Circadian clock network desynchrony promotes weight gain and alters glucose homeostasis in mice.

OBJECTIVE: A network of endogenous circadian clocks adapts physiology and behavior to recurring changes in environmental demands across the 24-hour day cycle. Circadian disruption promotes weight gain and type 2 diabetes development. In this study, we aim to dissect the roles of different tissue clocks in the regulation of energy metabolism. METHODS: We used mice with genetically ablated clock function in the circadian pacemaker of the suprachiasmatic nucleus (SCN) under different light and feeding conditions to study peripheral clock resetting and the role of the peripheral clock network in the regulation of glucose handling and metabolic homeostasis. RESULTS: In SCN clock-deficient mice, behavioral and non-SCN tissue clock rhythms are sustained under rhythmic lighting conditions but deteriorate quickly in constant darkness. In parallel to the loss of behavioral and molecular rhythms, the animals develop adiposity and impaired glucose utilization in constant darkness. Restoring peripheral clock rhythmicity and synchrony by time-restricted feeding normalizes body weight and glucose metabolism. CONCLUSIONS: These data reveal the importance of an overall synchronized circadian clockwork for the maintenance of metabolic homeostasis.

Artery-Associated Sympathetic Innervation Drives Rhythmic Vascular Inflammation of Arteries and Veins.

BACKGROUND: The incidence of acute cardiovascular complications is highly time-of-day dependent. However, the mechanisms driving rhythmicity of ischemic vascular events are unknown. Although enhanced numbers of leukocytes have been linked to an increased risk of cardiovascular complications, the role that rhythmic leukocyte adhesion plays in different vascular beds has not been studied. METHODS: We evaluated leukocyte recruitment in vivo by using real-time multichannel fluorescence intravital microscopy of a tumor necrosis factor-α-induced acute inflammation model in both murine arterial and venous macrovasculature and microvasculature. These approaches were complemented with genetic, surgical, and pharmacological ablation of sympathetic nerves or adrenergic receptors to assess their relevance for rhythmic leukocyte adhesion. In addition, we genetically targeted the key circadian clock gene Bmal1 (also known as Arntl) in a lineage-specific manner to dissect the importance of oscillations in leukocytes and components of the vessel wall in this process. RESULTS: In vivo quantitative imaging analyses of acute inflammation revealed a 24-hour rhythm in leukocyte recruitment to arteries and veins of the mouse macrovasculature and microvasculature. Unexpectedly, although in arteries leukocyte adhesion was highest in the morning, it peaked at night in veins. This phase shift was governed by a rhythmic microenvironment and a vessel type-specific oscillatory pattern in the expression of promigratory molecules. Differences in cell adhesion molecules and leukocyte adhesion were ablated when disrupting sympathetic nerves, demonstrating their critical role in this process and the importance of ß2-adrenergic receptor signaling. Loss of the core clock gene Bmal1 in leukocytes, endothelial cells, or arterial mural cells affected the oscillations in a vessel type-specific manner. Rhythmicity in the intravascular reactivity of adherent leukocytes resulted in increased interactions with platelets in the morning in arteries and in veins at night with a higher predisposition to acute thrombosis at different times as a consequence. CONCLUSIONS: Together, our findings point to an important and previously unrecognized role of artery-associated sympathetic innervation in governing rhythmicity in vascular inflammation in both arteries and veins and its potential implications in the occurrence of time-of-day-dependent vessel type-specific thrombotic events.

Chronodisruption, Metabolic Homeostasis, and the Regulation of Inflammation in Adipose Tissues.

Molecular circadian clocks align daily behavioral and metabolic rhythms with the external day-night cycle. Priming energy metabolism for recurring changes on a 24-hour basis, these clocks are deeply interlinked with metabolic homeostasis and health. Circadian rhythm disruptions, as occurring in shift work or sleep disorders, are often accompanied by metabolic disturbances - from the promotion of overweight and type-2 diabetes to the development of the metabolic syndrome. An important indicator of the adverse outcomes of overweight seems to be a systemic low-grade inflammation which is initially observed in adipose tissues and is promoted by circadian misalignment. Interestingly, the genetic disruption of circadian clocks in rodents leads to metabolic dysregulations very comparable to what is observed in shift workers and with the development of tissue specific clock gene knockout mice, the importance of single-tissue clocks for the metabolic regulation was further deciphered. In this review, we summarize the current knowledge on the role of mistimed behavior in metabolic health and outline behavioral interventions aiming at reducing the metabolic ramifications of chronodisruption.

Interplay of central and peripheral circadian clocks in energy metabolism regulation.

Metabolic health founds on a homeostatic balance that has to integrate the daily changes of rest/activity and feeding/fasting cycles. A network of endogenous 24-hour circadian clocks helps to anticipate daily recurring events and adjust physiology and behavioural functions accordingly. Circadian clocks are self-sustained cellular oscillators based on a set of clock genes/proteins organised in interlocked transcriptional-translational feedback loops. The body's clocks need to be regularly reset and synchronised with each other to achieve coherent rhythmic output signals. This synchronisation is achieved by interplay of a master clock, which resides in the suprachiasmatic nucleus, and peripheral tissue clocks. This clock network is reset by time signals such as the light/dark cycle, food intake and activity. The balanced interplay of clocks is easily disturbed in modern society by shiftwork or high-energy diets, which may further promote the development of metabolic disorders. In this review, we summarise the current model of central-peripheral clock interaction in metabolic health. Different established mouse models for central or peripheral clock disruption and their metabolic phenotypes are compared and the possible relevance of clock network interaction for the development of therapeutic approaches in humans is discussed.

Circadian period of luciferase expression shortens with age in human mature adipocytes from obese patients.

Daily rhythms in physiology and behavior change with age. An unresolved question is to what extent such age-related alterations in circadian organization are driven by the central clock in the suprachiasmatic nucleus (SCN), modifying timing signals to contributing peripheral tissue oscillators, and are mediated by underlying changes in the local cellular oscillators themselves. Using a bioluminescence reporter approach, we sought to determine whether circadian clock function in human adipocytes from subcutaneous (SAT) and visceral (VAT) adipose tissues changes with age. SAT and VAT biopsies were obtained from obese individuals during gastric bypass surgeries [ n = 16; body mass index: 44.8 ± 11.4 kg/m2; age: 44 ± 9 yr (range: 30-58)]. Cells were isolated and transduced with a lentiviral circadian reporter construct [brain and muscle aryl hydrocarbon receptor nuclear translocator-like:luciferase ( BMAL:LUC)], and bioluminescence was recorded over a period of 3 d. Human BMAL1:LUC adipocytes displayed a robust luminescence rhythm with comparable within-individual periods in mature and preadipocytes ( P > 0.05). With increasing age, the circadian period decreased in mature adipocytes ( P = 0.005) (ß = 4 min/yr; P < 0.05). Our ex vivo approach indicated that ageing changes the organization of endogenous circadian oscillators in human adipocytes, independent of SCN signaling.-Kolbe, I., Carrasco-Benso, M. P., López-Mínguez, J., Luján, J., Scheer, F. A. J. L., Oster, H., Garaulet, M. Circadian period of luciferase expression shortens with age in human mature adipocytes from obese patients.

Circadian clock rhythms in different adipose tissue model systems.

Circadian clock-controlled 24-h oscillations in adipose tissues play an important role in the regulation of energy homeostasis, thus representing a potential drug target for prevention and therapy of metabolic diseases. For pharmacological screens, scalable adipose model systems are needed that largely recapitulate clock properties observed in vivo. In this study, we compared molecular circadian clock regulation in different ex vivo and in vitro models derived from murine adipose tissues. Explant cultures from three different adipose depots of PER2::LUC circadian reporter mice revealed stable and comparable rhythms of luminescence ex vivo. Likewise, primary pre- and mature adipocytes from these mice displayed stable luminescence rhythms, but with strong damping in mature adipocytes. Stable circadian periods were also observed using Bmal1-luc and Per2-luc reporters after lentiviral transduction of wild-type pre-adipocytes. SV40 immortalized adipocytes of murine brown, subcutaneous and epididymal adipose tissue origin showed rhythmic mRNA expression of the core clock genes Bmal1, Per2, Dbp and REV-erbα in pre- and mature adipocytes, with a maturation-associated increase in overall mRNA levels and amplitudes. A comparison of clock gene mRNA rhythm phases revealed specific changes between in vivo and ex vivo conditions. In summary, our data indicate that adipose culture systems to a large extent mimic in vivo tissue clock regulation. Thus, both explant and cell systems may be useful tools for large-scale screens for adipose clock regulating factors.

Hepatic gene therapy rescues high-fat diet responses in circadian Clock mutant mice.

OBJECTIVE: Circadian Clock gene mutant mice show dampened 24-h feeding rhythms and an increased sensitivity to high-fat diet (HFD) feeding. Restricting HFD access to the dark phase counteracts its obesogenic effect in wild-type mice. The extent to which altered feeding rhythms are causative for the obesogenic phenotype of Clock mutant mice, however, remains unknown. METHODS: Metabolic parameters of wild-type (WT) and ClockΔ19 mutant mice (MT) were investigated under ad libitum and nighttime restricted HFD feeding. Liver circadian clock function was partially rescued by hydrodynamic tail vein delivery of WT-Clock DNA vectors in mutant mice and transcriptional, metabolic, endocrine and behavioral rhythms studied. RESULTS: Nighttime-restricted feeding restored food intake, but not body weight regulation in MT mice under HFD, suggesting Clock-dependent metabolic dysregulation downstream of circadian appetite control. Liver-directed Clock gene therapy partially restored liver circadian oscillator function and transcriptome regulation without affecting centrally controlled circadian behaviors. Under HFD, MT mice with partially restored liver clock function (MT-LR) showed normalized body weight gain, rescued 24-h food intake rhythms, and WT-like energy expenditure. This was associated with decreased nighttime leptin and daytime ghrelin levels, reduced hepatic lipid accumulation, and improved glucose tolerance. Transcriptome analysis revealed that hepatic Clock rescue in MT mice affected a range of metabolic pathways. CONCLUSION: Liver Clock gene therapy improves resistance against HFD-induced metabolic impairments in mice with circadian clock disruption. Restoring or stabilizing liver clock function might be a promising target for therapeutic interventions in obesity and metabolic disorders.

Circadian Rhythms in Adipose Tissue Physiology.

The different types of adipose tissues fulfill a wide range of biological functions-from energy storage to hormone secretion and thermogenesis-many of which show pronounced variations over the course of the day. Such 24-h rhythms in physiology and behavior are coordinated by endogenous circadian clocks found in all tissues and cells, including adipocytes. At the molecular level, these clocks are based on interlocked transcriptional-translational feedback loops comprised of a set of clock genes/proteins. Tissue-specific clock-controlled transcriptional programs translate time-of-day information into physiologically relevant signals. In adipose tissues, clock gene control has been documented for adipocyte proliferation and differentiation, lipid metabolism as well as endocrine function and other adipose oscillations are under control of systemic signals tied to endocrine, neuronal, or behavioral rhythms. Circadian rhythm disruption, for example, by night shift work or through genetic alterations, is associated with changes in adipocyte metabolism and hormone secretion. At the same time, adipose metabolic state feeds back to central and peripheral clocks, adjusting behavioral and physiological rhythms. In this overview article, we summarize our current knowledge about the crosstalk between circadian clocks and energy metabolism with a focus on adipose physiology. © 2017 American Physiological Society. Compr Physiol 7:383-427, 2017.

The SCN Clock Governs Circadian Transcription Rhythms in Murine Epididymal White Adipose Tissue.

The circadian master pacemaker in the suprachiasmatic nucleus (SCN) orchestrates peripheral clocks in various organs and synchronizes them with external time, including those in adipose tissue, which displays circadian oscillations in various metabolic and endocrine outputs. Because our knowledge about the instructive role of the SCN clock on peripheral tissue function is based mainly on SCN lesion studies, we here used an alternative strategy employing the Cre/ loxP system to functionally delete the SCN clock in mice. We performed whole-genome microarray hybridizations of murine epididymal white adipose tissue (eWAT) RNA preparations to characterize the role of the SCN clock in eWAT circadian transcriptome regulation. Most of the rhythmic transcripts in control animals were not rhythmic in SCN mutants, but a significant number of transcripts were rhythmic only in mutant eWAT. Core clock genes were rhythmic in both groups, but as was reported before for other tissues, rhythms were dampened and phase advanced in mutant animals. In SCN-mutant mice, eWAT lost the rhythm of metabolic pathway-related transcripts, while transcripts gaining rhythms in SCN-mutant mice were associated with various immune functions. These data reveal a complex interaction of SCN-derived and local circadian signals in the regulation of adipose transcriptome programs.

Circadian Clocks and the Interaction between Stress Axis and Adipose Function.

Many physiological processes and most endocrine functions show fluctuations over the course of the day. These so-called circadian rhythms are governed by an endogenous network of cellular clocks and serve as an adaptation to daily and, thus, predictable changes in the organism's environment. Circadian clocks have been described in several tissues of the stress axis and in adipose cells where they regulate the rhythmic and stimulated release of stress hormones, such as glucocorticoids, and various adipokine factors. Recent work suggests that both adipose and stress axis clock systems reciprocally influence each other and adrenal-adipose rhythms may be key players in the development and therapy of metabolic disorders. In this review, we summarize our current understanding of adrenal and adipose tissue rhythms and clocks and how they might interact to regulate energy homoeostasis and stress responses under physiological conditions. Potential chronotherapeutic strategies for the treatment of metabolic and stress disorders are discussed.

Interaction of circadian and stress systems in the regulation of adipose physiology.

Endogenous circadian clocks facilitate the adaptation of physiology and behavior to recurring environmental changes brought about by the Earth's rotation around its axis. Adipose tissues harbor intrinsic circadian oscillators based on interlocked transcriptional-translational feedback loops built from a set of clock genes that regulate important aspects of lipid metabolism and adipose endocrine function. These adipocyte clocks are reset via neuronal and endocrine pathways originating from a master circadian pacemaker residing in the hypothalamic suprachiasmatic nucleus. One important mediator of circadian output is the stress hormone cortisol, which, at the same time, is one of the major regulators of adipose physiology. In this review we summarize recent findings on the interaction between circadian and stress systems in the regulation of adipose physiology and discuss the implications of this crosstalk for the development of metabolic disorders associated with circadian disruption and/or chronic stress, for example in shift workers.