Multi-organ transcriptome atlas of a mouse model of relative energy deficiency in sport.

Insufficient energy intake to meet energy expenditure demands of physical activity can result in systemic neuroendocrine and metabolic abnormalities in activity-dependent anorexia and relative energy deficiency in sport (REDs). REDs affects >40% of athletes, yet the lack of underlying molecular changes has been a hurdle to have a better understanding of REDs and its treatment. To assess the molecular changes in response to energy deficiency, we implemented the "exercise-for-food" paradigm, in which food reward size is determined by wheel-running activity. By using this paradigm, we replicated several aspects of REDs in female and male mice with high physical activity and gradually reduced food intake, which results in weight loss, compromised bone health, organ-specific mass changes, and altered rest-activity patterns. By integrating transcriptomics of 19 different organs, we provide a comprehensive dataset that will guide future understanding of REDs and may provide important implications for metabolic health and (athletic) performance.

Time-Restricted Feeding Reduces Atherosclerosis in LDLR KO Mice but Not in ApoE Knockout Mice.

BACKGROUND: Dyslipidemia increases cardiovascular disease risk, the leading cause of death worldwide. Under time-restricted feeding (TRF), wherein food intake is restricted to a consistent window of <12 hours, weight gain, glucose intolerance, inflammation, dyslipidemia, and hypercholesterolemia are all reduced in mice fed an obesogenic diet. LDLR (low-density lipoprotein receptor) mutations are a major cause of familial hypercholesterolemia and early-onset cardiovascular disease. METHODS: We subjected benchmark preclinical models, mice lacking LDLR-knockout or ApoE knockout to ad libitum feeding of an isocaloric atherogenic diet either ad libitum or 9 hours TRF for up to 13 weeks and assessed disease development, mechanism, and global changes in hepatic gene expression and plasma lipids. In a regression model, a subset of LDLR-knockout mice were ad libitum fed and then subject to TRF. RESULTS: TRF could significantly attenuate weight gain, hypercholesterolemia, and atherosclerosis in mice lacking the LDLR-knockout mice under experimental conditions of both prevention and regression. In LDLR-knockout mice, increased hepatic expression of genes mediating ß-oxidation during fasting is associated with reduced VLDL (very-low-density lipoprotein) secretion and lipid accumulation. Additionally, increased sterol catabolism coupled with fecal loss of cholesterol and bile acids contributes to the atheroprotective effect of TRF. Finally, TRF alone or combined with a cholesterol-free diet can reduce atherosclerosis in LDLR-knockout mice. However, mice lacking ApoE, which is an important protein for hepatic lipoprotein reuptake do not respond to TRF. CONCLUSIONS: In a preclinical animal model, TRF is effective in both the prevention and regression of atherosclerosis in LDLR knockout mice. The results suggest TRF alone or in combination with a low-cholesterol diet can be a lifestyle intervention for reducing cardiovascular disease risk in humans.

Increased central obesity correlates with physical activity and food processing in recently diagnosed multiple sclerosis.

BACKGROUND: Environmental and lifestyle factors are associated with an increased risk of Multiple Sclerosis (MS). Metabolic syndrome (MetS) contributes to systemic inflammation, which is associated with poorer MS disease evolution. We compared persons with MS (PwMS) and controls to assess metabolic and lifestyle parameters associated with MS. METHODS: We pooled data from two prospective observational studies with the same eligibility criteria, matching PwMS and controls (1:2 ratio) by sex, age, and body mass index (BMI). We compared anthropometric, biological and lifestyle parameters, including sleep and physical activity. RESULTS: We included 53 PwMS and 106 controls with a median age of 35 years and 79% of women. PwMS had low Expanded Disability Status Scale (median 1.5). Compared to controls, PwMS had increased waist-to-hip (p<0.001) and waist-to-height (p=0.007) ratios, and practiced less physical activity (p=0.03). In regression models, lifestyle factors with the strongest factor loadings to predict central obesity were processed food consumption, and vigorous physical activity. DISCUSSION: Although both groups were matched by age, sex, and BMI, we found increased central obesity in PwMS. Even with minimal neurological impairment, PwMS practiced less physical activity. This suggests that improvement of lifestyle and metabolic parameters should be targeted in MS.

International consensus on fasting terminology.

Although fasting is increasingly applied for disease prevention and treatment, consensus on terminology is lacking. Using Delphi methodology, an international, multidisciplinary panel of researchers and clinicians standardized definitions of various fasting approaches in humans. Five online surveys and a live online conference were conducted with 38 experts, 25 of whom completed all 5 surveys. Consensus was achieved for the following terms: "fasting" (voluntary abstinence from some or all foods or foods and beverages), "modified fasting" (restriction of energy intake to max. 25% of energy needs), "fluid-only fasting," "alternate-day fasting," "short-term fasting" (lasting 2-3 days), "prolonged fasting" (≥4 consecutive days), and "religious fasting." "Intermittent fasting" (repetitive fasting periods lasting ≤48 h), "time-restricted eating," and "fasting-mimicking diet" were discussed most. This study provides expert recommendations on fasting terminology for future research and clinical applications, facilitating communication and cross-referencing in the field.

Diet and Meal Pattern Determinants of Glucose Levels and Variability in Adults with and without Prediabetes or Early-Onset Type 2 Diabetes: A Pilot Study.

This observational pilot study examined the association between diet, meal pattern and glucose over a 2-week period under free-living conditions in 26 adults with dysglycemia (D-GLYC) and 14 with normoglycemia (N-GLYC). We hypothesized that a prolonged eating window and late eating occasions (EOs), along with a higher dietary carbohydrate intake, would result in higher glucose levels and glucose variability (GV). General linear models were run with meal timing with time-stamped photographs in real time, and diet composition by dietary recalls, and their variability (SD), as predictors and glucose variables (mean glucose, mean amplitude of glucose excursions [MAGE], largest amplitude of glucose excursions [LAGE] and GV) as dependent variables. After adjusting for calories and nutrients, a later eating midpoint predicted a lower GV (ß = -2.3, SE = 1.0, p = 0.03) in D-GLYC, while a later last EO predicted a higher GV (ß = 1.5, SE = 0.6, p = 0.04) in N-GLYC. A higher carbohydrate intake predicted a higher MAGE (ß = 0.9, SE = 0.4, p = 0.02) and GV (ß = 0.4, SE = 0.2, p = 0.04) in N-GLYC, but not D-GLYC. In summary, our data suggest that meal patterns interact with dietary composition and should be evaluated as potential modifiable determinants of glucose in adults with and without dysglycemia. Future research should evaluate causality with controlled diets.

Effects of 3 months of 10-h per-day time-restricted eating and 3 months of follow-up on bodyweight and cardiometabolic health in Danish individuals at high risk of type 2 diabetes: the RESET single-centre, parallel, superiority, open-label, randomised controlled trial.

BACKGROUND: Time-restricted eating (TRE) has been suggested to be a simple, feasible, and effective dietary strategy for individuals with overweight or obesity. We aimed to investigate the effects of 3 months of 10-h per-day TRE and 3 months of follow-up on bodyweight and cardiometabolic risk factors in individuals at high risk of type 2 diabetes. METHODS: This was a single-centre, parallel, superiority, open-label randomised controlled clinical trial conducted at Steno Diabetes Center Copenhagen (Denmark). The inclusion criteria were age 30-70 years with either overweight (ie, BMI ≥25 kg/m2) and concomitant prediabetes (ie, glycated haemoglobin [HbA1c] 39-47 mmol/mol) or obesity (ie, BMI ≥30 kg/m2) with or without prediabetes and a habitual self-reported eating window (eating and drinking [except for water]) of 12 h per day or more every day and of 14 h per day or more at least 1 day per week. Individuals were randomly assigned 1:1 to 3 months of habitual living (hereafter referred to as the control group) or TRE, which was a self-selected 10-h per-day eating window placed between 0600 h and 2000 h. Randomisation was done in blocks varying in size and was open for participants and research staff, but outcome assessors were masked during statistical analyses. The randomisation list was generated by an external statistician. The primary outcome was change in bodyweight, assessed after 3 months (12 weeks) of the intervention and after 3 months (13 weeks) of follow-up. Adverse events were reported and registered at study visits or if participants contacted study staff to report events between visits. This trial is registered on ClinicalTrials.gov (NCT03854656). FINDINGS: Between March 12, 2019, and March 2, 2022, 100 participants (66 [66%] were female and 34 [34%] were male; median age 59 years [IQR 52-65]) were enrolled and randomly assigned (50 to each group). Of those 100, 46 (92%) in the TRE group and 46 (92%) in the control group completed the intervention period. After 3 months of the intervention, there was no difference in bodyweight between the TRE group and the control group (-0·8 kg, 95% CI -1·7 to 0·2; p=0·099). Being in the TRE group was not associated with a lower bodyweight compared with the control group after subsequent 3-month follow-up (-0·2 kg, -1·6 to 1·2). In the per-protocol analysis, participants who completed the intervention in the TRE group lost 1·0 kg (-1·9 to -0·0; p=0·040) bodyweight compared with the control group after 3 months of intervention, which was not maintained after the 3-month follow-up period (-0·4 kg, -1·8 to 1·0). During the trial and follow-up period, one participant in the TRE group reported a severe adverse event: development of a subcutaneous nodule and pain when the arm was in use. This side-effect was evaluated to be related to the trial procedures. INTERPRETATION: 3 months of 10-h per-day TRE did not lead to clinically relevant effects on bodyweight in middle-aged to older individuals at high risk of type 2 diabetes. FUNDING: Novo Nordisk Foundation, Aalborg University, Helsefonden, and Innovation Fund Denmark.

Diurnal expression of Dgat2 induced by time-restricted feeding maintains cardiac health in the Drosophila model of circadian disruption.

Circadian disruption is associated with an increased risk of cardiometabolic disorders and cardiac diseases. Time-restricted feeding/eating (TRF/TRE), restricting food intake within a consistent window of the day, has shown improvements in heart function from flies and mice to humans. However, whether and how TRF still conveys cardiac benefits in the context of circadian disruption remains unclear. Here, we demonstrate that TRF sustains cardiac performance, myofibrillar organization, and regulates cardiac lipid accumulation in Drosophila when the circadian rhythm is disrupted by constant light. TRF induces oscillations in the expression of genes associated with triglyceride metabolism. In particular, TRF induces diurnal expression of diacylglycerol O-acyltransferase 2 (Dgat2), peaking during the feeding period. Heart-specific manipulation of Dgat2 modulates cardiac function and lipid droplet accumulation. Strikingly, heart-specific overexpression of human Dgat2 at ZT 0-10 significantly improves cardiac performance in flies exposed to constant light. We have demonstrated that TRF effectively attenuates cardiac decline induced by circadian disruption. Moreover, our data suggests that diurnal expression of Dgat2 induced by TRF is beneficial for heart health under circadian disruption. Overall, our findings have underscored the relevance of TRF in preserving heart health under circadian disruptions and provided potential targets, such as Dgat2, and strategies for therapeutic interventions in mitigating cardiac aging, metabolic disorders, and cardiac diseases in humans.

Energy balance drives diurnal and nocturnal brain transcriptome rhythms.

Plasticity in daily timing of activity has been observed in many species, yet the underlying mechanisms driving nocturnality and diurnality are unknown. By regulating how much wheel-running activity will be rewarded with a food pellet, we can manipulate energy balance and switch mice to be nocturnal or diurnal. Here, we present the rhythmic transcriptome of 21 tissues, including 17 brain regions, sampled every 4 h over a 24-h period from nocturnal and diurnal male CBA/CaJ mice. Rhythmic gene expression across tissues comprised different sets of genes with minimal overlap between nocturnal and diurnal mice. We show that non-clock genes in the suprachiasmatic nucleus (SCN) change, and the habenula was most affected. Our results indicate that adaptive flexibility in daily timing of behavior is supported by gene expression dynamics in many tissues and brain regions, especially in the habenula, which suggests a crucial role for the observed nocturnal-diurnal switch.

Neuronal reprogramming of mouse and human fibroblasts using transcription factors involved in suprachiasmatic nucleus development.

The hypothalamic suprachiasmatic nucleus (SCN) is composed of heterogenous populations of neurons that express signaling peptides such as vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP) and regulate circadian rhythms in behavior and physiology. SCN neurons acquire functional and morphological specializations from waves of transcription factors (TFs) that are expressed during neurogenesis. However, the in vitro generation of SCN neurons has never been achieved. Here we supplemented a highly efficient neuronal conversion protocol with TFs that are expressed during SCN neurogenesis, namely Six3, Six6, Dlx2, and Lhx1. Neurons induced from mouse and human fibroblasts predominantly exhibited neuronal properties such as bipolar or multipolar morphologies, GABAergic neurons with expression of VIP. Our study reveals a critical contribution of these TFs to the development of vasoactive intestinal peptide (Vip) expressing neurons in the SCN, suggesting the regenerative potential of neuronal subtypes contained in the SCN for future SCN regeneration and in vitro disease remodeling.

Learning from circadian rhythm to transform cancer prevention, prognosis, and survivorship care.

Circadian timekeeping mechanisms and cell cycle regulation share thematic biological principles in responding to signals, repairing cellular damage, coordinating metabolism, and allocating cellular resources for optimal function. Recent studies show interactions between cell cycle regulators and circadian clock components, offering insights into potential cancer treatment approaches. Understanding circadian control of metabolism informs timing for therapies to reduce adverse effects and enhance treatment efficacy. Circadian adaptability to lifestyle factors, such as activity, sleep, and nutrition sheds light on their impact on cancer. Leveraging circadian regulatory mechanisms for cancer prevention and care is vital, as most risk stems from modifiable lifestyles. Monitoring circadian factors aids risk assessment and targeted interventions across the cancer care continuum.

Reciprocal regulation between the molecular clock and kidney injury.

Tubulointerstitial fibrosis is the common pathological substrate for many etiologies leading to chronic kidney disease. Although perturbations in the circadian rhythm have been associated with renal disease, the role of the molecular clock in the pathogenesis of fibrosis remains incompletely understood. We investigated the relationship between the molecular clock and renal damage in experimental models of injury and fibrosis (unilateral ureteral obstruction, folic acid, and adenine nephrotoxicity), using genetically modified mice with selective deficiencies of the clock components Bmal1, Clock, and Cry We found that the molecular clock pathway was enriched in damaged tubular epithelial cells with marked metabolic alterations. In human tubular epithelial cells, TGFß significantly altered the expression of clock components. Although Clock played a role in the macrophage-mediated inflammatory response, the combined absence of Cry1 and Cry2 was critical for the recruitment of neutrophils, correlating with a worsening of fibrosis and with a major shift in the expression of metabolism-related genes. These results support that renal damage disrupts the kidney peripheral molecular clock, which in turn promotes metabolic derangement linked to inflammatory and fibrotic responses.

Ultrastructure of Synaptic Connectivity within Subregions of the Suprachiasmatic Nucleus Revealed by a Genetically Encoded Tag and Serial Blockface Electron Microscopy.

The hypothalamic suprachiasmatic nucleus (SCN) is the central circadian pacemaker in vertebrates. The SCN receives photic information exclusively through melanopsin-expressing retinal ganglion cells (mRGCs) to synchronize circadian rhythms with the environmental light cycles. The SCN is composed of two major peptidergic neuron types in the core and shell regions of the SCN. Determining how mRGCs interact with the network of synaptic connections onto and between SCN neurons is key to understand how light regulates the circadian clock and to elucidate the relevant local circuits within the SCN. To map these connections, we used a newly developed Cre-dependent electron microscopy (EM) reporter, APEX2, to label the mitochondria of mRGC axons. Serial blockface scanning electron microscopy was then used to resolve the fine 3D structure of mRGC axons and synaptic boutons in the SCN of a male mouse. The resulting maps reveal patterns of connectomic organization in the core and shell of the SCN. We show that these regions are composed of different neuronal subtypes and differ with regard to the pattern of mRGC input, as the shell receives denser mRGC synaptic input compared with the core. This finding challenges the present view that photic information coming directly from the retina is received primarily by the core region of the SCN.

When a calorie is not just a calorie: Diet quality and timing as mediators of metabolism and healthy aging.

An epidemic of obesity has affected large portions of the world, increasing the risk of developing many different age-associated diseases, including cancer, cardiovascular disease, and diabetes. In contrast with the prevailing notion that "a calorie is just a calorie," there are clear differences, within and between individuals, in the metabolic response to different macronutrient sources. Recent findings challenge this oversimplification; calories from different macronutrient sources or consumed at different times of day have metabolic effects beyond their value as fuel. Here, we summarize discussions conducted at a recent NIH workshop that brought together experts in calorie restriction, macronutrient composition, and time-restricted feeding to discuss how dietary composition and feeding schedule impact whole-body metabolism, longevity, and healthspan. These discussions may provide insights into the long-sought molecular mechanisms engaged by calorie restriction to extend lifespan, lead to novel therapies, and potentially inform the development of a personalized food-as-medicine approach to healthy aging.

Targeting Energy Intake and Circadian Biology to Engage Mechanisms of Aging in Older Adults With Obesity: Calorie Restriction and Time-Restricted Eating.

With the rise in obesity across age groups, it has been a hindrance to engaging in physical activity and mobility in older adults. Daily calorie restriction (CR) up to 25% has been the cornerstone of obesity management even though the safety in older adults remains incompletely understood. Although some adults can follow CR with clinically significant weight loss and improved health metrics, CR faces 2 obstacles-many fail to adopt CR and even among those who can adopt it short term, long-term compliance can be difficult. Furthermore, there is a continuing debate about the net benefits of CR-induced weight loss in older adults because of the concern that CR may worsen sarcopenia, osteopenia, and frailty. The science of circadian rhythm and its plasticity toward the timing of nutrition offer promise to alleviate some challenges of CR. The new concept of Time-Restricted Feeding/Eating (TRF for animal studies and TRE for human studies) can be an actionable approach to sustaining the circadian regulation of physiology, metabolism, and behavior. TRE can often (not always) lead to CR. Hence, the combined effect of TRE through circadian optimization and CR can potentially reduce weight and improve cardiometabolic and functional health while lessening the detrimental effects of CR. However, the science and efficacy of TRE as a sustainable lifestyle in humans are in its infancy, whereas animal studies have offered many desirable outcomes and underlying mechanisms. In this article, we will discuss the scope and opportunities to combine CR, exercise, and TRE to improve functional capacity among older adults with obesity.

Dynamic activity in cis-regulatory elements of leukocytes identifies transcription factor activation and stratifies COVID-19 severity in ICU patients.

Transcription factor programs mediating the immune response to coronavirus disease 2019 (COVID-19) are not fully understood. Capturing active transcription initiation from cis-regulatory elements such as enhancers and promoters by capped small RNA sequencing (csRNA-seq), in contrast to capturing steady-state transcripts by conventional RNA-seq, allows unbiased identification of the underlying transcription factor activity and regulatory pathways. Here, we profile transcription initiation in critically ill COVID-19 patients, identifying transcription factor motifs that correlate with clinical lung injury and disease severity. Unbiased clustering reveals distinct subsets of cis-regulatory elements that delineate the cell type, pathway-specific, and combinatorial transcription factor activity. We find evidence of critical roles of regulatory networks, showing that STAT/BCL6 and E2F/MYB regulatory programs from myeloid cell populations are activated in patients with poor disease outcomes and associated with COVID-19 susceptibility genetic variants. More broadly, we demonstrate how capturing acute, disease-mediated changes in transcription initiation can provide insight into the underlying molecular mechanisms and stratify patient disease severity.

Time-restricted feeding promotes muscle function through purine cycle and AMPK signaling in Drosophila obesity models.

Obesity caused by genetic and environmental factors can lead to compromised skeletal muscle function. Time-restricted feeding (TRF) has been shown to prevent muscle function decline from obesogenic challenges; however, its mechanism remains unclear. Here we demonstrate that TRF upregulates genes involved in glycine production (Sardh and CG5955) and utilization (Gnmt), while Dgat2, involved in triglyceride synthesis is downregulated in Drosophila models of diet- and genetic-induced obesity. Muscle-specific knockdown of Gnmt, Sardh, and CG5955 lead to muscle dysfunction, ectopic lipid accumulation, and loss of TRF-mediated benefits, while knockdown of Dgat2 retains muscle function during aging and reduces ectopic lipid accumulation. Further analyses demonstrate that TRF upregulates the purine cycle in a diet-induced obesity model and AMPK signaling-associated pathways in a genetic-induced obesity model. Overall, our data suggest that TRF improves muscle function through modulations of common and distinct pathways under different obesogenic challenges and provides potential targets for obesity treatments.

Insulin-regulated serine and lipid metabolism drive peripheral neuropathy.

Diabetes represents a spectrum of disease in which metabolic dysfunction damages multiple organ systems including liver, kidneys and peripheral nerves1,2. Although the onset and progression of these co-morbidities are linked with insulin resistance, hyperglycaemia and dyslipidaemia3-7, aberrant non-essential amino acid (NEAA) metabolism also contributes to the pathogenesis of diabetes8-10. Serine and glycine are closely related NEAAs whose levels are consistently reduced in patients with metabolic syndrome10-14, but the mechanistic drivers and downstream consequences of this metabotype remain unclear. Low systemic serine and glycine are also emerging as a hallmark of macular and peripheral nerve disorders, correlating with impaired visual acuity and peripheral neuropathy15,16. Here we demonstrate that aberrant serine homeostasis drives serine and glycine deficiencies in diabetic mice, which can be diagnosed with a serine tolerance test that quantifies serine uptake and disposal. Mimicking these metabolic alterations in young mice by dietary serine or glycine restriction together with high fat intake markedly accelerates the onset of small fibre neuropathy while reducing adiposity. Normalization of serine by dietary supplementation and mitigation of dyslipidaemia with myriocin both alleviate neuropathy in diabetic mice, linking serine-associated peripheral neuropathy to sphingolipid metabolism. These findings identify systemic serine deficiency and dyslipidaemia as novel risk factors for peripheral neuropathy that may be exploited therapeutically.

Diurnal transcriptome landscape of a multi-tissue response to time-restricted feeding in mammals.

Time-restricted feeding (TRF) is an emerging behavioral nutrition intervention that involves a daily cycle of feeding and fasting. In both animals and humans, TRF has pleiotropic health benefits that arise from multiple organ systems, yet the molecular basis of TRF-mediated benefits is not well understood. Here, we subjected mice to isocaloric ad libitum feeding (ALF) or TRF of a western diet and examined gene expression changes in samples taken from 22 organs and brain regions collected every 2 h over a 24-h period. We discovered that TRF profoundly impacts gene expression. Nearly 80% of all genes show differential expression or rhythmicity under TRF in at least one tissue. Functional annotation of these changes revealed tissue- and pathway-specific impacts of TRF. These findings and resources provide a critical foundation for future mechanistic studies and will help to guide human time-restricted eating (TRE) interventions to treat various disease conditions with or without pharmacotherapies.

Daytime light exposure is a strong predictor of seasonal variation in sleep and circadian timing of university students.

In the absence of electric light, sleep for humans typically starts soon after dusk and at higher latitudes daily sleep timing changes seasonally as photoperiod changes. However, access to electric light shields humans from natural photoperiod changes, and whether seasonal changes in sleep occur despite this isolation from the natural light-dark cycle remains a matter of controversy. We measured sleep timing in over 500 university students living in the city of Seattle, WA (47.6°N) throughout the four seasons; we show that even when students are following a school schedule, sleep timing is delayed during the fall and winter. For instance, during the winter school days, students fell asleep 35 min later and woke up 27 min later (under daylight-savings time) than students during the summer school days, a change that is an hour larger relative to solar midnight. Furthermore, chronotype defined by mid-sleep on free days corrected for oversleep (MSFc), an indirect estimate of circadian phase, was more than 30 min later in the winter compared with the summer. Analysis of the effect of light exposure showed that the number of hours of light exposure to at least 50 lux during the daytime was a stronger predictor of MSFc than the exposure time to this illuminance after dusk. Specifically, MSFc was advanced by 30 min for each additional hour of light exposure during daytime and delayed by only 15 min for each additional hour of postdusk exposure to light. Additionally, the time of the day of exposure to high light intensities was more predictive of MSFc when daytime exposure was considered than when exposure for the full 24-h day was considered. Our results show that although sleep time is highly synchronized to social time, a delayed timing of sleep is evident during the winter months. They also suggest that daily exposure to daylight is key to prevent this delayed phase of the circadian clock and thus circadian disruption that is typically exacerbated in high-latitude winters.