Circadian mechanisms in adipose tissue bioenergetics and plasticity.

The circadian clock plays an essential role in coordinating feeding and metabolic rhythms with the light/dark cycle. Disruption of clocks is associated with increased adiposity and metabolic disorders, whereas aligning feeding time with cell-autonomous rhythms in metabolism improves health. Here, we provide a comprehensive overview of recent literature in adipose tissue biology as well as our understanding of molecular mechanisms underlying the circadian regulation of transcription, metabolism, and inflammation in adipose tissue. We highlight recent efforts to uncover the mechanistic links between clocks and adipocyte metabolism, as well as its application to dietary and behavioral interventions to improve health and mitigate obesity.

NAD+ Controls Circadian Reprogramming through PER2 Nuclear Translocation to Counter Aging.

Disrupted sleep-wake and molecular circadian rhythms are a feature of aging associated with metabolic disease and reduced levels of NAD+, yet whether changes in nucleotide metabolism control circadian behavioral and genomic rhythms remains unknown. Here, we reveal that supplementation with the NAD+ precursor nicotinamide riboside (NR) markedly reprograms metabolic and stress-response pathways that decline with aging through inhibition of the clock repressor PER2. NR enhances BMAL1 chromatin binding genome-wide through PER2K680 deacetylation, which in turn primes PER2 phosphorylation within a domain that controls nuclear transport and stability and that is mutated in human advanced sleep phase syndrome. In old mice, dampened BMAL1 chromatin binding, transcriptional oscillations, mitochondrial respiration rhythms, and late evening activity are restored by NAD+ repletion to youthful levels with NR. These results reveal effects of NAD+ on metabolism and the circadian system with aging through the spatiotemporal control of the molecular clock.

A window in time for ß-cell regeneration.

In vivo regeneration of ß cells provides hope for self-renewal of functional insulin-secreting cells following ß-cell failure, a historically fatal condition now sustainable only by administration of exogenous insulin. Despite advances in the treatment of diabetes mellitus, the path toward endogenous renewal of ß-cell populations has remained elusive. Intensive efforts have focused on elucidating pancreatic transcriptional programs that can drive the division and (trans-)differentiation of non-ß cells to produce insulin. A surprise has been the identification of an essential role of the molecular circadian clock in the regulation of competent insulin-producing ß cells. In this issue of Genes & Development, work by Petrenko and colleagues (pp. 1650-1665) now shows a requirement for the intrinsic clock in the regenerative capacity of insulin-producing cells following genetic ablation of ß cells. These studies raise the possibility that enhancing core clock activity may provide an adjuvant in cell replacement therapies.

A role for alternative splicing in circadian control of exocytosis and glucose homeostasis.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.

Neurogenetic basis for circadian regulation of metabolism by the hypothalamus.

Circadian rhythms are driven by a transcription-translation feedback loop that separates anabolic and catabolic processes across the Earth's 24-h light-dark cycle. Central pacemaker neurons that perceive light entrain a distributed clock network and are closely juxtaposed with hypothalamic neurons involved in regulation of sleep/wake and fast/feeding states. Gaps remain in identifying how pacemaker and extrapacemaker neurons communicate with energy-sensing neurons and the distinct role of circuit interactions versus transcriptionally driven cell-autonomous clocks in the timing of organismal bioenergetics. In this review, we discuss the reciprocal relationship through which the central clock drives appetitive behavior and metabolic homeostasis and the pathways through which nutrient state and sleep/wake behavior affect central clock function.

Engineered calprotectin-sensing probiotics for IBD surveillance in humans.

Inflammatory bowel disease (IBD) is a spectrum of autoimmune diseases affecting the gastrointestinal tract characterized by a relapsing and remitting course of gut mucosal inflammation. Disease flares can be difficult to predict, and the current practice of IBD disease activity surveillance through endoscopy is invasive and requires medical expertise. Recent advancements in synthetic biology raise the possibility that symbiotic microbes can be engineered to selectively detect disease biomarkers used in current clinical practice. Here, we introduce an engineered probiotic capable of detecting the clinical gold standard IBD biomarker, calprotectin, with sensitivity and specificity in IBD patients. Specifically, we identified a bacterial promoter in the probiotic strain Escherichia coli Nissle 1917 (EcN) which exhibits a specific expression increase in the presence of calprotectin. Using murine models of colitis, we show that the reporter signal is activated in vivo during transit of the GI tract following oral delivery. Furthermore, our engineered probiotic can successfully discriminate human patients with active IBD from those in remission and without IBD using patient stool samples, where the intensity of reporter signal quantitatively tracks with clinical laboratory-measured levels of calprotectin. Our pilot study sets the stage for probiotics that can be engineered to detect fecal calprotectin for precise noninvasive disease activity monitoring in IBD patients.

A day in the life of chromatin: how enhancer-promoter loops shape daily behavior.

Each spring, we get out of bed 1 h ahead of our biological wake-up time due to the misalignment of internal clocks with the light-dark cycle. Genetic discoveries revealed that clock genes encode transcription factors that are expressed throughout many tissues, yet a gap has remained in understanding the temporal dynamics of transcription. Two groups now apply circular chromosome conformation capture and high-throughput sequencing to dissect how "time of day"-dependent changes in chromatin drive core clock oscillations. A surprise is the finding that disruption of enhancer-promoter contacts within chromatin leads to an advance in the "wake-up" time of mice. Furthermore, the assembly of transcriptionally active domains of chromatin requires the ordered recruitment of core clock transcription factors each day. These studies show that waking up involves highly dynamic changes in the three-dimensional positioning of genes within the cell.

Requirement for NF-κB in maintenance of molecular and behavioral circadian rhythms in mice.

The mammalian circadian clock is encoded by an autoregulatory transcription feedback loop that drives rhythmic behavior and gene expression in the brain and peripheral tissues. Transcriptomic analyses indicate cell type-specific effects of circadian cycles on rhythmic physiology, although how clock cycles respond to environmental stimuli remains incompletely understood. Here, we show that activation of the inducible transcription factor NF-κB in response to inflammatory stimuli leads to marked inhibition of clock repressors, including the Period, Cryptochrome, and Rev-erb genes, within the negative limb. Furthermore, activation of NF-κB relocalizes the clock components CLOCK/BMAL1 genome-wide to sites convergent with those bound by NF-κB, marked by acetylated H3K27, and enriched in RNA polymerase II. Abrogation of NF-κB during adulthood alters the expression of clock repressors, disrupts clock-controlled gene cycles, and impairs rhythmic activity behavior, revealing a role for NF-κB in both unstimulated and activated conditions. Together, these data highlight NF-κB-mediated transcriptional repression of the clock feedback limb as a cause of circadian disruption in response to inflammation.

Circadian NAD(P)(H) cycles in cell metabolism.

Intrinsic circadian clocks are present in all forms of photosensitive life, enabling daily anticipation of the light/dark cycle and separation of energy storage and utilization cycles on a 24-h timescale. The core mechanism underlying circadian rhythmicity involves a cell-autonomous transcription/translation feedback loop that in turn drives rhythmic organismal physiology. In mammals, genetic studies have established that the core clock plays an essential role in maintaining metabolic health through actions within both brain pacemaker neurons and peripheral tissues and that disruption of the clock contributes to disease. Peripheral clocks, in turn, can be entrained by metabolic cues. In this review, we focus on the role of the nucleotide NAD(P)(H) and NAD+-dependent sirtuin deacetylases as integrators of circadian and metabolic cycles, as well as the implications for this interrelationship in healthful aging.

The meter of metabolism.

The circadian system orchestrates the temporal organization of many aspects of physiology, including metabolism, in synchrony with the 24 hr rotation of the Earth. Like the metabolic system, the circadian system is a complex feedback network that involves interactions between the central nervous system and peripheral tissues. Emerging evidence suggests that circadian regulation is intimately linked to metabolic homeostasis and that dysregulation of circadian rhythms can contribute to disease. Conversely, metabolic signals also feed back into the circadian system, modulating circadian gene expression and behavior. Here, we review the relationship between the circadian and metabolic systems and the implications for cardiovascular disease, obesity, and diabetes.

Exploring the variability of sarcopenia prevalence in a research population using different disease definitions.

BACKGROUND: Sarcopenia is the progressive loss of muscle mass and function with age. A number of different sarcopenia definitions have been proposed and utilised in research. This study aimed to investigate how the prevalence of sarcopenia in a research cohort of older adults is influenced by the use of independent aspects of these different definitions. METHODS: Data from 255 research participants were compiled. Defining criteria by the European Working Group on Sarcopenia in Older People, the International Working Group on Sarcopenia (IWGS), and the Foundation for the National Institutes of Health were applied. RESULTS: Prevalence of sarcopenia using muscle mass ranged from 4 to 22%. Gait speed and handgrip strength criteria identified 4-34% and 4-16% of participants as sarcopenic, respectively. CONCLUSION: Prevalence of sarcopenia differs substantially depending on the criteria used. Work is required to address the impact of this for sarcopenia research to be usefully translated to inform on clinical practice.

Motor unit dysregulation following 15 days of unilateral lower limb immobilisation.

Disuse atrophy, caused by situations of unloading such as limb immobilisation, causes a rapid yet diverging reduction in skeletal muscle function when compared to muscle mass. While mechanistic insight into the loss of mass is well studied, deterioration of muscle function with a focus towards the neural input to muscle remains underexplored. This study aimed to determine the role of motor unit adaptation in disuse-induced neuromuscular deficits. Ten young, healthy male volunteers underwent 15 days of unilateral lower limb immobilisation with intramuscular electromyography (iEMG) bilaterally recorded from the vastus lateralis (VL) during knee extensor contractions normalised to maximal voluntary contraction (MVC), pre and post disuse. Muscle cross-sectional area was determined by ultrasound. Individual MUs were sampled and analysed for changes in motor unit (MU) discharge and MU potential (MUP) characteristics. VL CSA was reduced by approximately 15% which was exceeded by a two-fold decrease of 31% in muscle strength in the immobilised limb, with no change in either parameter in the non-immobilised limb. Parameters of MUP size were reduced by 11% to 24% with immobilisation, while neuromuscular junction (NMJ) transmission instability remained unchanged, and MU firing rate decreased by 8% to 11% at several contraction levels. All adaptations were observed in the immobilised limb only. These findings highlight impaired neural input following immobilisation reflected by suppressed MU firing rate which may underpin the disproportionate reductions of strength relative to muscle size. KEY POINTS: Muscle mass and function decline rapidly in situations of disuse such as bed rest and limb immobilisation. The reduction in muscle function commonly exceeds that of muscle mass, which may be associated with the dysregulation of neural input to muscle. We have used intramuscular electromyography to sample individual motor unit and near fibre potentials from the vastus lateralis following 15 days of unilateral limb immobilisation. Following disuse, the disproportionate loss of muscle strength when compared to size coincided with suppressed motor unit firing rate. These motor unit adaptations were observed at multiple contraction levels and in the immobilised limb only. Our findings demonstrate neural dysregulation as a key component of functional loss following muscle disuse in humans.

Transcriptomic links to muscle mass loss and declines in cumulative muscle protein synthesis during short-term disuse in healthy younger humans.

Muscle disuse leads to a rapid decline in muscle mass, with reduced muscle protein synthesis (MPS) considered the primary physiological mechanism. Here, we employed a systems biology approach to uncover molecular networks and key molecular candidates that quantitatively link to the degree of muscle atrophy and/or extent of decline in MPS during short-term disuse in humans. After consuming a bolus dose of deuterium oxide (D2 O; 3 mL.kg-1 ), eight healthy males (22 ± 2 years) underwent 4 days of unilateral lower-limb immobilization. Bilateral muscle biopsies were obtained post-intervention for RNA sequencing and D2 O-derived measurement of MPS, with thigh lean mass quantified using dual-energy X-ray absorptiometry. Application of weighted gene co-expression network analysis identified 15 distinct gene clusters ("modules") with an expression profile regulated by disuse and/or quantitatively connected to disuse-induced muscle mass or MPS changes. Module scans for candidate targets established an experimentally tractable set of candidate regulatory molecules (242 hub genes, 31 transcriptional regulators) associated with disuse-induced maladaptation, many themselves potently tied to disuse-induced reductions in muscle mass and/or MPS and, therefore, strong physiologically relevant candidates. Notably, we implicate a putative role for muscle protein breakdown-related molecular networks in impairing MPS during short-term disuse, and further establish DEPTOR (a potent mTOR inhibitor) as a critical mechanistic candidate of disuse driven MPS suppression in humans. Overall, these findings offer a strong benchmark for accelerating mechanistic understanding of short-term muscle disuse atrophy that may help expedite development of therapeutic interventions.

Equipment-free, unsupervised high intensity interval training elicits significant improvements in the physiological resilience of older adults.

BACKGROUND: Reduced cardiorespiratory fitness (CRF) is an independent risk factor for dependency, cognitive impairment and premature mortality. High-intensity interval training (HIIT) is a proven time-efficient stimulus for improving both CRF and other facets of cardiometabolic health also known to decline with advancing age. However, the efficacy of equipment-free, unsupervised HIIT to improve the physiological resilience of older adults is not known. METHODS: Thirty independent, community-dwelling older adults (71(SD: 5) years) were randomised to 4 weeks (12 sessions) equipment-free, supervised (in the laboratory (L-HIIT)) or unsupervised (at home (H-HIIT)) HIIT, or a no-intervention control (CON). HIIT involved 5, 1-minute intervals of a bodyweight exercise each interspersed with 90-seconds recovery. CRF, exercise tolerance, blood pressure (BP), body composition, muscle architecture, circulating lipids and glucose tolerance were assessed at baseline and after the intervention period. RESULTS: When compared to the control group, both HIIT protocols improved the primary outcome of CRF ((via anaerobic threshold) mean difference, L-HIIT: +2.27, H-HIIT: +2.29, both p < 0.01) in addition to exercise tolerance, systolic BP, total cholesterol, non-HDL cholesterol and m. vastus lateralis pennation angle, to the same extent. There was no improvement in these parameters in CON. There was no change in diastolic BP, glucose tolerance, whole-body composition or HDL cholesterol in any of the groups. CONCLUSIONS: This is the first study to show that short-term, time-efficient, equipment-free, HIIT is able to elicit improvements in the CRF of older adults irrespective of supervision status. Unsupervised HIIT may offer a novel approach to improve the physiological resilience of older adults, combating age-associated physiological decline, the rise of inactivity and the additional challenges currently posed by the COVID-19 pandemic. TRIAL REGISTRATION: This study was registered at clinicaltrials.gov and coded: NCT03473990 .

Influence of sex on the age-related adaptations of neuromuscular function and motor unit properties in elite masters athletes.

KEY POINTS: Masters athletes maintain high levels of activity into older age and allow an examination of the effects of aging dissociated from the effects of increased sedentary behaviour. Evidence suggests masters athletes are more successful at motor unit remodelling, the reinnervation of denervated fibres acting to preserve muscle fibre number, but little data are available in females. Here we used intramuscular electromyography to demonstrate that motor units sampled from the tibialis anterior show indications of remodelling from middle into older age and which does not differ between males and females. The age-related trajectory of motor unit discharge characteristic differs according to sex, with female athletes progressing to a slower firing pattern that was not observed in males. Our findings indicate motor unit remodelling from middle to older age occurs to a similar extent in male and female athletes, with discharge rates progressively slowing in females only. ABSTRACT: Motor unit (MU) remodelling acts to minimise loss of muscle fibres following denervation in older age, which may be more successful in masters athletes. Evidence suggests performance and neuromuscular function decline with age in this population, although the majority of studies have focused on males, with little available data on female athletes. Functional assessments of strength, balance and motor control were performed in 30 masters athletes (16 male) aged 44-83 years. Intramuscular needle electrodes were used to sample individual motor unit potentials (MUPs) and near-fibre MUPs in the tibialis anterior (TA) during isometric contractions at 25% maximum voluntary contraction, and used to determine discharge characteristics (firing rate, variability) and biomarkers of peripheral MU remodelling (MUP size, complexity, stability). Multilevel mixed-effects linear regression models examined effects of age and sex. All aspects of neuromuscular function deteriorated with age (P < 0.05) with no age × sex interactions, although males were stronger (P < 0.001). Indicators of MU remodelling also progressively increased with age to a similar extent in both sexes (P < 0.05), whilst MU firing rate progressively decreased with age in females (p = 0.029), with a non-significant increase in males (p = 0.092). Masters athletes exhibit age-related declines in neuromuscular function that are largely equal across males and females. Notably, they also display features of MU remodelling with advancing age, probably acting to reduce muscle fibre loss. The age trajectory of MU firing rate assessed at a single contraction level differed between sexes, which may reflect a greater tendency for females to develop a slower muscle phenotype.

The mechanisms of skeletal muscle atrophy in response to transient knockdown of the vitamin D receptor in vivo.

KEY POINTS: Reduced vitamin D receptor (VDR) expression prompts skeletal muscle atrophy. Atrophy occurs through catabolic processes, namely the induction of autophagy, while anabolism remains unchanged. In response to VDR-knockdown mitochondrial function and related gene-set expression is impaired. In vitro VDR knockdown induces myogenic dysregulation occurring through impaired differentiation. These results highlight the autonomous role the VDR has within skeletal muscle mass regulation. ABSTRACT: Vitamin D deficiency is estimated to affect ∼40% of the world's population and has been associated with impaired muscle maintenance. Vitamin D exerts its actions through the vitamin D receptor (VDR), the expression of which was recently confirmed in skeletal muscle, and its down-regulation is linked to reduced muscle mass and functional decline. To identify potential mechanisms underlying muscle atrophy, we studied the impact of VDR knockdown (KD) on mature skeletal muscle in vivo, and myogenic regulation in vitro in C2C12 cells. Male Wistar rats underwent in vivo electrotransfer (IVE) to knock down the VDR in hind-limb tibialis anterior (TA) muscle for 10 days. Comprehensive metabolic and physiological analysis was undertaken to define the influence loss of the VDR on muscle fibre composition, protein synthesis, anabolic and catabolic signalling, mitochondrial phenotype and gene expression. Finally, in vitro lentiviral transfection was used to induce sustained VDR-KD in C2C12 cells to analyse myogenic regulation. Muscle VDR-KD elicited atrophy through a reduction in total protein content, resulting in lower myofibre area. Activation of autophagic processes was observed, with no effect upon muscle protein synthesis or anabolic signalling. Furthermore, RNA-sequencing analysis identified systematic down-regulation of multiple mitochondrial respiration-related protein and genesets. Finally, in vitro VDR-knockdown impaired myogenesis (cell cycling, differentiation and myotube formation). Together, these data indicate a fundamental regulatory role of the VDR in the regulation of myogenesis and muscle mass, whereby it acts to maintain muscle mitochondrial function and limit autophagy.

Roadmap on biology in time varying environments.

Biological organisms experience constantly changing environments, from sudden changes in physiology brought about by feeding, to the regular rising and setting of the Sun, to ecological changes over evolutionary timescales. Living organisms have evolved to thrive in this changing world but the general principles by which organisms shape and are shaped by time varying environments remain elusive. Our understanding is particularly poor in the intermediate regime with no separation of timescales, where the environment changes on the same timescale as the physiological or evolutionary response. Experiments to systematically characterize the response to dynamic environments are challenging since such environments are inherently high dimensional. This roadmap deals with the unique role played by time varying environments in biological phenomena across scales, from physiology to evolution, seeking to emphasize the commonalities and the challenges faced in this emerging area of research.

Mammalian and Invertebrate Models as Complementary Tools for Gaining Mechanistic Insight on Muscle Responses to Spaceflight.

Bioinformatics approaches have proven useful in understanding biological responses to spaceflight. Spaceflight experiments remain resource intensive and rare. One outstanding issue is how to maximize scientific output from a limited number of omics datasets from traditional animal models including nematodes, fruitfly, and rodents. The utility of omics data from invertebrate models in anticipating mammalian responses to spaceflight has not been fully explored. Hence, we performed comparative analyses of transcriptomes of soleus and extensor digitorum longus (EDL) in mice that underwent 37 days of spaceflight. Results indicate shared stress responses and altered circadian rhythm. EDL showed more robust growth signals and Pde2a downregulation, possibly underlying its resistance to atrophy versus soleus. Spaceflight and hindlimb unloading mice shared differential regulation of proliferation, circadian, and neuronal signaling. Shared gene regulation in muscles of humans on bedrest and space flown rodents suggest targets for mitigating muscle atrophy in space and on Earth. Spaceflight responses of C. elegans were more similar to EDL. Discrete life stages of D. melanogaster have distinct utility in anticipating EDL and soleus responses. In summary, spaceflight leads to shared and discrete molecular responses between muscle types and invertebrate models may augment mechanistic knowledge gained from rodent spaceflight and ground-based studies.

The vitamin D receptor regulates mitochondrial function in C2C12 myoblasts.

Vitamin D deficiency has been linked to a reduction in skeletal muscle function and oxidative capacity; however, the mechanistic bases of these impairments are poorly understood. The biological actions of vitamin D are carried out via the binding of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) to the vitamin D receptor (VDR). Recent evidence has linked 1α,25(OH)2D3 to the regulation of skeletal muscle mitochondrial function in vitro; however, little is known with regard to the role of the VDR in this process. To examine the regulatory role of the VDR in skeletal muscle mitochondrial function, we used lentivirus-mediated shRNA silencing of the VDR in C2C12 myoblasts (VDR-KD) and examined mitochondrial respiration and protein content compared with an shRNA scrambled control. VDR protein content was reduced by ~95% in myoblasts and myotubes (P < 0.001). VDR-KD myoblasts displayed a 30%, 30%, and 36% reduction in basal, coupled, and maximal respiration, respectively (P < 0.05). This phenotype was maintained in VDR-KD myotubes, displaying a 34%, 33%, and 48% reduction in basal, coupled, and maximal respiration (P < 0.05). Furthermore, ATP production derived from oxidative phosphorylation (ATPOx) was reduced by 20%, suggesting intrinsic impairments within the mitochondria following VDR-KD. However, despite the observed functional decrements, mitochondrial protein content, as well as markers of mitochondrial fission were unchanged. In summary, we highlight a direct role for the VDR in regulating skeletal muscle mitochondrial respiration in vitro, providing a potential mechanism as to how vitamin D deficiency might impact upon skeletal muscle oxidative capacity.