RESUMO
In mammals, endogenous circadian clocks sense and respond to daily feeding and lighting cues, adjusting internal â¼24 h rhythms to resonate with, and anticipate, external cycles of day and night. The mechanism underlying circadian entrainment to feeding time is critical for understanding why mistimed feeding, as occurs during shift work, disrupts circadian physiology, a state that is associated with increased incidence of chronic diseases such as type 2 (T2) diabetes. We show that feeding-regulated hormones insulin and insulin-like growth factor 1 (IGF-1) reset circadian clocks in vivo and in vitro by induction of PERIOD proteins, and mistimed insulin signaling disrupts circadian organization of mouse behavior and clock gene expression. Insulin and IGF-1 receptor signaling is sufficient to determine essential circadian parameters, principally via increased PERIOD protein synthesis. This requires coincident mechanistic target of rapamycin (mTOR) activation, increased phosphoinositide signaling, and microRNA downregulation. Besides its well-known homeostatic functions, we propose insulin and IGF-1 are primary signals of feeding time to cellular clocks throughout the body.
Assuntos
Relógios Circadianos/fisiologia , Comportamento Alimentar/fisiologia , Proteínas Circadianas Period/metabolismo , Animais , Ritmo Circadiano/fisiologia , Feminino , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor IGF Tipo 1/metabolismo , Transdução de SinaisRESUMO
Cellular circadian rhythms confer temporal organisation upon physiology that is fundamental to human health. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body, but their physiological function is poorly understood. Here, we present a novel biochemical assay for haemoglobin (Hb) oxidation status which relies on a redox-sensitive covalent haem-Hb linkage that forms during SDS-mediated cell lysis. Formation of this linkage is lowest when ferrous Hb is oxidised, in the form of ferric metHb. Daily haemoglobin oxidation rhythms are observed in mouse and human RBCs cultured in vitro, or taken from humans in vivo, and are unaffected by mutations that affect circadian rhythms in nucleated cells. These rhythms correlate with daily rhythms in core body temperature, with temperature lowest when metHb levels are highest. Raising metHb levels with dietary sodium nitrite can further decrease daytime core body temperature in mice via nitric oxide (NO) signalling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature.
Assuntos
Eritrócitos , Hemoglobinas , Humanos , Camundongos , Animais , Eritrócitos/metabolismo , Hemoglobinas/metabolismo , Oxirredução , Heme/metabolismo , Ritmo CircadianoRESUMO
Chronic inflammation underpins many human diseases. Morbidity and mortality associated with chronic inflammation are often mediated through metabolic dysfunction. Inflammatory and metabolic processes vary through circadian time, suggesting an important temporal crosstalk between these systems. Using an established mouse model of rheumatoid arthritis, we show that chronic inflammatory arthritis results in rhythmic joint inflammation and drives major changes in muscle and liver energy metabolism and rhythmic gene expression. Transcriptional and phosphoproteomic analyses revealed alterations in lipid metabolism and mitochondrial function associated with increased EGFR-JAK-STAT3 signaling. Metabolomic analyses confirmed rhythmic metabolic rewiring with impaired ß-oxidation and lipid handling and revealed a pronounced shunt toward sphingolipid and ceramide accumulation. The arthritis-related production of ceramides was most pronounced during the day, which is the time of peak inflammation and increased reliance on fatty acid oxidation. Thus, our data demonstrate that localized joint inflammation drives a time-of-daydependent build-up of bioactive lipid species driven by rhythmic inflammation and altered EGFR-STAT signaling.
Assuntos
Artrite , Relógios Circadianos , Ritmo Circadiano/fisiologia , Metabolismo Energético , Humanos , Inflamação/metabolismoRESUMO
The gut microbiota is important for host health and immune system function. Moreover autoimmune diseases, such as rheumatoid arthritis, are associated with significant gut microbiota dysbiosis, although the causes and consequences of this are not fully understood. It has become clear that the composition and metabolic outputs of the microbiome exhibit robust 24 h oscillations, a result of daily variation in timing of food intake as well as rhythmic circadian clock function in the gut. Here, we report that experimental inflammatory arthritis leads to a re-organization of circadian rhythmicity in both the gut and associated microbiome. Mice with collagen induced arthritis exhibited extensive changes in rhythmic gene expression in the colon, and reduced barrier integrity. Re-modeling of the host gut circadian transcriptome was accompanied by significant alteration of the microbiota, including widespread loss of rhythmicity in symbiont species of Lactobacillus, and alteration in circulating microbial derived factors, such as tryptophan metabolites, which are associated with maintenance of barrier function and immune cell populations within the gut. These findings highlight that altered circadian rhythmicity during inflammatory disease contributes to dysregulation of gut integrity and microbiome function.
Assuntos
Artrite Experimental , Microbioma Gastrointestinal , Microbiota , Camundongos , Animais , Microbioma Gastrointestinal/fisiologia , Disbiose/etiologia , Artrite Experimental/complicações , ColágenoRESUMO
Mammalian circadian rhythms are orchestrated by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN), which receives information about the 24 h light-dark cycle from the retina. The accepted function of this light signal is to reset circadian phase in order to ensure appropriate synchronization with the celestial day. Here, we ask whether light also impacts another key property of the circadian oscillation, its amplitude. To this end, we measured circadian rhythms in behavioral activity, body temperature, and SCN electrophysiological activity in the diurnal murid rodent Rhabdomys pumilio following stable entrainment to 12:12 light-dark cycles at four different daytime intensities (ranging from 18 to 1,900 lx melanopic equivalent daylight illuminance). R. pumilio showed strongly diurnal activity and body temperature rhythms in all conditions, but measures of rhythm robustness were positively correlated with daytime irradiance under both entrainment and subsequent free run. Whole-cell and extracellular recordings of electrophysiological activity in ex vivo SCN revealed substantial differences in electrophysiological activity between dim and bright light conditions. At lower daytime irradiance, daytime peaks in SCN spontaneous firing rate and membrane depolarization were substantially depressed, leading to an overall marked reduction in the amplitude of circadian rhythms in spontaneous activity. Our data reveal a previously unappreciated impact of daytime light intensity on SCN physiology and the amplitude of circadian rhythms and highlight the potential importance of daytime light exposure for circadian health.
Assuntos
Ritmo Circadiano , Luz , Mamíferos/fisiologia , Animais , Neurônios/fisiologia , Reprodutibilidade dos Testes , Núcleo Supraquiasmático/fisiologiaRESUMO
The nuclear receptor REVERBα is a core component of the circadian clock and proposed to be a dominant regulator of hepatic lipid metabolism. Using antibody-independent ChIP-sequencing of REVERBα in mouse liver, we reveal a high-confidence cistrome and define direct target genes. REVERBα-binding sites are highly enriched for consensus RORE or RevDR2 motifs and overlap with corepressor complex binding. We find no evidence for transcription factor tethering and DNA-binding domain-independent action. Moreover, hepatocyte-specific deletion of Reverbα drives only modest physiological and transcriptional dysregulation, with derepressed target gene enrichment limited to circadian processes. Thus, contrary to previous reports, hepatic REVERBα does not repress lipogenesis under basal conditions. REVERBα control of a more extensive transcriptional program is only revealed under conditions of metabolic perturbation (including mistimed feeding, which is a feature of the global Reverbα-/- mouse). Repressive action of REVERBα in the liver therefore serves to buffer against metabolic challenge, rather than drive basal rhythmicity in metabolic activity.
Assuntos
Metabolismo Energético , Fígado/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Motivos de Aminoácidos , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relógios Circadianos , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/química , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genéticaRESUMO
Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinß1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.
Assuntos
Proteínas CLOCK/antagonistas & inibidores , Relógios Circadianos/fisiologia , Fibroblastos/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Animais , Bleomicina/efeitos adversos , Proteínas CLOCK/genética , Proteínas CLOCK/uso terapêutico , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fibrose Pulmonar Idiopática , Integrinas , Pulmão/patologia , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Knockout , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , TranscriptomaRESUMO
The circadian timing system governs daily biological rhythms, synchronising physiology and behaviour to the temporal world. External time cues, including the light-dark cycle and timing of food intake, provide daily signals for entrainment of the central, master circadian clock in the hypothalamic suprachiasmatic nuclei (SCN), and of metabolic rhythms in peripheral tissues, respectively. Chrono-nutrition is an emerging field building on the relationship between temporal eating patterns, circadian rhythms, and metabolic health. Evidence from both animal and human research demonstrates adverse metabolic consequences of circadian disruption. Conversely, a growing body of evidence indicates that aligning food intake to periods of the day when circadian rhythms in metabolic processes are optimised for nutrition may be effective for improving metabolic health. Circadian rhythms in glucose and lipid homeostasis, insulin responsiveness and sensitivity, energy expenditure, and postprandial metabolism, may favour eating patterns characterised by earlier temporal distribution of energy. This review details the molecular basis for metabolic clocks, the regulation of feeding behaviour, and the evidence for meal timing as an entraining signal for the circadian system in animal models. The epidemiology of temporal eating patterns in humans is examined, together with evidence from human intervention studies investigating the metabolic effects of morning compared to evening energy intake, and emerging chrono-nutrition interventions such as time-restricted feeding. Chrono-nutrition may have therapeutic application for individuals with and at-risk of metabolic disease and convey health benefits within the general population.
Assuntos
Ritmo Circadiano/fisiologia , Metabolismo Energético/fisiologia , Comportamento Alimentar/fisiologia , Homeostase/fisiologia , Neurônios/fisiologia , Animais , Humanos , FotoperíodoRESUMO
BACKGROUND: Daily variations in mammalian physiology are under control of a central clock in the suprachiasmatic nucleus (SCN). SCN timing signals are essential for coordinating cellular clocks and associated circadian variations in cell and tissue function across the body; however, direct SCN projections primarily target a restricted set of hypothalamic and thalamic nuclei involved in physiological and behavioural control. The role of the SCN in driving rhythmic activity in these targets remains largely unclear. Here, we address this issue via multielectrode recording and manipulations of SCN output in adult mouse brain slices. RESULTS: Electrical stimulation identifies cells across the midline hypothalamus and ventral thalamus that receive inhibitory input from the SCN and/or excitatory input from the retina. Optogenetic manipulations confirm that SCN outputs arise from both VIP and, more frequently, non-VIP expressing cells and that both SCN and retinal projections almost exclusively target GABAergic downstream neurons. The majority of midline hypothalamic and ventral thalamic neurons exhibit circadian variation in firing and those receiving inhibitory SCN projections consistently exhibit peak activity during epochs when SCN output is low. Physical removal of the SCN confirms that neuronal rhythms in ~ 20% of the recorded neurons rely on central clock input but also reveals many neurons that can express circadian variation in firing independent of any SCN input. CONCLUSIONS: We identify cell populations across the midline hypothalamus and ventral thalamus exhibiting SCN-dependent and independent rhythms in neural activity, providing new insight into the mechanisms by which the circadian system generates daily physiological rhythms.
Assuntos
Ritmo Circadiano/fisiologia , Hipotálamo/fisiologia , Neurônios/fisiologia , Núcleo Supraquiasmático/fisiologia , Tálamo/fisiologia , Animais , CamundongosRESUMO
Twilight is characterised by changes in both quantity ("irradiance") and quality ("colour") of light. Animals use the variation in irradiance to adjust their internal circadian clocks, aligning their behaviour and physiology with the solar cycle. However, it is currently unknown whether changes in colour also contribute to this entrainment process. Using environmental measurements, we show here that mammalian blue-yellow colour discrimination provides a more reliable method of tracking twilight progression than simply measuring irradiance. We next use electrophysiological recordings to demonstrate that neurons in the mouse suprachiasmatic circadian clock display the cone-dependent spectral opponency required to make use of this information. Thus, our data show that some clock neurons are highly sensitive to changes in spectral composition occurring over twilight and that this input dictates their response to changes in irradiance. Finally, using mice housed under photoperiods with simulated dawn/dusk transitions, we confirm that spectral changes occurring during twilight are required for appropriate circadian alignment under natural conditions. Together, these data reveal a new sensory mechanism for telling time of day that would be available to any mammalian species capable of chromatic vision.
Assuntos
Relógios Circadianos/fisiologia , Cor , Animais , Camundongos , Camundongos Knockout , Estimulação Luminosa , Opsinas de Bastonetes/fisiologia , Núcleo Supraquiasmático/fisiologiaRESUMO
There is strong diurnal variation in the symptoms and severity of chronic inflammatory diseases, such as rheumatoid arthritis. In addition, disruption of the circadian clock is an aggravating factor associated with a range of human inflammatory diseases. To investigate mechanistic links between the biological clock and pathways underlying inflammatory arthritis, mice were administered collagen (or saline as a control) to induce arthritis. The treatment provoked an inflammatory response within the limbs, which showed robust daily variation in paw swelling and inflammatory cytokine expression. Inflammatory markers were significantly repressed during the dark phase. Further work demonstrated an active molecular clock within the inflamed limbs and highlighted the resident inflammatory cells, fibroblast-like synoviocytes (FLSs), as a potential source of the rhythmic inflammatory signal. Exposure of mice to constant light disrupted the clock in peripheral tissues, causing loss of the nighttime repression of local inflammation. Finally, the results show that the core clock proteins cryptochrome (CRY) 1 and 2 repressed inflammation within the FLSs, and provide novel evidence that a CRY activator has anti-inflammatory properties in human cells. We conclude that under chronic inflammatory conditions, the clock actively represses inflammatory pathways during the dark phase. This interaction has exciting potential as a therapeutic avenue for treatment of inflammatory disease.-Hand, L. E., Hopwood, T. W., Dickson, S. H., Walker, A. L., Loudon, A. S. I., Ray, D. W., Bechtold, D. A., Gibbs, J. E. The circadian clock regulates inflammatory arthritis.
Assuntos
Artrite Reumatoide/metabolismo , Proteínas CLOCK/metabolismo , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Animais , Artrite Reumatoide/terapia , Proteínas CLOCK/genética , Modelos Animais de Doenças , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/terapia , Masculino , CamundongosRESUMO
Coordinated daily rhythms are evident in most aspects of our physiology, driven by internal timing systems known as circadian clocks. Our understanding of how biological clocks are built and function has grown exponentially over the past 20 years. With this has come an appreciation that disruption of the clock contributes to the pathophysiology of numerous diseases, from metabolic disease to neurological disorders to cancer. However, it remains to be determined whether it is the disruption of our rhythmic physiology per se (loss of timing itself), or altered functioning of individual clock components that drive pathology. Here, we review the importance of circadian rhythms in terms of how we (and other organisms) relate to the external environment, but also in relation to how internal physiological processes are coordinated and synchronized. These issues are of increasing importance as many aspects of modern life put us in conflict with our internal clockwork.
Assuntos
Relógios Circadianos , Animais , Ritmo Circadiano , Humanos , Transtornos do Sono do Ritmo Circadiano/fisiopatologia , Núcleo Supraquiasmático/fisiopatologiaRESUMO
Circadian and homeostatic neural circuits organize the temporal architecture of physiology and behavior, but knowledge of their interactions is imperfect. For example, neurons containing the neuropeptide orexin homeostatically control arousal and appetitive states, while neurons in the suprachiasmatic nuclei (SCN) function as the brain's master circadian clock. The SCN regulates orexin neurons so that they are much more active during the circadian night than the circadian day, but it is unclear whether the orexin neurons reciprocally regulate the SCN clock. Here we show both orexinergic innervation and expression of genes encoding orexin receptors (OX1 and OX2) in the mouse SCN, with OX1 being upregulated at dusk. Remarkably, we find through in vitro physiological recordings that orexin predominantly suppresses mouse SCN Period1 (Per1)-EGFP-expressing clock cells. The mechanisms underpinning these suppressions vary across the circadian cycle, from presynaptic modulation of inhibitory GABAergic signaling during the day to directly activating leak K(+) currents at night. Orexin also augments the SCN clock-resetting effects of neuropeptide Y (NPY), another neurochemical correlate of arousal, and potentiates NPY's inhibition of SCN Per1-EGFP cells. These results build on emerging literature that challenge the widely held view that orexin signaling is exclusively excitatory and suggest new mechanisms for avoiding conflicts between circadian clock signals and homeostatic cues in the brain.
Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neuropeptídeos/fisiologia , Receptores de Orexina/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptores de Orexina/deficiência , Orexinas , Técnicas de Cultura de Órgãos , Transdução de Sinais/fisiologia , Núcleo Supraquiasmático/metabolismo , Fatores de TempoRESUMO
Ob/ob mice provide an animal model for non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) in patients with obesity and type-2 diabetes. Low liver copper has been linked to hepatic lipid build-up (steatosis) in animals with systemic copper deficiency caused by low-copper diets. However, hepatic copper status in patients with NAFLD or NASH is uncertain, and a validated animal model useful for the study of hepatic copper regulation in common forms of metabolic liver disease is lacking. Here, we report parallel measurements of essential metal levels in whole-liver tissue and defatted-dried liver tissue from ob/ob and non-obese control mice. Measurements in whole-liver tissue from ob/ob mice at an age when they have developed NAFLD/NASH, provide compelling evidence for factitious lowering of copper and all other essential metals by steatosis, and so cannot be used to study hepatic metal regulation in this model. By marked contrast, metal measurements in defatted-dried liver samples reveal that most essential metals were actually normal and indicate specific lowering of copper in ob/ob mice, consistent with hepatic copper deficiency. Thus ob/ob mice can provide a model useful for the study of copper regulation in NAFLD and NASH, provided levels are measured in defatted-dried liver tissue.
Assuntos
Cobre/metabolismo , Gorduras/isolamento & purificação , Hepatopatias/metabolismo , Fígado/metabolismo , Animais , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Rheumatoid arthritis is a chronic inflammatory disease that shows characteristic diurnal variation in symptom severity, where joint resident fibroblast-like synoviocytes (FLS) act as important mediators of arthritis pathology. We investigate the role of FLS circadian clock function in directing rhythmic joint inflammation in a murine model of inflammatory arthritis. We demonstrate FLS time-of-day-dependent gene expression is attenuated in arthritic joints, except for a subset of disease-modifying genes. The deletion of essential clock gene Bmal1 in FLS reduced susceptibility to collagen-induced arthritis but did not impact symptomatic severity in affected mice. Notably, FLS Bmal1 deletion resulted in loss of diurnal expression of disease-modulating genes across the joint, and elevated production of MMP3, a prognostic marker of joint damage in inflammatory arthritis. This work identifies the FLS circadian clock as an influential driver of daily oscillations in joint inflammation, and a potential regulator of destructive pathology in chronic inflammatory arthritis.
Assuntos
Fatores de Transcrição ARNTL , Artrite Experimental , Ritmo Circadiano , Fibroblastos , Sinoviócitos , Animais , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Camundongos , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Artrite Experimental/patologia , Artrite Experimental/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Relógios Circadianos/genética , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Camundongos Knockout , Modelos Animais de Doenças , Regulação da Expressão Gênica , MasculinoRESUMO
Within the core molecular clock, protein phosphorylation and degradation play a vital role in determining circadian period. The 'after-hours' (Afh) mutation in mouse slows the degradation of the core clock protein Cryptochrome, lengthening the period of the molecular clock in the suprachiasmatic nuclei (SCN) and behavioural wheel-running rhythms. However, we do not yet know how the Afh mutation affects other aspects of physiology or the activity of circadian oscillators in other brain regions. Here we report that daily rhythms of metabolism and ingestive behaviours are altered in these animals, as are PERIOD2::LUCIFERASE (PER2::LUC) rhythms in mediobasal hypothalamic nuclei, which influence these behaviours. Overall there is a trend towards period lengthening and a decrease in amplitude of PER2::LUC rhythms throughout the brain. Imaging of single cells from the arcuate and dorsomedial hypothalamic nuclei revealed this reduction in tissue oscillator amplitude to be due to a decrease in the amplitude, rather than a desynchrony, of single cells. Consistent with existing models of oscillator function, this cellular phenotype was associated with a greater susceptibility to phase-shifting stimuli in vivo and in vitro, with light evoking high-amplitude Type 0 resetting in Afh mutant mice. Together, these findings reveal unexpected consequences of the Afh mutation on the amplitude and synchrony of individual cellular oscillators in the SCN.
Assuntos
Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/fisiologia , Ritmo Circadiano/fisiologia , Animais , Comportamento Animal/fisiologia , Relógios Circadianos/fisiologia , Comportamento de Ingestão de Líquido/fisiologia , Metabolismo Energético , Camundongos , Camundongos Mutantes , Mutação , Corrida/fisiologia , Núcleo Supraquiasmático/fisiologiaRESUMO
Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) epsilon or delta can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1epsilon and CK1delta using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1delta is the principal regulator of the clock period: pharmacological inhibition of CK1delta, but not CK1epsilon, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1delta inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1delta inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2(-/-) mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2(-/-) mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1delta offers an avenue for therapeutic modulation of perturbed circadian behavior.
Assuntos
Caseína Quinase 1 épsilon/antagonistas & inibidores , Caseína Quinase Idelta/antagonistas & inibidores , Ritmo Circadiano/fisiologia , Animais , Sequência de Bases , Caseína Quinase 1 épsilon/fisiologia , Caseína Quinase Idelta/deficiência , Caseína Quinase Idelta/genética , Caseína Quinase Idelta/fisiologia , Ritmo Circadiano/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Técnicas In Vitro , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Circadianas Period/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/deficiência , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/fisiologiaRESUMO
BACKGROUND: The three-ringed polycyclic aromatic hydrocarbon (PAH) phenanthrene (Phe) has been implicated in the cardiotoxicity of petroleum-based pollution in aquatic systems, where it disrupts the contractile and electrical function of the fish heart. Phe is also found adsorbed to particulate matter and in the gas phase of air pollution, but to date, no studies have investigated the impact of Phe on mammalian cardiac function. OBJECTIVES: Our objectives were to determine the arrhythmogenic potential of acute Phe exposure on mammalian cardiac function and define the underlying mechanisms to provide insight into the toxicity risk to humans. METHODS: Ex vivo Langendorff-perfused mouse hearts were used to test the arrhythmogenic potential of Phe on myocardial function, and voltage- and current-clamp recordings were used to define underlying cellular mechanisms in isolated cardiomyocytes. RESULTS: Mouse hearts exposed to â¼8µM Phe for 15-min exhibited a significantly slower heart rate (p=0.0006, N=10 hearts), a prolonged PR interval (p=0.036, N=8 hearts), and a slower conduction velocity (p=0.0143, N=7 hearts). Whole-cell recordings from isolated cardiomyocytes revealed action potential (AP) duration prolongation (at 80% repolarization; p=0.0408, n=9 cells) and inhibition of key murine repolarizing currents-transient outward potassium current (Ito) and ultrarapid potassium current (IKur)-following Phe exposure. A significant reduction in AP upstroke velocity (p=0.0445, n=9 cells) and inhibition of the fast sodium current (INa; p=0.001, n=8 cells) and calcium current (ICa; p=0.0001) were also observed, explaining the slowed conduction velocity in intact hearts. Finally, acute exposure to â¼8µM Phe significantly increased susceptibility to arrhythmias (p=0.0455, N=9 hearts). DISCUSSION: To the best of our knowledge, this is the first evidence of direct inhibitory effects of Phe on mammalian cardiac electrical activity at both the whole-heart and cell levels. This electrical dysfunction manifested as an increase in arrhythmia susceptibility due to impairment of both conduction and repolarization. Similar effects in humans could have serious health consequences, warranting greater regulatory attention and toxicological investigation into this ubiquitous PAH pollutant generated from fossil-fuel combustion. https://doi.org/10.1289/EHP12775.
Assuntos
Poluentes Atmosféricos , Fenantrenos , Humanos , Camundongos , Animais , Poluentes Atmosféricos/toxicidade , Arritmias Cardíacas/induzido quimicamente , Miócitos Cardíacos , Potenciais de Ação , Modelos Animais de Doenças , Fenantrenos/toxicidade , Potássio/farmacologia , MamíferosRESUMO
Disruption of brain-expressed G protein-coupled receptor-10 (GPR10) causes obesity in animals. Here, we identify multiple rare variants in GPR10 in people with severe obesity and in normal weight controls. These variants impair ligand binding and G protein-dependent signalling in cells. Transgenic mice harbouring a loss of function GPR10 variant found in an individual with obesity, gain excessive weight due to decreased energy expenditure rather than increased food intake. This evidence supports a role for GPR10 in human energy homeostasis. Therapeutic targeting of GPR10 may represent an effective weight-loss strategy.
Assuntos
Obesidade , Receptores Acoplados a Proteínas G , Animais , Humanos , Camundongos , Metabolismo Energético , Camundongos Transgênicos , Obesidade/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Aumento de Peso/genéticaRESUMO
The glucocorticoid receptor (GR) is a nuclear receptor critical to the regulation of energy metabolism and inflammation. The actions of GR are dependent on cell type and context. Here, we demonstrate the role of liver lineage-determining factor hepatocyte nuclear factor 4A (HNF4A) in defining liver specificity of GR action. In mouse liver, the HNF4A motif lies adjacent to the glucocorticoid response element (GRE) at GR binding sites within regions of open chromatin. In the absence of HNF4A, the liver GR cistrome is remodeled, with loss and gain of GR recruitment evident. Loss of chromatin accessibility at HNF4A-marked sites associates with loss of GR binding at weak GRE motifs. GR binding and chromatin accessibility are gained at sites characterized by strong GRE motifs, which show GR recruitment in non-liver tissues. The functional importance of these HNF4A-regulated GR sites is indicated by an altered transcriptional response to glucocorticoid treatment in the Hnf4a-null liver.