Abstract Cold acclimation holds therapeutic potential for improving metabolic health. We previously demonstrated that repeated cold-induced shivering enhances insulin sensitivity in humans. However, the molecular pathways that underlie the skeletal muscle shivering response, and how these relate to beneficial physiological effects, remain poorly understood. In this study, we combined complementary bioinformatics approaches to allow in-depth analysis of the transcriptomic response of human skeletal muscle to repeated shivering. We identified a robust transcriptional signature and show a sex-specific component in the shivering skeletal muscle response, which seemed to diminish following cold adaptation. Our findings provide mechanistic insights into cold-induced muscle adaptations, shed light on potential interesting molecular targets for further investigation, and emphasize the importance of including both sexes in future cold acclimation studies.

We previously showed that nocturnal fat oxidation is reduced in older individuals with overweight/obesity and impaired glucose tolerance and insulin sensitivity compared with young lean individuals. Here, we compared nocturnal energy expenditure and substrate oxidation across groups varying in age, body composition and metabolic status to unravel factors underlying variations in nocturnal substrate metabolism. Data were collected from 18 previously conducted human clinical studies (N=187), all performed under conditions of energy balance with similar diet composition and meal timing. Individuals were categorised into four groups: young lean (YL); older lean (OL); older with overweight/obesity (OBE); and older with overweight/obesity and type 2 diabetes (T2D). Nocturnal energy expenditure and substrate oxidation were determined by whole-room indirect calorimetry, body composition was assessed by air-displacement plethysmography or dual energy x-ray absorptiometry, and glucose, insulin, HOMA-IR, NEFA and triglycerides were measured from fasted blood samples. Group comparisons for nocturnal energy expenditure and substrate oxidation were performed using Kruskal–Wallis tests, and over time using linear mixed models including group × time interactions, with Bonferroni correction applied to both analyses. Multivariate linear regression analysis was applied to identify whether age, sex, HOMA-IR, fasting NEFA, fasting triglycerides, fat mass and fat-free mass were independent factors of nocturnal energy expenditure and substrate oxidation. Nocturnal energy expenditure, adjusted for fat-free mass, was higher in OBE compared with YL and OL (p<0.01 for both); it was also higher in T2D compared with OL (p<0.01). Nocturnal fat oxidation, expressed as a percentage of energy expenditure, was lower in OBE (median: 46.28

Exercise is fundamental to healthy aging, yet how it mitigates age-related molecular changes and how fitness level shapes exercise responses remain unclear. To address these questions, we performed transcriptomics, lipidomics and metabolomics on skeletal muscle of young and older adults with differing physical function, both before and after an acute bout of submaximal exercise. At baseline, older adults exhibited reduced expression of genes associated with cellular respiration and energy metabolism compared to young adults with comparable activity levels. Here we found that 50% of these age-related differences were absent in trained older adults, resulting in profiles resembling those of young adults. Although all participants displayed transcriptional immune and stress responses upon acute exercise, the magnitude of these responses in older adults was positively correlated with their physical fitness. Integrated multiomic analyses further revealed links among mitochondrial respiration, lipid metabolism, stress responses and NAD+ biology. These findings demonstrate that sustained physical training transforms age-related molecular profiles and provide a molecular atlas for study of fitness-dependent aging mechanisms.

Physical exercise (PE) may be important for glucose metabolism. Therefore, this systematic review and meta-analysis aims to investigate the effect of PE performed in the morning versus afternoon or evening on glycemia parameters in human intervention studies. MEDLINE and Embase.com were searched until February 2023 for intervention studies in the general adult population, examining the effect the timing of PE on glycemia parameters after one or multiple bouts of exercise. Results were meta-analysed using a random-effects models where appropriate or were described using qualitative synthesis. 55,569 publications were screened for a series of reviews, of which 20 studies (680 participants) were included in this review. In studies including one bout of PE (n = 12), glucose levels measured directly after PE were 0.26 mmol/L (95

Abstract Impaired post-prandial skeletal muscle glucose uptake plays a pivotal role in the development of type 2 diabetes mellitus (T2DM), yet pharmacological strategies to enhance muscle glucose uptake are limited. Previous (pre)clinical research revealed that β2-adrenergic receptor (β2-AR) stimulation enhances glucose uptake, but its clinical relevance in individuals susceptible to developing T2DM is unknown. Here we determined in a double-blinded, placebo-controlled, crossover study (ClinicalTrials.gov-identifier: NCT04921306), the effects of a 4-week treatment with the β2-adrenergic agonist clenbuterol (40 μg/day) on insulin-stimulated glucose uptake in the quadriceps muscle (primary outcome) and brown adipose tissue (BAT) (secondary outcome) using 18F-FDG PET-MRI during a hyperinsulinemic-euglycemic clamp in individuals with overweight or obesity (age: 40-70 years, BMI: 25-35 kg/m2). A total of 14 participants were recruited and randomized. Insulin-stimulated glucose uptake tended to improve in vastus lateralis (15%, p = 0.072) and increased significantly in the hamstring (13%, p = 0.039) muscle, while BAT uptake (p = 0.720) remained unaffected. These findings suggest potential therapeutic benefits of β2-AR stimulation for improving muscle-specific glucose uptake in individuals with or at risk for developing diabetes.

AIMS:This systematic review and meta-analysis aims to summarize the effect of the timing of meal intake on glycaemic outcomes in human intervention studies. DATA SYNTHESIS:Medline and Embase.com were searched for intervention studies in the adult population directly comparing the timing of meal intake on glycaemic outcomes. Results were meta-analysed in random-effects models or qualitatively described using vote counting. Of 55,569 publications screened, 44 studies were included. Study duration was up to one week (n = 37) or 2-12 weeks (n = 7). Shorter studies demonstrated that early meal timing, compared to late meal timing, significantly lowers postprandial 2-h glucose (-1.51 [-2.03, -0.99] mmol/L, I2 = 79%) and postprandial peak glucose (-1.03 [-1.63, -0.44] mmol/L, I2 = 61%). Mean glucose over 24-h, largest glucose peak over 24-h, fasting glucose, fasting insulin and postprandial 2-h insulin also showed the same direction, albeit not significant. In longer studies, fasting glucose seemed lower in early meal timing (-0.05 [-0.13, 0.02] mmol/L, I2 = 27%). Conversely, fasting insulin and HOMA-IR showed no differences. CONCLUSIONS:This systematic review and meta-analysis shows physiological plausibility that early meal intake may be beneficial for glycaemic control, compared to late meal intake. However, this is only seen in studies with a duration of up to one week. Moreover, the high heterogeneity between study designs, interventions, and methods precludes definite conclusions and clinical recommendations. Larger and longer trials, that control for total energy intake, weight loss, and rhythms of other Zeitgebers, are needed before chrono-nutrition principles can be translated into clinical dietary guidelines for glycaemic control.

Repeated cold exposure with shivering has been proposed as a potential strategy to enhance glucose metabolism by increasing energy expenditure and substrate utilisation. However, acute effects/benefits of cold-induced shivering on glucose homeostasis in metabolically compromised individuals are unknown. Here, we aimed to determine whether cold exposure at two different intensities improves 24 h glucose homeostasis in individuals with prediabetes and type 2 diabetes. In a randomised crossover trial conducted in the South Limburg/Maastricht region of the Netherlands, men and postmenopausal women with prediabetes (n=12) and stable type 2 diabetes (n=12), aged 40–75 years, body mass index ≥27 and ≤35 kg/m2, non-smoking and sedentary, underwent two whole-body cold exposure sessions using a water-perfused suit. Session order was randomised using an online randomisation tool (randomizer.org); participants were masked to the cold exposure intensity received, but investigators were not. Sessions were designed to elicit 1.5-fold (mild, 15°C) and 2.5-fold (moderate, 4°C) increases in resting metabolic rate (RMR). Continuous glucose monitoring assessed interstitial glucose concentrations over 24 h periods before and after each intervention, with controlled diet and activity. Shivering was confirmed via indirect calorimetry and electromyography. In both study groups and periods, RMR increased significantly vs baseline (p<0.001 for all). In prediabetes, the increase in the final 1 h of cold was 1.53 × RMR in mild and 1.94 × RMR in moderate cold. In type 2 diabetes, the increase was 1.57 × RMR and 2.09 × RMR in the final 1 h of mild and moderate cold, respectively. In prediabetes, neither mild nor moderate cold exposure altered mean 24 h glucose levels. In contrast, after mild cold exposure the type 2 diabetes group exhibited a significant reduction in mean 24 h glucose levels (−0.6 ± 0.5 mmol/l, p=0.003) and fasting glucose (−0.6 ± 0.8 mmol/l, p=0.019), as well as an increase in time in normal range (+8.8 ± 10.3

AIMS:Circadian regulation of metabolism is an important factor in metabolic health, yet the role of rhythmic metabolites in Type 2 diabetes development remains poorly understood. This study investigated associations between circulating rhythmic metabolites and incident Type 2 diabetes risk and evaluated causal relationships using two-sample Mendelian randomisation. MATERIALS AND METHODS:We analysed longitudinal data from 9992 community-dwelling adults aged 45-85 years (49.1% male) in the Canadian Longitudinal Study on Aging with baseline (2012-2015) serum metabolomics data. Untargeted metabolomics profiling was conducted using ultrahigh-performance liquid chromatography-tandem mass spectrometry. Incident Type 2 diabetes at 3-year follow-up was assessed based on diabetes medication use and HbA1c level. Associations between rhythmic metabolites and diabetes risk were evaluated using multivariable binomial regression. Pathway and network analyses were conducted to explore underlying biological mechanisms. Causality was assessed using two-sample Mendelian randomisation for rhythmic metabolites significantly associated with diabetes risk. RESULTS:Altogether, 20 rhythmic metabolites were associated with Type 2 diabetes risk, including a subset overlapping with genetic predisposition to chronotype, suggesting potential circadian regulation. Key pathways included leucine, isoleucine and valine biosynthesis and degradation, and glycine, serine and threonine metabolism. Mendelian randomisation analyses revealed causal associations between higher levels of mannose, valine, isoleucine, threonine and sphingomyelin (d18:0/18:0, d19:0/17:0) and higher Type 2 diabetes risk, whereas creatine, glycine, 1-linoleoyl-GPC (18:2), 1-palmitoyl-2-oleoyl-GPE, 1-palmitoyl-2-linoleoyl-GPE (16:0/18:2) and 1-stearoyl-2-oleoyl-GPE (18:0/18:1) were protective. CONCLUSIONS:Disruptions in rhythmic metabolites are implicated in Type 2 diabetes pathophysiology through specific metabolic pathways, highlighting the potential for biomarkers to support circadian-based prevention strategies.

BACKGROUND:Misalignment of the endogenous circadian system may contribute to the risk of type 2 diabetes. This systematic review and meta-analysis examined the association between clock gene polymorphisms and glycemic parameters and type 2 diabetes. METHODS:Embase, Medline, and Web of Science databases were searched from inception to August 20, 2024. Empirical studies examining the association between core clock gene polymorphisms and type 2 diabetes and glycemic parameters, and studies examining non-core clock genes with information on environmental factors were included. A multi-level meta-analytical approach was used, and a weighted odds ratio was reported (PROSPERO, CRD42022337706). RESULTS:In total, 37 studies comprising 535,063 participants were included. CRY2 was associated with higher fasting blood glucose (OR: 1.07, 95 % CI: 1.03-1.11) and impaired glucose tolerance (OR: 1.02, CI: 1.00-1.04). Polymorphisms in MTNR1B were associated with a greater risk of type 2 diabetes. CLOCK was associated with lower risk of type 2 diabetes (OR: 0.94, CI: 0.89-1.00), and PER3 was associated with lower fasting insulin (OR: 0.94, CI: 0.91-0.97) and lower risk of insulin resistance (OR: 0.92, CI: 0.88-0.95). These associations reflect pooled variant-level effects within genes, and the effects of certain variants were modified by diet, alcohol consumption, physical activity, sleep, and length of daylight. CONCLUSIONS:Specific polymorphisms in circadian genes, including CRY2, MTNR1B, CLOCK, and PER3, were associated with glycemic parameters and type 2 diabetes risk. These associations may be influenced by lifestyle and environmental factors, and interventions targeting circadian alignment could potentially modify diabetes risk, although further research is needed.

Measuring biological age typically requires invasive and costly procedures. To address this, the MoveIt! Age Score was developed: a simple, scalable, and interpretable aging clock that predicts biological age using only wearable-derived steps data. MoveIt! Age was trained on steps data from the United States National Health and Nutrition Examination Survey (NHANES), using chronological age, maximum step count, and step count variability to predict PhenoAge, a blood biochemistry biological age score. MoveIt! Age performance was evaluated in two independent cohorts: Mitochondria and Muscle Health in Elderly (MitoHealth; N = 55; healthy young adults or older adults from the Netherlands) and Restoring Health of Acutely Unwell Adults (RESORT; N = 145; geriatric rehabilitation inpatients from Australia). In RESORT, MoveIt! Age was assessed and compared to SenoClock-BloodAge and PhenoAge (hematological aging clocks). Delta age was the predicted biological age minus chronological age. In the NHANES testing dataset, MoveIt! Age demonstrated high predictive accuracy of chronological age (r = 0.97, RMSE = 5.4 years) and was more significantly associated with mortality than PhenoAge. In MitoHealth, delta MoveIt! Age showed differences between young adults and older adults who were normal, healthy, or health-impaired, with MoveIt! Age more significantly associated with muscle NAD+ levels (r = −0.37, p = 0.023) than chronological age (p = 0.416). Delta MoveIt! Age associated more strongly than other clocks with physical function outcomes, including frailty, handgrip strength, and functional performance. These findings support MoveIt! Age as a practical tool to gain insights into biological age in both clinical and community settings.

BACKGROUND:Exercising at a specific time of day has the potential to mitigate the negative effects of disrupted circadian rhythms caused by irregular work and sleep schedules on the development of chronic diseases. Afternoon/evening exercise is postulated to be superior to morning exercise for various health outcomes, but patient acceptance of timed exercise remains unclear. The aim of this systematic review was to assess the impact of exercise timing on patient-reported outcomes (PROMs). METHODS:We conducted a systematic review, following Cochrane and PRISMA guidelines (PROSPERO: CRD42022322646). We systematically searched databases including MEDLINE, SCOPUS, Embase, APA PsycInfo, CINAHL, and Web of Science, to identify studies which reported on PROMs related to timed exercise interventions: either acutely after a bout of exercise or following extended training (>1 month). Studies were included if they reported primary data from randomized or non-randomized experiments of timed exercise interventions (against any comparator), published in English until August 2023 and reporting on any PROM. Machine-learning software (AR Reviews) was used to aid in abstract screening. Subsequently, two independent reviewers reviewed the included full texts, extracted study details (participants, interventions, outcomes), and evaluated the risk of bias using Cochrane tools (ROB-2 and ROBINS-I). Exercise interventions were summarized using the TIDieR reporting method and results were presented in accordance with the Synthesis Without Meta-analysis (SWiM) guidelines for systematic reviews. RESULTS:Seventeen studies with 403 participants were included in the review. The interventions varied widely in exercise modality, duration, and participant characteristics, contributing to substantial heterogeneity in the findings. Most studies found no significant impact of exercise timing on PROMs. There was some inconsistency between studies for certain outcomes. DISCUSSION:The review suggests that there are no clear detrimental effects of afternoon or evening exercise on PROMs compared to morning exercise. However, the lack of homogeneity in study populations and small sample sizes resulting in low power for PROM outcomes are major limitations of the research in this field. If future research confirms the metabolic advantages of afternoon/evening exercise, this may be an acceptable alternative for individuals.

Because 80%-90% of our time is spent indoors and daylight is the main synchronizer of the central biological clock, the chronic lack of daylight is increasingly considered as a risk factor for metabolic diseases, such as type 2 diabetes. In a randomized crossover design (NCT05263232), 13 individuals with type 2 diabetes were exposed to natural daylight facilitated through windows vs. constant artificial lighting during office hours for 4.5 consecutive days. Continuous glucose monitoring revealed that participants spent more time in the normal glucose range, and whole-body substrate metabolism shifted toward a greater reliance on fat oxidation during daylight. Primary myotubes cultured from skeletal muscle biopsies displayed a phase advance after daylight exposure. Multi-omic analyses revealed daylight-induced differences in serum metabolites, lipids, and monocyte transcripts. Our findings suggest that natural daylight exposure has a positive metabolic impact on individuals with type 2 diabetes and could support the treatment of metabolic diseases.

Objectives:To examine whether the amount and timing of moderate-to-vigorous physical activity (MVPA) was associated with sleep quality and duration in the general population. Methods:This is a cross-sectional analysis of data of a Dutch cohort collected between 2008 and 2012. Timing of physical activity (measured using an accelerometer) was categorized as performing most MVPA in morning (06:00-12:00), afternoon (12:00-18:00), evening (18:00-00:00), or even distribution of MVPA over the day (reference). Sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI). We estimated OR with 95 % CI of a poor score on individual PSQI components and global PSQI score using logistic regression while adjusting for relevant covariates. Results:We analyzed 736 participants, of whom 57 % women, aged 56 (6) years, BMI 26.1 (4.2) kg/m2). Amount of MVPA (hours/day) was associated with lower odds of fatigue-related dysfunction during daytime (OR: 0.54 0.32-0.94), but not with global PSQI score. Participants who performed most MVPA in the morning were less likely to report sleep disturbances (OR: 0.23, 95 % CI: 0.09-0.60), compared to participants with an even distribution of. Timing of MVPA was not associated with global PSQI score nor other components and CI were large. Conclusions:Differences in sleep quality are unlikely to be biological mechanisms underlying the previously shown associations between timing of physical activity and metabolic health.

NADH and NAD+ act as electron donors and acceptors and NAD+ was shown to stimulate mitochondrial biogenesis and metabolic health. We here develop a non-invasive Phosphorous Magnetic Resonance Spectroscopy (31P-MRS) method to quantify these metabolites in human skeletal muscle on a clinical 3 T MRI scanner. This new MR-sequence enables NADH and NAD+ quantification by suppressing alpha-ATP signal, normally overlapping with NADH and NAD+. The sequence is based on a double spin echo in combination with a modified z-Filter achieving strong alpha-ATP suppression with little effect on NAD+ and NADH. Here we test and validate it in phantoms and in humans by measuring reproducibility and detecting a physiological decrease in NAD+ and increase in NADH induced by ischemia. Furthermore, the 31P-MRS outcomes are compared to analysis in biopsies. Additionally, we show higher NAD+ and lower NADH content in physically active older adults compared to sedentary individuals, reflecting increased metabolic health. NADH and NAD+ act as electron donors and acceptors and NAD+ was shown to stimulate mitochondrial biogenesis and metabolic health. Here, the researchers developed and validated a non-invasive Phosphorous Magnetic Resonance Spectroscopy method to non-invasively quantify NAD+ and NADH in muscle on a clinical 3 T MRI scanner.

AIMS:Alterations in sleep timing can lead to disturbances in glycaemic control, although the evidence is inconsistent. Therefore, this systematic review summarizes results from human intervention studies of altered sleep timing on glycaemic outcomes. MATERIALS AND METHODS:As part of a broader search on the effect of altering timing of sleep, physical activity and dietary intake, Medline and Embase were searched from inception to February 2023, and subsequent reference searches were done. With the help of a machine learning-aided program 'ASReview', we selected any type of intervention study in the general adult population, which acutely delayed sleep by ≥2 h for at least one night, while the total time in bed was the same between early and late sleep. Quality assessment was done using the quality assessment tool for quantitative studies. RESULTS:In total, 14 studies (159 adults with normal or increased weight) were identified. Methodological quality was high (n = 4), moderate (n = 7) or low (n = 3). Acute delays of sleep onset showed unfavourable effects in 10 out of 27 measured glycaemic outcomes (one-six studies reported on each outcome) with outcomes mostly measured in the postprandial period, compared to (early) nighttime sleep. CONCLUSIONS:Acutely delaying sleep timing might have unfavourable effects on glycaemic outcomes, compared to (early) nighttime sleep. Future research does however need better controlled trials, also measuring and controlling sleep quantity, sleep quality, physical activity and dietary intake, with longer follow-up periods, consistent outcomes and designs and more diverse populations to provide targeted advice regarding the optimal timing for sleep. PROTOCOL REGISTRATION:This review is part of a larger search 'The effect of altering timing of physical activity, sleep and energy intake on glycaemia and Type 2 Diabetes risk in humans', of which the protocol was registered in the PROSPERO database on 27 November 2021 under number: CRD42021287828.

Complex lipids, essential components in biological processes, exhibit conserved age-related changes that alter membrane properties and cellular functions and are implicated as biomarkers and contributors to longevity and age-related diseases. While physical activity alleviates age-related comorbidities and physical impairments, comprehensive exploration of the underlying biological mechanisms, particularly at the level of complex lipids, remains limited. However, clinical studies suggest that physical activity may counteract these age-related lipidomic changes, presenting a promising avenue for intervention. We performed lipidomic profiling of plasma from an extensively characterized cohort of young and aged individuals. Annotating 1446 unique lipid species across 24 lipid classes, we found the most prominent difference in older adults was an accumulation of triacylglycerols (TGs), with lower physical activity levels associated with higher TG levels in plasma and reduced physical functionality. Remarkably, lipid species in the TG class did not accumulate uniformly. Rather, our study unveiled a negative correlation between higher physical activity levels and TGs with shorter chain lengths and more double bonds in this demographic. Overall, our research highlights that plasma TG length and saturation level can help mark healthy aging groups in humans. These findings deepen our understanding of how aging affects complex lipids and the influence of physical activity on this process.

ObjectiveSimultaneous activation of β2- and β3-adrenoceptors (ARs) improves whole-body metabolism via beneficial effects in skeletal muscle and brown adipose tissue (BAT). Nevertheless, high-efficacy agonists simultaneously targeting these receptors whilst limiting activation of β1-ARs – and thus inducing cardiovascular complications – are currently non-existent. Therefore, we here developed and evaluated the therapeutic potential of a novel β2- and β3-AR, named ATR-127, for the treatment of obesity and its associated metabolic perturbations in preclinical models.MethodsIn the developmental phase, we assessed the impact of ATR-127's on cAMP accumulation in relation to the non-selective β-AR agonist isoprenaline across various rodent β-AR subtypes, including neonatal rat cardiomyocytes. Following these experiments, L6 muscle cells were stimulated with ATR-127 to assess the impact on GLUT4-mediated glucose uptake and intramyocellular cAMP accumulation. Additionally, in vitro, and in vivo assessments are conducted to measure ATR-127's effects on BAT glucose uptake and thermogenesis. Finally, diet-induced obese mice were treated with 5mg/Kg ATR-127 for 21 days to investigate the effects on glucose homeostasis, body weight, fat mass, skeletal muscle glucose uptake, BAT thermogenesis and hepatic steatosis.ResultsExposure of L6 muscle cells to ATR-127 robustly enhanced GLUT4-mediated glucose uptake despite low intramyocellular cAMP accumulation. Similarly, ATR-127 markedly increased BAT glucose uptake and thermogenesis both in vitro and in vivo. Prolonged treatment of diet-induced obese mice with ATR-127 dramatically improved glucose homeostasis, an effect accompanied by decreases in body weight and fat mass. These effects were paralleled by an enhanced skeletal muscle glucose uptake, BAT thermogenesis, and improvements in hepatic steatosis.ConclusionsOur results demonstrate that ATR-127 is a highly effective, novel β2- and β3-ARs agonist holding great therapeutic promise for the treatment of obesity and its comorbidities, whilst potentially limiting cardiovascular complications. As such, the therapeutic effects of ATR-127 should be investigated in more detail in clinical studies.

Cold acclimation increases insulin sensitivity, and some level of muscle contraction appears to be needed for provoking this effect. Here 15 men and (postmenopausal) women with overweight or obesity, the majority of whom had impaired glucose tolerance, were intermittently exposed to cold to induce 1 h of shivering per day over 10 days. We determined the effect of cold acclimation with shivering on overnight fasted oral glucose tolerance (primary outcome) and on skeletal muscle glucose transporter 4 translocation (secondary outcome). We find that cold acclimation with shivering improves oral glucose tolerance, fasting glucose, triglycerides, non-esterified fatty acid concentrations and blood pressure. Cold acclimation with shivering may thus represent an alternative lifestyle approach for the prevention and treatment of obesity-related metabolic disorders. ClinicalTrials.gov registration: NCT04516018 .