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Chromatin immunoprecipitation (ChIP) coupled to qPCR or sequencing is a crucial experiment to determine direct transcriptional regulation under the control of specific transcriptional factors or co-regulators at loci-specific or pan-genomic levels.Here we provide a reliable method for processing ChIP from adipocytes or frozen adipose tissue collection, isolation of nuclei, cross-linking of protein-DNA complexes, chromatin shearing, immunoprecipitation, and DNA purification. We also discuss critical steps for optimizing the experiment to perform a successful ChIP in lipid-rich cells/tissues.
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Adipocitos , Tejido Adiposo , Inmunoprecipitación de Cromatina , ADN , Factores de Transcripción , Adipocitos/metabolismo , Adipocitos/citología , Tejido Adiposo/metabolismo , Tejido Adiposo/citología , Inmunoprecipitación de Cromatina/métodos , ADN/metabolismo , ADN/genética , Factores de Transcripción/metabolismo , Humanos , Animales , Unión Proteica , Cromatina/metabolismo , Cromatina/genéticaRESUMEN
Background: Timing drug administration to endogenous circadian rhythms may enhance treatment efficacy. In the Chronotype sub-study of the Treatment in Morning versus Evening (TIME) clinical trial we examined whether timing of usual antihypertensive medications according to patient chronotype (a behavioural marker of personal circadian rhythm) may influence clinical cardiovascular outcomes. Methods: This was a cohort sub-study of TIME, a prospective, randomised, open-label, blinded-endpoint, UK clinical trial of morning versus evening dosing of usual antihypertensive medications and cardiovascular outcomes. On August 3rd, 2020, all active TIME participants were invited to complete a validated chronotype questionnaire. Chronotype was quantitatively assessed as the mid sleep time on free days corrected for sleep debt on workdays (MSFsc). We analysed associations between chronotype and antihypertensive dosing time and explored their combined effect on cardiovascular outcomes (a composite endpoint of hospitalisation for non-fatal myocardial infarction (MI) or non-fatal stroke, and single components) using proportional hazard time-to-event models adjusted for baseline covariates. These were used to specifically test for interactions between dosing time and chronotype. Findings: Between August 3, 2020, and March 31, 2021, 5358 TIME participants completed the online questionnaire. 2778 were previously randomised to morning dosing and 2580 to evening dosing of their usual antihypertensives. Chronotype was symmetrically distributed around a median MSFsc of 3:07 am. The composite endpoint increased for later MSFsc (later chronotype) dosed in the morning but not in those dosed in the evening (hazard ratios 1.46 [95% CI 1.14-1.86] and 0.96 [95% CI 0.70-1.30] per hour of MSFsc, respectively; interaction p = 0.036). Later chronotype was associated with increased risk of hospitalisation for non-fatal MI in the morning dosing group, and reduced risk in the evening dosing group (hazard ratios 1.62 [95% CI 1.18-2.22] and 0.66 [95% CI 0.44-1.00] per hour of MSFsc, respectively; interaction p < 0.001). No interaction between chronotype and antihypertensive dosing time was observed for stroke events. Interpretation: Alignment of dosing time of usual antihypertensives with personal chronotype could lower the incidence of non-fatal MI compared to a 'misaligned' dosing time regimen. Future studies are warranted to establish whether synchronizing administration time of antihypertensive therapy with individual chronotype reduces risk of MI. Funding: The TIME study was funded by the British Heart Foundation (CS/14/1/30659) with support from the British and Irish Hypertension Society.
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Viewing metabolism through the lens of exercise biology has proven an accessible and practical strategy to gain new insights into local and systemic metabolic regulation. Recent methodological developments have advanced understanding of the central role of skeletal muscle in many exercise-associated health benefits and have uncovered the molecular underpinnings driving adaptive responses to training regimens. In this Review, we provide a contemporary view of the metabolic flexibility and functional plasticity of skeletal muscle in response to exercise. First, we provide background on the macrostructure and ultrastructure of skeletal muscle fibres, highlighting the current understanding of sarcomeric networks and mitochondrial subpopulations. Next, we discuss acute exercise skeletal muscle metabolism and the signalling, transcriptional and epigenetic regulation of adaptations to exercise training. We address knowledge gaps throughout and propose future directions for the field. This Review contextualizes recent research of skeletal muscle exercise metabolism, framing further advances and translation into practice.
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Epigénesis Genética , Ejercicio Físico , Ejercicio Físico/fisiología , Adaptación Fisiológica/fisiología , Mitocondrias/metabolismo , Músculo Esquelético/metabolismoRESUMEN
OBJECTIVE: The circadian clock aligns physiology with the 24-hour rotation of Earth. Light and food are the main environmental cues (zeitgebers) regulating circadian rhythms in mammals. Yet, little is known about the interaction between specific dietary components and light in coordinating circadian homeostasis. Herein, we focused on the role of essential amino acids. METHODS: Mice were fed diets depleted of specific essential amino acids and their behavioral rhythms were monitored and tryptophan was selected for downstream analyses. The role of tryptophan metabolism in modulating circadian homeostasis was studied using isotope tracing as well as transcriptomic- and metabolomic- analyses. RESULTS: Dietary tryptophan depletion alters behavioral rhythms in mice. Furthermore, tryptophan metabolism was shown to be regulated in a time- and light- dependent manner. A multi-omics approach and combinatory diet/light interventions demonstrated that tryptophan metabolism modulates temporal regulation of metabolism and transcription programs by buffering photic cues. Specifically, tryptophan metabolites regulate central circadian functions of the suprachiasmatic nucleus and the core clock machinery in the liver. CONCLUSIONS: Tryptophan metabolism is a modulator of circadian homeostasis by integrating environmental cues. Our findings propose tryptophan metabolism as a potential point for pharmacologic intervention to modulate phenotypes associated with disrupted circadian rhythms.
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Relojes Circadianos , Ritmo Circadiano , Animales , Ritmo Circadiano/fisiología , Hígado/metabolismo , Mamíferos , Ratones , Núcleo Supraquiasmático/metabolismo , Triptófano/metabolismoRESUMEN
Oxidative stress participates in the development and exacerbation of cardiovascular diseases (CVD). The ability to promptly quantify an imbalance in an individual reductive-oxidative (RedOx) state could improve cardiovascular risk assessment and management. Derivatives-reactive oxygen metabolites (d-ROMs) are an emerging biomarker of oxidative stress quantifiable in minutes through standard biochemical analysers or by a bedside point-of-care test. The current review evaluates available data on the prognostic value of d-ROMs for CVD events and mortality in individuals with known and unknown CVD. Outcome studies involving small and large cohorts were analysed and hazard ratio, risk ratio, odds ratio, and mean differences were used as measures of effect. High d-ROM plasma levels were found to be an independent predictor of CVD events and mortality. Risk begins increasing at d-ROM levels higher than 340 UCARR and rises considerably above 400 UCARR. Conversely, low d-ROM plasma levels are a good negative predictor for CVD events in patients with coronary artery disease and heart failure. Moreover, combining d-ROMs with other relevant biomarkers routinely used in clinical practice might support a more precise cardiovascular risk assessment. We conclude that d-ROMs represent an emerging oxidative-stress-related biomarker with the potential for better risk stratification both in primary and secondary cardiovascular prevention.
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The gut microbiome influences cognition and behavior in mammals, yet its metabolic impact on the brain is only starting to be defined. Using metabolite profiling of antibiotics-treated mice, we reveal the microbiome as a key input controlling circadian metabolic cycles in the brain. Intra and inter-region analyses characterise the influence of the microbiome on the suprachiasmatic nucleus, containing the central clockwork, as well as the hippocampus and cortex, regions involved in learning and behavior.
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Antibacterianos , Microbioma Gastrointestinal , Animales , Antibacterianos/farmacología , Encéfalo/metabolismo , Mamíferos , Ratones , Núcleo SupraquiasmáticoRESUMEN
Resistance training promotes metabolic health and stimulates muscle hypertrophy, but the precise routes by which resistance exercise (RE) conveys these health benefits are largely unknown. AIM: To investigate how acute RE affects human skeletal muscle metabolism. METHODS: We collected vastus lateralis biopsies from six healthy male untrained volunteers at rest, before the first of 13 RE training sessions, and 45 min after the first and last bouts of RE. Biopsies were analysed using untargeted mass spectrometry-based metabolomics. RESULTS: We measured 617 metabolites covering a broad range of metabolic pathways. In the untrained state RE altered 33 metabolites, including increased 3-methylhistidine and N-lactoylvaline, suggesting increased protein breakdown, as well as metabolites linked to ATP (xanthosine) and NAD (N1-methyl-2-pyridone-5-carboxamide) metabolism; the bile acid chenodeoxycholate also increased in response to RE in muscle opposing previous findings in blood. Resistance training led to muscle hypertrophy, with slow type I and fast/intermediate type II muscle fibre diameter increasing by 10.7% and 10.4%, respectively. Comparison of post-exercise metabolite levels between trained and untrained state revealed alterations of 46 metabolites, including decreased N-acetylated ketogenic amino acids and increased beta-citrylglutamate which might support growth. Only five of the metabolites that changed after acute exercise in the untrained state were altered after chronic training, indicating that training induces multiple metabolic changes not directly related to the acute exercise response. CONCLUSION: The human skeletal muscle metabolome is sensitive towards acute RE in the trained and untrained states and reflects a broad range of adaptive processes in response to repeated stimulation.
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Tissue sensitivity and response to exercise vary according to the time of day and alignment of circadian clocks, but the optimal exercise time to elicit a desired metabolic outcome is not fully defined. To understand how tissues independently and collectively respond to timed exercise, we applied a systems biology approach. We mapped and compared global metabolite responses of seven different mouse tissues and serum after an acute exercise bout performed at different times of the day. Comparative analyses of intra- and inter-tissue metabolite dynamics, including temporal profiling and blood sampling across liver and hindlimb muscles, uncovered an unbiased view of local and systemic metabolic responses to exercise unique to time of day. This comprehensive atlas of exercise metabolism provides clarity and physiological context regarding the production and distribution of canonical and novel time-dependent exerkine metabolites, such as 2-hydroxybutyrate (2-HB), and reveals insight into the health-promoting benefits of exercise on metabolism.
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Relojes Circadianos , Condicionamiento Físico Animal , Animales , Ritmo Circadiano , Homeostasis , Hígado/metabolismo , Metabolómica , RatonesRESUMEN
The mammalian circadian clock, expressed throughout the brain and body, controls daily metabolic homeostasis. Clock function in peripheral tissues is required, but not sufficient, for this task. Because of the lack of specialized animal models, it is unclear how tissue clocks interact with extrinsic signals to drive molecular oscillations. Here, we isolated the interaction between feeding and the liver clock by reconstituting Bmal1 exclusively in hepatocytes (Liver-RE), in otherwise clock-less mice, and controlling timing of food intake. We found that the cooperative action of BMAL1 and the transcription factor CEBPB regulates daily liver metabolic transcriptional programs. Functionally, the liver clock and feeding rhythm are sufficient to drive temporal carbohydrate homeostasis. By contrast, liver rhythms tied to redox and lipid metabolism required communication with the skeletal muscle clock, demonstrating peripheral clock cross-talk. Our results highlight how the inner workings of the clock system rely on communicating signals to maintain daily metabolism.
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AIM: Resistance exercise increases muscle mass over time. However, the early signalling events leading to muscle growth are not yet well-defined. Here, we aim to identify new signalling pathways important for muscle remodelling after exercise. METHODS: We performed a phosphoproteomics screen after a single bout of exercise in mice. As an exercise model we used unilateral electrical stimulation in vivo and treadmill running. We analysed muscle biopsies from human subjects to verify if our findings in murine muscle also translate to exercise in humans. RESULTS: We identified a new phosphorylation site on Myocardin-Related Transcription Factor B (MRTF-B), a co-activator of serum response factor (SRF). Phosphorylation of MRTF-B is required for its nuclear translocation after exercise and is accompanied by the transcription of the SRF target gene Fos. In addition, high-intensity exercise also remodels chromatin at specific SRF target gene loci through the phosphorylation of histone 3 on serine 10 in myonuclei of both mice and humans. Ablation of the MAP kinase member MSK1/2 is sufficient to prevent this histone phosphorylation, reduce induction of SRF-target genes, and prevent increases in protein synthesis after exercise. CONCLUSION: Our results identify a new exercise signalling fingerprint in vivo, instrumental for exercise-induced protein synthesis and potentially muscle growth.
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Cromatina/química , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal , Factor de Respuesta Sérica , Transducción de Señal , Factores de Transcripción/metabolismo , Animales , Ejercicio Físico , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Biosíntesis de Proteínas , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismoRESUMEN
During ß-adrenergic stimulation of brown adipose tissue (BAT), p38 phosphorylates the activating transcription factor 2 (ATF2) which then translocates to the nucleus to activate the expression of Ucp1 and Pgc-1α. The mechanisms underlying ATF2 target activation are unknown. Here we demonstrate that p62 (Sqstm1) binds to ATF2 to orchestrate activation of the Ucp1 enhancer and Pgc-1α promoter. P62Δ69-251 mice show reduced expression of Ucp1 and Pgc-1α with impaired ATF2 genomic binding. Modulation of Ucp1 and Pgc-1α expression through p62 regulation of ATF2 signaling is demonstrated in vitro and in vivo in p62Δ69-251 mice, global p62-/- and Ucp1-Cre p62flx/flx mice. BAT dysfunction resulting from p62 deficiency is manifest after birth and obesity subsequently develops despite normal food intake, intestinal nutrient absorption and locomotor activity. In summary, our data identify p62 as a master regulator of BAT function in that it controls the Ucp1 pathway through regulation of ATF2 genomic binding.
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Factor de Transcripción Activador 2/metabolismo , Proteína Sequestosoma-1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adipogénesis/fisiología , Tejido Adiposo Pardo/diagnóstico por imagen , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/diagnóstico por imagen , Tejido Adiposo Blanco/metabolismo , Animales , Núcleo Celular/metabolismo , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Tomografía Computarizada por Tomografía de Emisión de Positrones , Unión Proteica , Proteína Sequestosoma-1/genética , Proteína Desacopladora 1/metabolismoRESUMEN
Metabolic diseases are often characterized by circadian misalignment in different tissues, yet how altered coordination and communication among tissue clocks relate to specific pathogenic mechanisms remains largely unknown. Applying an integrated systems biology approach, we performed 24-hr metabolomics profiling of eight mouse tissues simultaneously. We present a temporal and spatial atlas of circadian metabolism in the context of systemic energy balance and under chronic nutrient stress (high-fat diet [HFD]). Comparative analysis reveals how the repertoires of tissue metabolism are linked and gated to specific temporal windows and how this highly specialized communication and coherence among tissue clocks is rewired by nutrient challenge. Overall, we illustrate how dynamic metabolic relationships can be reconstructed across time and space and how integration of circadian metabolomics data from multiple tissues can improve our understanding of health and disease.
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Relojes Circadianos/fisiología , Metaboloma , Animales , Dieta Alta en Grasa , Metabolismo Energético , Hígado/metabolismo , Masculino , Redes y Vías Metabólicas , Metabolómica , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Corteza Prefrontal/metabolismo , Núcleo Supraquiasmático/metabolismo , Proteína Desacopladora 1/metabolismoRESUMEN
PURPOSE OF REVIEW: The review is focused on the unexpected role of myogenic regulatory factor 4 (MRF4) in controlling muscle mass by repressing myocyte enhancer binding factor 2 (MEF2) activity in adult skeletal muscle, and on the emerging role of MEF2 in skeletal muscle growth. RECENT FINDINGS: The MRF4s of the MyoD family (MyoD, MYF5, MRF4, myogenin) and the MEF2 factors are known to play a major role in embryonic myogenesis. However, their function in adult muscle tissue is not known. A recent study shows that MRF4 loss in adult skeletal muscle causes muscle hypertrophy and prevents denervation atrophy. This effect is mediated by MEF2 factors that promote muscle growth, with MRF4 acting as a repressor of MEF2 activity. The role of MEF2 in skeletal muscle growth is supported by the finding that muscle regeneration is impaired by muscle-specific triple knockout of Mef2a, c, and d genes. SUMMARY: The finding that the MRF4-MEF2 axis controls muscle growth opens a new perspective for preventing muscle wasting. A unique feature of this pathway is that MRF4 is exclusively expressed in skeletal muscle, thus reducing the risk that interventions aimed at down-regulating MRF4 or interfering with the interaction between MRF4 and MEF2 may have off-target effects in other tissues.
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Factores de Transcripción MEF2/metabolismo , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Enfermedades Musculares/metabolismo , Factores Reguladores Miogénicos/metabolismo , Síndrome Debilitante/metabolismo , Animales , Humanos , Atrofia Muscular/prevención & control , Miogenina/metabolismo , Síndrome Debilitante/prevención & controlRESUMEN
Skeletal muscle mass is a result of the balance between protein breakdown and protein synthesis. It has been shown that multiple conditions of muscle atrophy are characterized by the common regulation of a specific set of genes, termed atrogenes. It is not known whether various models of muscle hypertrophy are similarly regulated by a common transcriptional program. Here, we characterized gene expression changes in three different conditions of muscle growth, examining each condition during acute and chronic phases. Specifically, we compared the transcriptome of Extensor Digitorum Longus (EDL) muscles collected (1) during the rapid phase of postnatal growth at 2 and 4 weeks of age, (2) 24 h or 3 weeks after constitutive activation of AKT, and (3) 24 h or 3 weeks after overload hypertrophy caused by tenotomy of the Tibialis Anterior muscle. We observed an important overlap between significantly regulated genes when comparing each single condition at the two different timepoints. Furthermore, examining the transcriptional changes occurring 24 h after a hypertrophic stimulus, we identify an important role for genes linked to a stress response, despite the absence of muscle damage in the AKT model. However, when we compared all different growth conditions, we did not find a common transcriptional fingerprint. On the other hand, all conditions showed a marked increase in mTORC1 signaling and increased ribosome biogenesis, suggesting that muscle growth is characterized more by translational, than transcriptional regulation.
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Circadian rhythms are widely known to govern human health and disease, but specific pathogenic mechanisms linking circadian disruption to metabolic diseases are just beginning to come to light. This is thanks in part to the development and application of various "omics"-based tools in biology and medicine. Current high-throughput technologies allow for the simultaneous monitoring of multiple dynamic cellular events over time, ranging from gene expression to metabolite abundance and sub-cellular localization. These fundamental temporal and spatial perspectives have allowed for a more comprehensive understanding of how various dynamic cellular events and biochemical processes are related in health and disease. With advances in technology, metabolomics has become a more routine "omics" approach for studying metabolism, and "circadian metabolomics" (i.e., studying the 24-h metabolome) has recently been undertaken by several groups. To date, circadian metabolomes have been reported for human serum, saliva, breath, and urine, as well as tissues from several species under specific disease or mutagenesis conditions. Importantly, these studies have consistently revealed that 24-h rhythms are prevalent in almost every tissue and metabolic pathway. Furthermore, these circadian rhythms in tissue metabolism are ultimately linked to and directed by internal 24-h biological clocks. In this review, we will attempt to put these data-rich circadian metabolomics experiments into perspective to find out what they can tell us about metabolic health and disease, and what additional biomarker potential they may reveal.
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The circadian oscillations of muscle genes are controlled either directly by the intrinsic muscle clock or by extrinsic factors, such as feeding, hormonal signals, or neural influences, which are in turn regulated by the central pacemaker, the suprachiasmatic nucleus of the hypothalamus. A unique feature of circadian rhythms in skeletal muscle is motor neuron-dependent contractile activity, which can affect the oscillation of a number of muscle genes independently of the muscle clock. The role of the intrinsic muscle clock has been investigated using different Bmal1 knockout (KO) models. A comparative analysis of these models reveals that the dramatic muscle wasting and premature aging caused by global conventional KO are not present in muscle-specific Bmal1 KO or in global Bmal1 KO induced in the adult, therefore must reflect the loss of Bmal1 function during development in non-muscle tissues. On the other hand, muscle-specific Bmal1 knockout causes impaired muscle glucose uptake and metabolism, supporting a major role of the muscle clock in anticipating the sleep-to-wake transition, when glucose becomes the predominant fuel for the skeletal muscle.
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Factores de Transcripción ARNTL/fisiología , Relojes Circadianos , Músculo Esquelético/fisiología , Factores de Transcripción ARNTL/genética , Animales , Expresión Génica , Glucosa/metabolismo , Ratones Noqueados , Actividad Motora , Músculo Esquelético/metabolismo , Miocardio/metabolismoRESUMEN
The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.
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Envejecimiento/metabolismo , Factores de Transcripción MEF2/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Factores Reguladores Miogénicos/metabolismo , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Histona Desacetilasas/metabolismo , Humanos , Hipertrofia , Masculino , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Especificidad de Órganos/genética , Unión Proteica , Biosíntesis de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas Wistar , Proteínas Represoras/metabolismo , Transcripción Genética , Regulación hacia Arriba/genéticaRESUMEN
OBJECTIVE: Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle. METHODS: We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression. RESULTS: We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. CONCLUSIONS: We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca(2+)-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression.
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[This corrects the article DOI: 10.1016/j.molmet.2013.10.005.].
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Circadian rhythms control metabolism and energy homeostasis, but the role of the skeletal muscle clock has never been explored. We generated conditional and inducible mouse lines with muscle-specific ablation of the core clock gene Bmal1. Skeletal muscles from these mice showed impaired insulin-stimulated glucose uptake with reduced protein levels of GLUT4, the insulin-dependent glucose transporter, and TBC1D1, a Rab-GTPase involved in GLUT4 translocation. Pyruvate dehydrogenase (PDH) activity was also reduced due to altered expression of circadian genes Pdk4 and Pdp1, coding for PDH kinase and phosphatase, respectively. PDH inhibition leads to reduced glucose oxidation and diversion of glycolytic intermediates to alternative metabolic pathways, as revealed by metabolome analysis. The impaired glucose metabolism induced by muscle-specific Bmal1 knockout suggests that a major physiological role of the muscle clock is to prepare for the transition from the rest/fasting phase to the active/feeding phase, when glucose becomes the predominant fuel for skeletal muscle.