P < 0.001). Insulin, C-peptide, glucose-dependent insulinotropic peptide and leptin exhibited significant diurnal rhythms in all study groups (P < 0.05). Other hormones were only rhythmic in 1 or 2 groups. In every group, hormones associated with glucose regulation (insulin, C-peptide, glucose-dependent insulinotropic peptide, ghrelin and plasminogen activator inhibitor-1), triacylglycerol and glucose peaked in the afternoon, whereas glucagon and hormones associated with appetite and inflammation peaked at night. Thus being OW with or without type 2 diabetes significantly affected hormone concentrations but did not affect the timing of the hormonal rhythms.

Exploring Rates of Adherence and Barriers to Time-Restricted 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.

Human glucose rhythms and subjective hunger anticipate meal timing.

Circadian rhythms, metabolism, and nutrition are closely linked.1 Timing of a three-meal daily feeding pattern synchronizes some human circadian rhythms.2 Despite animal data showing anticipation of food availability, linked to a food-entrainable oscillator,3 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 6 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-h constant routine. Interstitial glucose was measured every 15 min 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.

Timing of daily calorie loading affects appetite and hunger responses without changes in energy metabolism in healthy subjects with obesity.

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.

The [13 C]octanoic acid breath test for gastric emptying quantification: A focus on nutrition and modeling.

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 CO2 breath test using the stable isotope [13 C]octanoic acid with semi-mechanistic modeling could foster a wider application in nutritional studies to further understand the metabolic response to food. Here, we discuss the use of the [13 C]octanoic acid breath test to label the solid phase of a meal, and the factors that influence GE to support mechanistic studies. Furthermore, we give an overview of existing mathematical models for the interpretation of the breath test data and how much nutritional studies could benefit from a physiological based pharmacokinetic model approach.

In vitro characterisation of murine pre-adipose nucleated cells reveals electrophysiological cycles associated with biological clocks.

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.

Pericytes' Circadian Clock Affects Endothelial Cells' Synchronization and Angiogenesis in a 3D Tissue Engineered Scaffold.

Angiogenesis, the formation of new capillaries from existing ones, is a fundamental process in regenerative medicine and tissue engineering. While it is known to be affected by circadian rhythms in vivo, its peripheral regulation within the vasculature and the role it performs in regulating the interplay between vascular cells have not yet been investigated. Peripheral clocks within the vasculature have been described in the endothelium and in smooth muscle cells. However, to date, scarce evidence has been presented regarding pericytes, a perivascular cell population deeply involved in the regulation of angiogenesis and vessel maturation, as well as endothelial function and homeostasis. More crucially, pericytes are also a promising source of cells for cell therapy and tissue engineering. Here, we established that human primary pericytes express key circadian genes and proteins in a rhythmic fashion upon synchronization. Conversely, we did not detect the same patterns in cultured endothelial cells. In line with these results, pericytes' viability was disproportionately affected by circadian cycle disruption, as compared to endothelial cells. Interestingly, endothelial cells' rhythm could be induced following exposure to synchronized pericytes in a contact co-culture. We propose that this mechanism could be linked to the altered release/uptake pattern of lactate, a known mediator of cell-cell interaction which was specifically altered in pericytes by the knockout of the key circadian regulator Bmal1. In an angiogenesis assay, the maturation of vessel-like structures was affected only when both endothelial cells and pericytes did not express Bmal1, indicating a compensation system. In a 3D tissue engineering scaffold, a synchronized clock supported a more structured organization of cells around the scaffold pores, and a maturation of vascular structures. Our results demonstrate that pericytes play a critical role in regulating the circadian rhythms in endothelial cells, and that silencing this system disproportionately affects their pro-angiogenic function. Particularly, in the context of tissue engineering and regenerative medicine, considering the effect of circadian rhythms may be critical for the development of mature vascular structures and to obtain the maximal reparative effect.

Circadian Rhythms in Resting Metabolic Rate Account for Apparent Daily Rhythms in the Thermic Effect of Food.

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 ±â€…5.6) (mean ±â€…SEM) was 1.6 times greater than TEF at lunch (36.3 kcal ±â€…8.4) and 2.4 times greater than dinner TEF (25.2 kcal ±â€…9.6) (P = .022). However, adjusting for modeled circadian RMR nullified any differences between breakfast (54.1 kcal ±â€…30.8), lunch (49.5 kcal ±â€…29.4), and dinner (49.1 kcal ±â€…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.

Identification of factors influencing motivation to undertake time-restricted feeding in humans.

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.