More than the clock: distinct regulation of muscle function and metabolism by PER2 and RORα.

Circadian rhythms, governed by the dominant central clock, in addition to various peripheral clocks, regulate almost all biological processes, including sleep-wake cycles, hormone secretion and metabolism. In certain contexts, the regulation and function of the peripheral oscillations can be decoupled from the central clock. However, the specific mechanisms underlying muscle-intrinsic clock-dependent modulation of muscle function and metabolism remain unclear. We investigated the outcome of perturbations of the primary and secondary feedback loops of the molecular clock in skeletal muscle by specific gene ablation of Period circadian regulator 2 (Per2) and RAR-related orphan receptor alpha (Rorα), respectively. In both models, a dampening of core clock gene oscillation was observed, while the phase was preserved. Moreover, both loops seem to be involved in the homeostasis of amine groups. Highly divergent outcomes were seen for overall muscle gene expression, primarily affecting circadian rhythmicity in the PER2 knockouts and non-oscillating genes in the RORα knockouts, leading to distinct outcomes in terms of metabolome and phenotype. These results highlight the entanglement of the molecular clock and muscle plasticity and allude to specific functions of different clock components, i.e. the primary and secondary feedback loops, in this context. The reciprocal interaction between muscle contractility and circadian clocks might therefore be instrumental to determining a finely tuned adaptation of muscle tissue to perturbations in health and disease. KEY POINTS: Specific perturbations of the primary and secondary feedback loop of the molecular clock result in specific outcomes on muscle metabolism and function. Ablation of Per2 (primary loop) or Rorα (secondary loop) blunts the amplitude of core clock genes, in absence of a shift in phase. Perturbation of the primary feedback loop by deletion of PER2 primarily affects muscle gene oscillation. Knockout of RORα and the ensuing modulation of the secondary loop results in the aberrant expression of a large number of non-clock genes and proteins. The deletion of PER2 and RORα affects muscle metabolism and contractile function in a circadian manner, highlighting the central role of the molecular clock in modulating muscle plasticity.

Daily running enhances molecular and physiological circadian rhythms in skeletal muscle.

OBJECTIVE: Exercise is a critical component of a healthy lifestyle and a key strategy for the prevention and management of metabolic disease. Identifying molecular mechanisms underlying adaptation in response to chronic physical activity is of critical interest in metabolic physiology. Circadian rhythms broadly modulate metabolism, including muscle substrate utilization and exercise capacity. Here, we define the molecular and physiological changes induced across the daily cycle by voluntary low intensity daily exercise. METHODS: Wildtype C57BL6/J male and female mice were housed with or without access to a running wheel for six weeks. Maximum running speed was measured at four different zeitgeber times (ZTs, hours after lights on) using either electrical or manual stimulation to motivate continued running on a motorized treadmill. RNA isolated from plantaris muscles at six ZTs was sequenced to establish the impact of daily activity on genome-wide transcription. Patterns of gene expression were analyzed using Gene Set Enrichment Analysis (GSEA) and Detection of Differential Rhythmicity (DODR). Blood glucose, lactate, and ketones, and muscle and liver glycogen were measured before and after exercise. RESULTS: We demonstrate that the use of mild electrical shocks to motivate running negatively impacts maximum running speed in mice, and describe a manual method to motivate running in rodent exercise studies. Using this method, we show that time of day influences the increase in exercise capacity afforded by six weeks of voluntary wheel running: when maximum running speed is measured at the beginning of the nighttime active period in mice, there is no measurable benefit from a history of daily voluntary running, while maximum increase in performance occurs at the end of the night. We show that daily voluntary exercise dramatically remodels the murine muscle circadian transcriptome. Finally, we describe daily rhythms in carbohydrate metabolism associated with the time-dependent response to moderate daily exercise in mice. CONCLUSIONS: Collectively, these data indicate that chronic nighttime physical activity dramatically remodels daily rhythms of murine muscle gene expression, which in turn support daily fluctuations in exercise performance.

Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle.

Preserving skeletal muscle function is essential to maintain life quality at high age. Calorie restriction (CR) potently extends health and lifespan, but is largely unachievable in humans, making "CR mimetics" of great interest. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, is considered a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Here we show that long-term CR and rapamycin unexpectedly display distinct gene expression profiles in geriatric mouse skeletal muscle, despite both benefiting aging muscles. Furthermore, CR improves muscle integrity in mice with nutrient-insensitive, sustained muscle mTORC1 activity and rapamycin provides additive benefits to CR in naturally aging mouse muscles. We conclude that rapamycin and CR exert distinct, compounding effects in aging skeletal muscle, thus opening the possibility of parallel interventions to counteract muscle aging.

Transcriptomic, proteomic and phosphoproteomic underpinnings of daily exercise performance and zeitgeber activity of training in mouse muscle.

KEY POINTS: Maximal endurance performance is greater in the early daytime. Timed exercise differentially alters the muscle transcriptome and (phospho)-proteome. Early daytime exercise triggers energy provisioning and tissue regeneration. Early night-time exercise activates stress-related and catabolic pathways. Scheduled training has limited effects on the muscle and liver circadian clocks. ABSTRACT: Timed physical activity might potentiate the health benefits of training. The underlying signalling events triggered by exercise at different times of day are, however, poorly understood. Here, we found that time-dependent variations in maximal treadmill exercise capacity of naïve mice were associated with energy stores, mostly hepatic glycogen levels. Importantly, running at different times of day resulted in a vastly different activation of signalling pathways, e.g. related to stress response, vesicular trafficking, repair and regeneration. Second, voluntary wheel running at the opposite phase of the dark, feeding period surprisingly revealed a minimal zeitgeber (i.e. phase-shifting) effect of training on the muscle clock. This integrated study provides important insights into the circadian regulation of endurance performance and the control of the circadian clock by exercise. In future studies, these results could contribute to better understanding circadian aspects of training design in athletes and the application of chrono-exercise-based interventions in patients.

RNA-bound PGC-1α controls gene expression in liquid-like nuclear condensates.

Plasticity of cells, tissues, and organs is controlled by the coordinated transcription of biological programs. However, the mechanisms orchestrating such context-specific transcriptional networks mediated by the dynamic interplay of transcription factors and coregulators are poorly understood. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a prototypical master regulator of adaptive transcription in various cell types. We now uncovered a central function of the C-terminal domain of PGC-1α to bind RNAs and assemble multiprotein complexes including proteins that control gene transcription and RNA processing. These interactions are important for PGC-1α recruitment to chromatin in transcriptionally active liquid-like nuclear condensates. Notably, such a compartmentalization of active transcription mediated by liquid-liquid phase separation was observed in mouse and human skeletal muscle, revealing a mechanism by which PGC-1α regulates complex transcriptional networks. These findings provide a broad conceptual framework for context-dependent transcriptional control of phenotypic adaptations in metabolically active tissues.

PGC-1ß-expressing POMC neurons mediate the effect of leptin on thermoregulation in the mouse.

The arcuate nucleus (ARC) of the hypothalamus is a key regulator of food intake, brown adipose tissue (BAT) thermogenesis, and locomotor activity. Whole-body deficiency of the transcriptional coactivator peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1ß (PGC-1ß) disrupts mouse circadian locomotor activity and BAT-regulated thermogenesis, in association with altered gene expression at the central level. We examined whether PGC-1ß expression in the ARC is required for proper energy balance and locomotor behavior by generating mice lacking the PGC-1ß gene specifically in pro-opiomelanocortin (POMC) neurons. POMC neuron-specific deletion of PGC-1ß did not impact locomotor behavior, food intake, body composition, energy fuel utilization and metabolic rate in fed, 24-h fasted and 24-h refed conditions. In contrast, in the fed state, deletion of PGC-1ß in POMC cells elevated core body temperature during the nighttime period. Importantly, this higher body temperature is not associated with changes in BAT function and gene expression. Conversely, we provide evidence that mice lacking PGC-1ß in POMC neurons are more sensitive to the effect of leptin on heat dissipation. Our data indicate that PGC-1ß-expressing POMC neurons are part of a circuit controlling body temperature homeostasis and that PGC-1ß function in these neurons is involved in the thermoregulatory effect of leptin.

The neuromuscular junction is a focal point of mTORC1 signaling in sarcopenia.

With human median lifespan extending into the 80s in many developed countries, the societal burden of age-related muscle loss (sarcopenia) is increasing. mTORC1 promotes skeletal muscle hypertrophy, but also drives organismal aging. Here, we address the question of whether mTORC1 activation or suppression is beneficial for skeletal muscle aging. We demonstrate that chronic mTORC1 inhibition with rapamycin is overwhelmingly, but not entirely, positive for aging mouse skeletal muscle, while genetic, muscle fiber-specific activation of mTORC1 is sufficient to induce molecular signatures of sarcopenia. Through integration of comprehensive physiological and extensive gene expression profiling in young and old mice, and following genetic activation or pharmacological inhibition of mTORC1, we establish the phenotypically-backed, mTORC1-focused, multi-muscle gene expression atlas, SarcoAtlas (https://sarcoatlas.scicore.unibas.ch/), as a user-friendly gene discovery tool. We uncover inter-muscle divergence in the primary drivers of sarcopenia and identify the neuromuscular junction as a focal point of mTORC1-driven muscle aging.

JAK2-mutant hematopoietic cells display metabolic alterations that can be targeted to treat myeloproliferative neoplasms.

Increased energy requirement and metabolic reprogramming are hallmarks of cancer cells. We show that metabolic alterations in hematopoietic cells are fundamental to the pathogenesis of mutant JAK2-driven myeloproliferative neoplasms (MPNs). We found that expression of mutant JAK2 augmented and subverted metabolic activity of MPN cells, resulting in systemic metabolic changes in vivo, including hypoglycemia, adipose tissue atrophy, and early mortality. Hypoglycemia in MPN mouse models correlated with hyperactive erythropoiesis and was due to a combination of elevated glycolysis and increased oxidative phosphorylation. Modulating nutrient supply through high-fat diet improved survival, whereas high-glucose diet augmented the MPN phenotype. Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2-mutant hematopoietic stem and progenitor cells that were altered in comparison with wild-type controls. We studied the consequences of elevated levels of Pfkfb3, a key regulatory enzyme of glycolysis, and found that pharmacological inhibition of Pfkfb3 with the small molecule 3PO reversed hypoglycemia and reduced hematopoietic manifestations of MPNs. These effects were additive with the JAK1/2 inhibitor ruxolitinib in vivo and in vitro. Inhibition of glycolysis by 3PO altered the redox homeostasis, leading to accumulation of reactive oxygen species and augmented apoptosis rate. Our findings reveal the contribution of metabolic alterations to the pathogenesis of MPNs and suggest that metabolic dependencies of mutant cells represent vulnerabilities that can be targeted for treating MPNs.

Peroxisome proliferator-activated receptor γ coactivator 1α regulates mitochondrial calcium homeostasis, sarcoplasmic reticulum stress, and cell death to mitigate skeletal muscle aging.

Age-related impairment of muscle function severely affects the health of an increasing elderly population. While causality and the underlying mechanisms remain poorly understood, exercise is an efficient intervention to blunt these aging effects. We thus investigated the role of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a potent regulator of mitochondrial function and exercise adaptation, in skeletal muscle during aging. We demonstrate that PGC-1α overexpression improves mitochondrial dynamics and calcium buffering in an estrogen-related receptor α-dependent manner. Moreover, we show that sarcoplasmic reticulum stress is attenuated by PGC-1α. As a result, PGC-1α prevents tubular aggregate formation and cell death pathway activation in old muscle. Similarly, the pro-apoptotic effects of ceramide and thapsigargin were blunted by PGC-1α in muscle cells. Accordingly, mice with muscle-specific gain-of-function and loss-of-function of PGC-1α exhibit a delayed and premature aging phenotype, respectively. Together, our data reveal a key protective effect of PGC-1α on muscle function and overall health span in aging.

BDNF is a mediator of glycolytic fiber-type specification in mouse skeletal muscle.

Brain-derived neurotrophic factor (BDNF) influences the differentiation, plasticity, and survival of central neurons and likewise, affects the development of the neuromuscular system. Besides its neuronal origin, BDNF is also a member of the myokine family. However, the role of skeletal muscle-derived BDNF in regulating neuromuscular physiology in vivo remains unclear. Using gain- and loss-of-function animal models, we show that muscle-specific ablation of BDNF shifts the proportion of muscle fibers from type IIB to IIX, concomitant with elevated slow muscle-type gene expression. Furthermore, BDNF deletion reduces motor end plate volume without affecting neuromuscular junction (NMJ) integrity. These morphological changes are associated with slow muscle function and a greater resistance to contraction-induced fatigue. Conversely, BDNF overexpression promotes a fast muscle-type gene program and elevates glycolytic fiber number. These findings indicate that BDNF is required for fiber-type specification and provide insights into its potential modulation as a therapeutic target in muscle diseases.

Endocrine Crosstalk Between Skeletal Muscle and the Brain.

Skeletal muscle is an essential regulator of energy homeostasis and a potent coordinator of exercise-induced adaptations in other organs including the liver, fat or the brain. Skeletal muscle-initiated crosstalk with other tissues is accomplished though the secretion of myokines, protein hormones which can exert autocrine, paracrine and long-distance endocrine effects. In addition, the enhanced release or uptake of metabolites from and into contracting muscle cells, respectively, likewise can act as a powerful mediator of tissue interactions, in particular in regard to the central nervous system. The present review will discuss the current stage of knowledge regarding how exercise and the muscle secretome improve a broad range of brain functions related to vascularization, neuroplasticity, memory, sleep and mood. Even though the molecular and cellular mechanisms underlying the communication between muscle and brain is still poorly understood, physical activity represents one of the most effective strategies to reduce the prevalence and incidence of depression, cognitive, metabolic or degenerative neuronal disorders, and thus warrants further study.

PGC-1α expression in murine AgRP neurons regulates food intake and energy balance.

OBJECTIVE: Food intake and whole-body energy homeostasis are controlled by agouti-related protein (AgRP) and pro-opiomelanocortin (POMC) neurons located in the arcuate nucleus of the hypothalamus. Key energy sensors, such as the AMP-activated protein kinase (AMPK) or sirtuin 1 (SIRT1), are essential in AgRP and POMC cells to ensure proper energy balance. In peripheral tissues, the transcriptional coactivator PGC-1α closely associates with these sensors to regulate cellular metabolism. The role of PGC-1α in the ARC nucleus, however, remains unknown. METHODS: Using AgRP and POMC neurons specific knockout (KO) mouse models we studied the consequences of PGC-1α deletion on metabolic parameters during fed and fasted states and on ghrelin and leptin responses. We also took advantage of an immortalized AgRP cell line to assess the impact of PGC-1α modulation on fasting induced AgRP expression. RESULTS: PGC-1α is dispensable for POMC functions in both fed and fasted states. In stark contrast, mice carrying a specific deletion of PGC-1α in AgRP neurons display increased adiposity concomitant with significantly lower body temperature and RER values during nighttime. In addition, the absence of PGC-1α in AgRP neurons reduces food intake in the fed and fasted states and alters the response to leptin. Finally, both in vivo and in an immortalized AgRP cell line, PGC-1α modulates AgRP expression induction upon fasting. CONCLUSIONS: Collectively, our results highlight a role for PGC-1α in the regulation of AgRP neuronal functions in the control of food intake and peripheral metabolism.

Rev-erbα in the brain is essential for circadian food entrainment.

Foraging is costly in terms of time and energy. An endogenous food-entrainable system allows anticipation of predictable changes of food resources in nature. Yet the molecular mechanism that controls food anticipation in mammals remains elusive. Here we report that deletion of the clock component Rev-erbα impairs food entrainment in mice. Rev-erbα global knockout (GKO) mice subjected to restricted feeding showed reduced elevations of locomotor activity and body temperature prior to mealtime, regardless of the lighting conditions. The failure to properly anticipate food arrival was accompanied by a lack of phase-adjustment to mealtime of the clock protein PERIOD2 in the cerebellum, and by diminished expression of phosphorylated ERK 1/2 (p-ERK) during mealtime in the mediobasal hypothalamus and cerebellum. Furthermore, brain-specific knockout (BKO) mice for Rev-erbα display a defective suprachiasmatic clock, as evidenced by blunted daily activity under a light-dark cycle, altered free-running rhythm in constant darkness and impaired clock gene expression. Notably, brain deletion of Rev-erbα totally prevented food-anticipatory behaviour and thermogenesis. In response to restricted feeding, brain deletion of Rev-erbα impaired changes in clock gene expression in the hippocampus and cerebellum, but not in the liver. Our findings indicate that Rev-erbα is required for neural network-based prediction of food availability.

The nuclear receptor REV-ERBα is required for the daily balance of carbohydrate and lipid metabolism.

Mutations of clock genes can lead to diabetes and obesity. REV-ERBα, a nuclear receptor involved in the circadian clockwork, has been shown to control lipid metabolism. To gain insight into the role of REV-ERBα in energy homeostasis in vivo, we explored daily metabolism of carbohydrates and lipids in chow-fed, unfed, or high-fat-fed Rev-erbα(-/-) mice and their wild-type littermates. Chow-fed Rev-erbα(-/-) mice displayed increased adiposity (2.5-fold) and mild hyperglycemia (∼10%) without insulin resistance. Indirect calorimetry indicates that chow-fed Rev-erbα(-/-) mice utilize more fatty acids during daytime. A 24-h nonfeeding period in Rev-erbα(-/-) animals favors further fatty acid mobilization at the expense of glycogen utilization and gluconeogenesis, without triggering hypoglycemia and hypothermia. High-fat feeding in Rev-erbα(-/-) mice amplified metabolic disturbances, including expression of lipogenic factors. Lipoprotein lipase (Lpl) gene, critical in lipid utilization/storage, is triggered in liver at night and constitutively up-regulated (∼2-fold) in muscle and adipose tissue of Rev-erbα(-/-) mice. We show that CLOCK, up-regulated (2-fold) at night in Rev-erbα(-/-) mice, can transactivate Lpl. Thus, overexpression of Lpl facilitates muscle fatty acid utilization and contributes to fat overload. This study demonstrates the importance of clock-driven Lpl expression in energy balance and highlights circadian disruption as a potential cause for the metabolic syndrome.

Interactions between metabolism and circadian clocks: reciprocal disturbances.

Obesity is a medical condition of excess body fat, recognized as a global epidemic. Besides genetic factors, overconsumption of high-energy food and a sedentary lifestyle are major obesogenic causes. A newly identified determinant is altered circadian rhythmicity. To anticipate and adapt to daily changes in the environment, organisms have developed an endogenous circadian timing system, comprising a main circadian clock, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, principally synchronized to the light-dark cycle. Secondary peripheral clocks are found in various tissues, such as the liver, pancreas, and adipose tissue. These clocks control the rhythmic patterns of myriad metabolic processes. We will review the evidence that metabolic dysfunction is associated with circadian disturbances at both central and peripheral levels and, conversely, that disruption of circadian clock functioning can lead to obesity. The roots of these reciprocal interactions will be illustrated by transcriptional crosstalk between metabolic and circadian systems. Chronotherapeutic approaches of dieting to maintain or restore a proper circadian alignment could be useful to limit the magnitude of metabolic risks.