In vivo monitoring of circadian timing in freely moving mice

In mammals, the principal circadian pacemaker driving daily physiology and behavioral rhythms is located in the suprachiasmatic nucleus (SCN) in the anterior hypothalamus. The neural output of SCN is essential for the circadian regulation of behavioral activity. Although remarkable progress has been made in revealing the molecular basis of circadian rhythm generation within the SCN, the output pathways by which the SCN exert control over circadian rhythms are not well understood. Most SCN efferents target the subparaventricular zone (SPZ), which resides just dorsal to the SCN. This output pathway has been proposed as a major component involved in the outflow for circadian regulation. We have examined the downstream pathway of the central clock by means of multiunit neural activity (MUA) in freely moving mice. SCN neural activity is tightly coupled to environmental photic input and anticorrelated with MUA rhythm in the SPZ. In Clock mutant mice exhibiting attenuated circadian locomotor rhythmicity, MUA rhythmicity in the SCN and SPZ is similarly blunted. These results suggest that the SPZ plays a functional role in relaying circadian and photic signals to centers involved in generating behavioral activity.     

Rhythms of mammalian body temperature can sustain peripheral circadian clocks

BACKGROUND: Low-amplitude temperature oscillations can entrain the phase of circadian rhythms in several unicellular and multicellular organisms, including Neurospora and Drosophila. Because mammalian body temperature is subject to circadian variations of 1 degrees C-4 degrees C, we wished to determine whether these temperature cycles could serve as a Zeitgeber for circadian gene expression in peripheral cell types. RESULTS: In RAT1 fibroblasts cultured in vitro, circadian gene expression could be established by a square wave temperature rhythm with a (Delta)T of 4 degrees C (12 hr 37 degrees C/12 hr 33 degrees C). To examine whether natural body temperature rhythms can also affect circadian gene expression, we first measured core body temperature cycles in the peritoneal cavities of mice by radiotelemetry. We then reproduced these rhythms with high precision in the liquid medium of cultured fibroblasts for several days by means of a homemade computer-driven incubator. While these "in vivo" temperature rhythms were incapable of establishing circadian gene expression de novo, they could maintain previously induced rhythms for multiple days; by contrast, the rhythms of control cells kept at constant temperature rapidly dampened. Moreover, circadian oscillations of environmental temperature could reentrain circadian clocks in the livers of mice, probably via the changes they imposed upon both body temperature and feeding behavior. Interestingly, these changes in ambient temperature did not affect the phase of the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus. CONCLUSIONS: We postulate that both endogenous and environmental temperature cycles can participate in the synchronization of peripheral clocks in mammals.     

Timing to perfection: the biology of central and peripheral circadian clocks

The mammalian circadian system, which is comprised of multiple cellular clocks located in the organs and tissues, orchestrates their regulation in a hierarchical manner throughout the 24?hr of the day. At the top of the hierarchy are the suprachiasmatic nuclei, which synchronize subordinate organ and tissue clocks using electrical, endocrine, and metabolic signaling pathways that impact the molecular mechanisms of cellular clocks. The interplay between the central neural and peripheral tissue clocks is not fully understood and remains a major challenge in determining how neurological and metabolic homeostasis is achieved across the sleep-wake cycle. Disturbances in the communication between the plethora of body clocks can desynchronize the circadian system, which is believed to contribute to the development of diseases such as obesity and neuropsychiatric disorders. This review will highlight the relationship between clocks and metabolism, and describe how cues such as light, food, and reward mediate entrainment of the circadian system.     

MicroRNAs function as cis- and trans-acting modulators of peripheral circadian clocks

Based on their extracellular expression and targeting of the clock gene Bmal1, miR-142-3p and miR-494 were analyzed for evidence of vesicle-mediated communication between cells and intracellular functional activity. Our studies demonstrate that: miR-142-3p + miR-494 overexpression decreases endogenous BMAL1 levels, increases the period of Per2 oscillations, and increases extracellular miR-142-3p/miR-494 abundance in conditioned medium; miRNA-enriched medium increases intracellular expression of miR-142-3p and represses Bmal1 3′-UTR activity in naïve cells; and inhibitors of vesicular trafficking modulate intercellular communication of these miRNAs and ensemble Per2 rhythms. Thus, miR-142-3p and miR-494 may function as cis- and trans-acting signals contributing to local temporal coordination of cell-autonomous circadian clocks.     

Mammalian cultured cells as a model system of peripheral circadian clocks

The mammalian circadian system consists of multiple oscillators with basically hierarchical relationship, in which the hypothalamic suprachiasmatic nucleus (SCN) is the master pacemaker and the other oscillators in the periphery are subordinate. Although peripheral oscillators have been preceded by the SCN in circadian studies, accumulating data have revealed the importance and characteristics of peripheral oscillators. Cultured cell lines have also provided valuable information about intracellular mechanisms of circadian rhythms. This review outlines the properties of peripheral clocks in several perspectives such as the mechanisms of autonomous oscillations, the clock resetting, and the clock outputs, and describes the usefulness of immortalized cultured cells as a model system of mammalian circadian clocks by introducing some fruits of related works.     

Pineal circadian clocks gate arylalkylamine N-acetyltransferase gene expression in the mouse pineal gland

In mammals it has been thought that the circadian clock localizes only in the suprachiasmatic nucleus of the hypothalamus. Recent studies have revealed that certain brain regions and peripheral tissues may also have intrinsic circadian clocks. However, the roles played by 'peripheral circadian clocks' have not been fully elucidated. In this study, we investigated their function using mouse pineal glands, and found that expression of the arylalkylamine N-acetyltransferase (Aa-Nat, EC 2.3.1.87, the rate-limiting enzyme of melatonin synthesis) gene after adrenergic receptor stimulation depended on the time of day even in vitro (gating). Phase-dependent Aa-Nat responses were observed in both melatonin-proficient and melatonin-deficient mouse pineal glands. Phosphodiesterases are unlikely to suppress Aa-Nat induction because a phosphodiesterase inhibitor itself had no effect on the mRNA levels. Puromycin was ineffective in inducing Aa-Nat mRNA levels in either the presence or absence of isoproterenol, suggesting that newly synthesized proteins may not be necessary to gate the Aa-Nat responses. We also discovered circadian dependence of the expression of Period1-luminescence in Period1-luciferase transgenic mouse pineal glands: circadian clocks may be functional in culture. Aa-Nat mRNA levels showed no significant circadian rhythms in the absence of isoproterenol, thus suggesting that Aa-Nat mRNA levels are induced by adrenergic mechanisms, not by a pineal circadian clock. Our results suggest that the pineal circadian clock may determine timing when Aa-Nat gene expression can respond to inputs from the master circadian clock in the suprachiasmatic nucleus, e.g. adrenergic stimulation.     

In vivo monitoring of pancreatic beta-cells in a transgenic mouse model

We generated a transgenic mouse model (RIP-luc) for the in vivo monitoring of pancreatic islet mass and function in response to metabolic disease. Using the rat insulin promoter fused to firefly luciferase, and noninvasive technology to detect luciferase activity, we tracked changes in reporter signal during metabolic disease states and correlated the changes in luciferase signal with metabolic status of the mouse. Transgene expression was found to be specific to the pancreatic islets in this transgenic model. Basal transgene expression was tracked in male and female mice fed either a chow or a high-fat diet and in response to treatment with streptozotocin. Pancreatic bioluminescent signal increased in mice fed a high-fat diet compared with chow-fed animals. In a model of chemically induced diabetes, the bioluminescent signal decreased in accordance with the onset of diabetes and reduction of islet beta-cell number. Preliminary studies using islets transplanted from this transgenic model suggest that in vivo image analysis can also be used to monitor transplanted islet viability and survival in the host. This transgenic model is a useful tool for in vivo studies of pancreatic beta-cells and as a donor for islet transplantation studies.     

Adaptive significance of circadian clocks

Circadian clocks are ubiquitous and are found in organisms ranging from bacteria to mammals. This ubiquity of occurrence implies adaptive significance, but to date there has been no rigorous empirical evidence to support this. It is believed that an organism possessing circadian clocks gains fitness advantage in two ways: (i) by synchronizing its behavioral and physiological processes to cyclic environmental factors (extrinsic adaptive value); (ii) by coordinating its internal metabolic processes (intrinsic adaptive value). There is preliminary circumstantial evidence to support both. Several studies using organisms living in constant environments have shown that these organisms possess functional circadian clocks, suggesting that circadian clocks may have some intrinsic adaptive value. Studies to assess the adaptive value of circadian clocks in periodic environments suggest that organisms may have a fitness advantage in those periodic environments, which closely match their own intrinsic periodicity. Furthermore, evidence from organisms living in the wild, selection studies, and studies on latitudinal clines suggest that circadian clocks may have an extrinsic adaptive value as well. In this paper, I have presented several hypotheses for the emergence of circadian clocks and have reviewed some major empirical studies suggesting adaptive significance of circadian clocks.     

Precise circadian clocks in prokaryotic cyanobacteria

Prokaryotic cyanobacteria express robust circadian (daily) rhythms under the control of a timing mechanism that is independent of the cell division cycle. This biological clock orchestrates global regulation of gene expression and controls the timing of cell division. Proteins that may be involved in input pathways have been identified. Mutational screening has identified three clock genes that are organized as a gene cluster. The structure of cyanobacterial clock proteins, their phosphorylation, and regulation is described. A new model for the core clockwork in cyanobacteria proposes that rhythmic changes in the status of the chromosome underlie the rhythms of gene expression. Mixed-strain experiments demonstrate that this timekeeper confers adaptive value when different strains compete against each other.     

Phase resetting of circadian peripheral clocks using human and rodent diets in mouse models of type 2 diabetes and chronic kidney disease

The expression rhythms of clock genes, such as Per1, Per2, Bmal1, and Rev-erb α, in mouse peripheral clocks, are entrained by a scheduled feeding paradigm. In terms of food composition, a carbohydrate-containing diet is reported to cause strong entrainment through insulin secretion. However, it is unknown whether human diets entrain peripheral circadian clocks. In this study, we used freeze-dried diets for type 2 diabetes (DB) and chronic kidney disease (CKD), as well as low-carbohydrate diets. After 24 h of fasting, PER2::LUC knock-in mice were given access to food for 2 days during inactive periods, and bioluminescence rhythm was then measured using an in vivo imaging system. AIN-93M, the control mouse diet with a protein:fat:carbohydrate (PFC) ratio of 14.7:9.5:75.8, caused a significant phase advance (7.3 h) in the liver clock compared with that in 24 h fasted mice, whereas human diets caused significant but smaller phase advances (4.7–6.2 h). Compared with healthy and high fat/sucrose-induced DB mice, adenine-induced CKD mice showed attenuation of a phase-advance with a normal diet. There were no significant differences in phase-advance values between human diets (normal, DB, and CKD). In addition, a normal-carbohydrate diet (PFC ratio of 20.3:23.3:56.4) and a low-carbohydrate diet (PFC ratio of 36.4:42.9:20.7) caused similar phase advances in peripheral clocks. The present results strongly suggest that scheduled feeding with human diets can cause phase advances in the peripheral clocks of not only healthy, but also DB and CKD mice. This discovery provides support to the food-induced entrainment of peripheral clocks in human clinical trials.     

The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks

Background  

Circadian rhythms are endogenous, self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes. The circadian organization of these processes in mammals is governed by the master oscillator within the suprachiasmatic nuclei (SCN) of the hypothalamus. Recent findings revealed that circadian oscillators exist in most organs, tissues, and even in immortalized cells, and that the oscillators in peripheral tissues are likely to be coordinated by SCN, the master oscillator. Some candidates for endogenous entrainment factors have sporadically been reported, however, their details remain mainly obscure.     

The circadian clocks of plants and cyanobacteria

Classical research on the circadian rhythms of plants helped to demonstrate that all living organisms utilize circadian clocks to adapt their day–night cycles and that the clock is the basis for photoperiodic time measurements. Molecular models for the circadian oscillator have now been elucidated in Drosophila, Neurospora, mice and cyanobacteria. All share a similar feedback structure, but key proteins in each of the oscillators are different. A plant clock model has yet to be proposed, but clock mutants of Arabidopsis are expected to reveal key proteins in the mechanism. Here we discuss how a self-sustained oscillation is established in eukaryotic and prokaryotic models, and the polyphyletic evolution of these clock systems.