Involvement of the Clock Gene Rev-erb alpha in the Regulation of Glucagon Secretion in Pancreatic Alpha-Cells

Disruption of pancreatic clock genes impairs pancreatic beta-cell function, leading to the onset of diabetes. Despite the importance of pancreatic alpha-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erb alpha as a new intracellular regulator of glucagon secretion. Rev-erb alpha down-regulation by siRNA (60–70% inhibition) in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05) and led to a decrease in key genes of the exocytotic machinery. The Rev-erb alpha agonist GSK4112 increased glucagon secretion (1.6 fold) and intracellular calcium signals in alphaTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erb alpha antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erb alpha expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001). High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05). AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01) and glucagon release (p<0.05). These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway.     

Rev-erb alpha gives a time cue to metabolism

Normal physiological processes are under control of circadian rhythms. Moreover, certain pathological events, such as cardiovascular accidents (myocardial infarction, stroke) occur more frequently at specific times of the day. Recent observations demonstrate a causal relationship between alterations in circadian rhythmicity and metabolic disorders. Disruption of clock genes results in dyslipidemia, insulin resistance and obesity, all predisposing to atherosclerosis. The nuclear receptor Rev-erb alpha is part of the clock circuitry and plays an important role in keeping proper timing of the clock. Rev-erb alpha also regulates lipid metabolism, adipogenesis and vascular inflammation. Interestingly, Rev-erb alpha also cross-talks with several other nuclear receptors involved in energy homeostasis. Therefore Rev-erb alpha may serve to couple metabolic and circadian signals.     

Circadian disruption alters mouse lung clock gene expression and lung mechanics

Most aspects of human physiology and behavior exhibit 24-h rhythms driven by a master circadian clock in the brain, which synchronizes peripheral clocks. Lung function and ventilation are subject to circadian regulation and exhibit circadian oscillations. Sleep disruption, which causes circadian disruption, is common in those with chronic lung disease, and in the general population; however, little is known about the effect on the lung of circadian disruption. We tested the hypothesis circadian disruption alters expression of clock genes in the lung and that this is associated with altered lung mechanics. Female and male mice were maintained on a 12:12-h light/dark cycle (control) or exposed for 4 wk to a shifting light regimen mimicking chronic jet lag (CJL). Airway resistance (Rn), tissue damping (G), and tissue elastance (H) did not differ between control and CJL females. Rn at positive end-expiratory pressure (PEEP) of 2 and 3 cmH(2)O was lower in CJL males compared with controls. G, H, and G/H did not differ between CJL and control males. Among CJL females, expression of clock genes, Bmal1 and Rev-erb alpha, was decreased; expression of their repressors, Per2 and Cry 2, was increased. Among CJL males, expression of Clock was decreased; Per 2 and Rev-erb alpha expression was increased. We conclude circadian disruption alters lung mechanics and clock gene expression and does so in a sexually dimorphic manner.     

Proxisome proliferator-activated receptor-α mediates high-fat, diet-enhanced daily oscillation of plasminogen activator inhibitor-1 activity in mice

Acute thrombotic events frequently occur in the early morning among hyperlipidemic patients. The activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the fibrinolytic system, oscillates daily, and this is considered one mechanism that underlies the morning onset of acute thrombotic events in hyperlipidemia. Although several studies have reported the expression of the PAI-1 gene is under the control of the circadian clock system, the molecular mechanism of the circadian transactivation of PAI-1 gene under hyperlipidemic conditions remains to be elucidated. Here, the authors investigated whether hyperlipidemia induced by a high-fat diet (HFD) enhances the daily oscillation of plasma PAI-1 activity in mice. The mRNA levels of the PAI-1 gene were increased and rhythmically fluctuated with high-oscillation amplitude in the livers of wild-type mice fed with the HFD. Circadian expression of proxisome proliferator-activated receptor-α (PPARα) mRNA was also augmented as well as that of PAI-1. Chromatin immunoprecipitation showed the HFD-induced hyperlipidemia significantly increased the binding of PPARα to the PAI-1 promoter. Luciferase reporter analysis using primary hepatocytes revealed CLOCK/BMAL1-mediated PAI-1 promoter activity was synergistically enhanced by cotransfection with PPARα/retinoid X receptor-α (RXRα), and this synergistic transactivation was repressed by negative limbs of the circadian clock, PERIOD2 and CRYPTOCHROME1. As expected, HFD-induced PAI-1 mRNA expression was significantly attenuated in PPARα-null mice. These results suggest a molecular link between the circadian clock and lipid metabolism system in the regulation of PAI-1 gene expression, and provide an aid for understanding why hyperlipidemia increases the risk of acute thrombotic events in the morning.     

The frequency gene is required for temperature-dependent regulation of many clock-controlled genes in Neurospora crassa

The circadian clock of Neurospora broadly regulates gene expression and is synchronized with the environment through molecular responses to changes in ambient light and temperature. It is generally understood that light entrainment of the clock depends on a functional circadian oscillator comprising the products of the wc-1 and wc-2 genes as well as those of the frq gene (the FRQ/WCC oscillator). However, various models have been advanced to explain temperature regulation. In nature, light and temperature cues reinforce one another such that transitions from dark to light and/or cold to warm set the clock to subjective morning. In some models, the FRQ/WCC circadian oscillator is seen as essential for temperature-entrained clock-controlled output; alternatively, this oscillator is seen exclusively as part of the light pathway mediating entrainment of a cryptic "driving oscillator" that mediates all temperature-entrained rhythmicity, in addition to providing the impetus for circadian oscillations in general. To identify novel clock-controlled genes and to examine these models, we have analyzed gene expression on a broad scale using cDNA microarrays. Between 2.7 and 5.9% of genes were rhythmically expressed with peak expression in the subjective morning. A total of 1.4-1.8% of genes responded consistently to temperature entrainment; all are clock controlled and all required the frq gene for this clock-regulated expression even under temperature-entrainment conditions. These data are consistent with a role for frq in the control of temperature-regulated gene expression in N. crassa and suggest that the circadian feedback loop may also serve as a sensor for small changes in ambient temperature.     

Spinal astrocytes contribute to the circadian oscillation of glutamine synthase, cyclooxygenase-1 and clock genes in the lumbar spinal cord of mice

Spinal astrocytes have key roles in the regulation of pain transmission. However, the relationship between astrocytes and the circadian system in the spinal cord remains poorly defined. In the current study, the circadian variations in the expression of several clock genes in the lumbar spinal cord of mice were examined by using real-time PCR. The expression of Period1, Period2 and Cryptochrome1 showed significant circadian oscillations, each gene peaking in the early evening. The expression of Bmal1 mRNA also exhibited a circadian pattern, peaking from around midnight to early morning. The mRNA levels of Cryptochrome2 were slightly, but not significantly altered. Molecules related to pain transmission were also investigated. The mRNA expression of glutamine synthase (GS), and cyclooxygenases (COXs), known to be involved in various spinal sensory functions, showed rhythmicity with a peak in the early evening, although the expression of the neurokinin-1 receptor, subunits of the N-methyl-d-aspartate receptor, and glutamate transporters did not change. In addition, we found that protein levels of GS and COX-1 were also high at midnight compared with midday. Furthermore, we examined the effect of intrathecal fluorocitrate (100pmol), an inhibitor of astrocytic metabolism, on the expression of oscillating genes in lumbar spinal cord. Fluorocitrate significantly suppressed astrocyte function. Furthermore, the circadian oscillation of clock gene expression and GS and COX-1 expression were suppressed. Together, these results suggest that a significant circadian rhythmicity of the expression of clock genes is present in the spinal cord and that the components of the circadian clock timed by astrocytes might contribute to spinal functions, including nociceptive processes.     

Mobilized hematopoietic stem cell yield depends on species-specific circadian timing

Endogenous rhythmicity likely evolved as a mechanism allowing organisms to anticipate predictable daily changes in the environment (Rutter et al., 2002). Under homeostasis, murine hematopoietic stem cell (HSC) egress is orchestrated by rhythmic beta 3 adrenergic signals delivered by the sympathetic nervous system (SNS) that regulate Cxcl12 expression in stromal cells (Mendez-Ferrer et al., 2008). Here, we show that CXCR4 is also regulated under circadian control whose rhythm is synchronized with its ligand, CXCL12, to optimize HSC trafficking. These circadian oscillations are inverted in humans compared to the mouse and continue to influence the yield even when stem cell mobilization is enforced. Our results suggest that the human HSC yield for clinical transplantation might be significantly greater if patients were harvested during the evening compared to the morning.     

Melatonin in the circadian system

In Mammals, the master circadian clock is located in the suprachiasmatic nuclei of the hypothalamus. This clock is synchronized with the astronomical time, essentially by the light/dark cycle. The different zeitgebers studied act on the Per1 and/or Per2 genes from the main molecular loop which initiates the circadian oscillations. Once synchronized with the environment, circadian oscillations are distributed through the organism by efferent signals, and the complex interaction of neural, hormonal and behavioural outputs from the circadian clock drive circadian expression of events, either directly or through coordination of the timing of peripheral oscillators. Melatonin, one of the endocrine output signals of the clock, provides the organism with circadian information, and can be considered as an endogenous synchronizer. Melatonin receptors are present in the suprachiasmatic nuclei which allows the hormone to feed back on the clock. To day, the physiological role of this peculiar feed-back has not yet been established. However, the presence of these receptors indicates that through an action on the circadian clock, exogenous melatonin can affect all levels of the circadian network and its capacity to entrain circadian rhythms to 24 h has been demonstrated. Melatonin is thus a zeitgeber. However, surprisingly, and different from the action mechanism of other zeitgebers on the clock, the chronobiotic effect of melatonin does not implicate Per1 and/or Per2. Rather, Rev-erb alpha could be the link between the physiological action of melatonin and the core of the molecular circadian clock.     

PROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-α MEDIATES HIGH-FAT,DIET-ENHANCED DAILY OSCILLATION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 ACTIVITY IN MICE

Acute thrombotic events frequently occur in the early morning among hyperlipidemic patients. The activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the fibrinolytic system, oscillates daily, and this is considered one mechanism that underlies the morning onset of acute thrombotic events in hyperlipidemia. Although several studies have reported the expression of the PAI-1 gene is under the control of the circadian clock system, the molecular mechanism of the circadian transactivation of PAI-1 gene under hyperlipidemic conditions remains to be elucidated. Here, the authors investigated whether hyperlipidemia induced by a high-fat diet (HFD) enhances the daily oscillation of plasma PAI-1 activity in mice. The mRNA levels of the PAI-1 gene were increased and rhythmically fluctuated with high-oscillation amplitude in the livers of wild-type mice fed with the HFD. Circadian expression of proxisome proliferator-activated receptor-α (PPARα) mRNA was also augmented as well as that of PAI-1. Chromatin immunoprecipitaion showed the HFD-induced hyperlipidemia significantly increased the binding of PPARα to the PAI-1 promoter. Luciferase reporter analysis using primary hepatocytes revealed CLOCK/BMAL1-mediated PAI-1 promoter activity was synergistically enhanced by cotransfection with PPARα/retinoid X receptor-α (RXRα), and this synergistic transactivation was repressed by negative limbs of the circadian clock, PERIOD2 and CRYPTOCHROME1. As expected, HFD-induced PAI-1 mRNA expression was significantly attenuated in PPARα-null mice. These results suggest a molecular link between the circadian clock and lipid metabolism system in the regulation of PAI-1 gene expression, and provide an aid for understanding why hyperlipidemia increases the risk of acute thrombotic events in the morning. (Author correspondence: ssoeda@fukuoka-u.ac.jp)     

Differential Involvement of the Circadian Clock in the Expression of Genes Required for Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Synthesis,Assembly, and Activation in Arabidopsis thaliana

We have investigated the role of the circadian clock in the regulation of expression of genes required for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) synthesis, assembly, and activation. Circadian oscillations in RCA (the gene encoding Rubisco activase) and RBCS (the gene encoding Rubisco small subunit) mRNA accumulation, with peak abundance occurring soon after dawn, occur in Arabidopsis thaliana grown in a light-dark (LD) photoperiod. These oscillations persist in plants that have been transferred from LD to either continuous darkness (DD) or continuous light (LL). In contrast, CPN60[alpha] (the gene encoding [alpha]-chaperonin) and CPN60[beta] (the gene encoding [beta]-chaperonin) mRNA abundance oscillates in a diurnal, but not in a circadian, fashion. Although rapid damping of the circadian oscillation in RCA mRNA abundance is observed in Arabidopsis that have been grown in LD and then transferred to DD for 2 d, the circadian oscillations in RCA and RBCS mRNA abundance persist for at least five continuous cycles in LL, demonstrating the robustness of the circadian oscillator.