Metabolism as an integral cog in the mammalian circadian clockwork

Abstract

Circadian rhythms are an integral part of life. These rhythms are apparent in virtually all biological processes studies to date, ranging from the individual cell (e.g. DNA synthesis) to the whole organism (e.g. behaviors such as physical activity). Oscillations in metabolism have been characterized extensively in various organisms, including mammals. These metabolic rhythms often parallel behaviors such as sleep/wake and fasting/feeding cycles that occur on a daily basis. What has become increasingly clear over the past several decades is that many metabolic oscillations are driven by cell-autonomous circadian clocks, which orchestrate metabolic processes in a temporally appropriate manner. During the process of identifying the mechanisms by which clocks influence metabolism, molecular-based studies have revealed that metabolism should be considered an integral circadian clock component. The implications of such an interrelationship include the establishment of a vicious cycle during cardiometabolic disease states, wherein metabolism-induced perturbations in the circadian clock exacerbate metabolic dysfunction. The purpose of this review is therefore to highlight recent insights gained regarding links between cell-autonomous circadian clocks and metabolism and the implications of clock dysfunction in the pathogenesis of cardiometabolic diseases.     

Network quantitative trait loci mapping of circadian clock outputs identifies metabolic pathway-to-clock linkages in Arabidopsis

Modern systems biology permits the study of complex networks, such as circadian clocks, and the use of complex methodologies, such as quantitative genetics. However, it is difficult to combine these approaches due to factorial expansion in experiments when networks are examined using complex methods. We developed a genomic quantitative genetic approach to overcome this problem, allowing us to examine the function(s) of the plant circadian clock in different populations derived from natural accessions. Using existing microarray data, we defined 24 circadian time phase groups (i.e., groups of genes with peak phases of expression at particular times of day). These groups were used to examine natural variation in circadian clock function using existing single time point microarray experiments from a recombinant inbred line population. We identified naturally variable loci that altered circadian clock outputs and linked these circadian quantitative trait loci to preexisting metabolomics quantitative trait loci, thereby identifying possible links between clock function and metabolism. Using single-gene isogenic lines, we found that circadian clock output was altered by natural variation in Arabidopsis thaliana secondary metabolism. Specifically, genetic manipulation of a secondary metabolic enzyme led to altered free-running rhythms. This represents a unique and valuable approach to the study of complex networks using quantitative genetics.     

Social jetlag and obesity

Obesity has reached crisis proportions in industrialized societies. Many factors converge to yield increased body mass index (BMI). Among these is sleep duration. The circadian clock controls sleep timing through the process of entrainment. Chronotype describes individual differences in sleep timing, and it is determined by genetic background, age, sex, and environment (e.g., light exposure). Social jetlag quantifies the discrepancy that often arises between circadian and social clocks, which results in chronic sleep loss. The circadian clock also regulates energy homeostasis, and its disruption-as with social jetlag-may contribute to weight-related pathologies. Here, we report the results from a large-scale epidemiological study, showing that, beyond sleep duration, social jetlag is associated with increased BMI. Our results demonstrate that living "against the clock" may be a factor contributing to the epidemic of obesity. This is of key importance in pending discussions on the implementation of Daylight Saving Time and on work or school times, which all contribute to the amount of social jetlag accrued by an individual. Our data suggest that improving the correspondence between biological and social clocks will contribute to the management of obesity.     

Exploring the role of circadian clock gene and association with cancer pathophysiology

ABSTRACT

Most of the processes that occur in the mind and body follow natural rhythms. Those with a cycle length of about one day are called circadian rhythms. These rhythms are driven by a system of self-sustained clocks and are entrained by environmental cues such as light-dark cycles as well as food intake. In mammals, the circadian clock system is hierarchically organized such that the master clock in the suprachiasmatic nuclei of the hypothalamus integrates environmental information and synchronizes the phase of oscillators in peripheral tissues.

The circadian system is responsible for regulating a variety of physiological and behavioral processes, including feeding behavior and energy metabolism. Studies revealed that the circadian clock system consists primarily of a set of clock genes. Several genes control the biological clock, including BMAL1, CLOCK (positive regulators), CRY1, CRY2, PER1, PER2, and PER3 (negative regulators) as indicators of the peripheral clock.

Circadian has increasingly become an important area of medical research, with hundreds of studies pointing to the body’s internal clocks as a factor in both health and disease. Thousands of biochemical processes from sleep and wakefulness to DNA repair are scheduled and dictated by these internal clocks. Cancer is an example of health problems where chronotherapy can be used to improve outcomes and deliver a higher quality of care to patients.

In this article, we will discuss knowledge about molecular mechanisms of the circadian clock and the role of clocks in physiology and pathophysiology of concerns.     

A randomized,double-blind and placebo-controlled crossover trial on the effect of l-ornithine ingestion on the human circadian clock

ABSTRACT

In mammals, daily physiological events are regulated by the circadian rhythm, which comprises two types of internal clocks: the central clock and peripheral clocks. Circadian rhythm plays an important role in maintaining physiological functions including the sleep-wake cycle, body temperature, metabolism and organ functions. Circadian rhythm disorder, which is caused, for example, by an irregular lifestyle or long-haul travel, increases the risk of developing disease; therefore, it is important to properly maintain the rhythm of the circadian clock. Food and the circadian clock system are known to be closely linked. Studies on rodents suggest that ingesting specific food ingredients, such as the flavonoid nobiletin, fish oil, the polyphenol resveratrol and the amino acid L-ornithine affects the circadian clock. However, there are few reports on the foods that affect these circadian clocks in humans. In this study, therefore, we examined whether L-ornithine affects the human central clock in a crossover design placebo-controlled human trial. In total, 28 healthy adults (i.e. ≥20 years) were randomly divided into two groups and completed the study protocol. In the 1st intake period, participants were asked to take either L-ornithine (400 mg) capsules or placebo capsules for 7 days. After 7 days’ interval, they then took the alternative test capsules for 7 days in the 2nd intake period. On the final day of each intake period, saliva was sampled at various time points in the dim light condition, and the concentration of melatonin was quantified to evaluate the phase of the central clock. The results revealed that dim light melatonin onset, a recognized marker of central circadian phase, was delayed by 15 min after ingestion of L-ornithine. Not only is this finding an indication that L-ornithine affects the human central clock, but it also demonstrates that the human central clock can be regulated by food ingredients.     

昼夜节律与能量代谢

地球上大多数生物存在内源性的昼夜节律生物钟,它使得生物个体能够预知环境中由于地球自转产生的周期性昼夜变化。这种预知性使得生物个体的内在生理节律与周围环境的变化周期保持一致,从而能够更有效地从周围环境中摄取能量,在体内更高效地利用能量,亦即更好的适应环境以获得进化上的优势。生物钟能够广泛调控哺乳动物的睡眠、进食和代谢等多个方面的行为和生理功能,生物钟的破坏与多种代谢疾病相关;同时代谢过程和进食行为也能反过来调控生物钟。近年来对生物钟的不断研究加深了人们对肥胖和糖尿病等代谢疾病的理解,为这些疾病的治疗提供了新的思路和方法。本文主要综述哺乳动物生物钟与能量代谢之间的关系及研究进展。     

Lipoic acid entrains the hepatic circadian clock and lipid metabolic proteins that have been desynchronized with advanced age

It is well established that lipid metabolism is controlled, in part, by circadian clocks. However, circadian clocks lose temporal precision with age and correlates with elevated incidence in dyslipidemia and metabolic syndrome in older adults. Because our lab has shown that lipoic acid (LA) improves lipid homeostasis in aged animals, we hypothesized that LA affects the circadian clock to achieve these results. We fed 24 month old male F344 rats a diet supplemented with 0.2% (w/w) LA for 2 weeks prior to sacrifice and quantified hepatic circadian clock protein levels and clock-controlled lipid metabolic enzymes. LA treatment caused a significant phase-shift in the expression patterns of the circadian clock proteins Period (Per) 2, Brain and Muscle Arnt-Like1 (BMAL1), and Reverse Erythroblastosis virus (Rev-erb) β without altering the amplitude of protein levels during the light phase of the day. LA also significantly altered the oscillatory patterns of clock-controlled proteins associated with lipid metabolism. The level of peroxisome proliferator-activated receptor (PPAR) α was significantly increased and acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were both significantly reduced, suggesting that the LA-supplemented aged animals are in a catabolic state. We conclude that LA remediates some of the dyslipidemic processes associated with advanced age, and this mechanism may be at least partially through entrainment of circadian clocks.     

Circadiane Uhren und Energiemetabolismus

Chrononutrition – circadian clocks and energy metabolism Genetically encoded endogenous clocks regulate 24‐hour rhythms of physiology and behavior. A central pacemaker residing in the suprachiasmatic nucleus synchronizes peripheral clocks found in all tissues with each other and with the external day‐night cycle. One function of circadian clocks is the regulation of energy metabolism via rhythmic activation of tissue‐specific clock‐controlled genes. In the liver, genes involved in glucose and lipid metabolism are regulated in this fashion, while in adipocytes, fatty acid release and adipokine secretion are controlled by the circadian clock. Disruption of circadian rhythms as seen, for example, in shift workers promotes the development of metabolic disorders such as obesity and type‐2 diabetes.     

Feeding and circadian clocks

The mammalian genome encodes at least a dozen of genes directly involved in the regulation of the feedback loops constituting the circadian clock. The circadian system is built up on a multitude of oscillators organized according to a hierarchical model in which neurons of the suprachiasmatic nuclei of the hypothalamus may drive the central circadian clock and all the other somatic cells may possess the molecular components allowing tissues and organs to constitute peripheral clocks. Suprachiasmatic neurons are driving the central circadian clock which is reset by lighting cues captured and integrated by the melanopsin cells of the retina and define the daily rhythms of locomotor activity and associated physiological regulatory pathways like feeding and metabolism. This central clock entrains peripheral clocks which can be synchronized by non-photic environmental cues and uncoupled from the central one depending on the nature and the strength of the circadian signal. The human circadian clock and its functioning in central or peripheral tissues are currently being explored to increase the therapeutic efficacy of timed administration of drugs or radiation, and to offer better advice on lighting and meal timing useful for frequent travelers suffering from jet lag and for night workers' comfort. However, the molecular mechanism driving and coordinating the central and peripheral clocks through a wide range of synchronizers (lighting, feeding, physical or social activities) remains a mystery.     

Familial advanced sleep-phase syndrome: A short-period circadian rhythm variant in humans.

Biological circadian clocks oscillate with an approximately 24-hour period, are ubiquitous, and presumably confer a selective advantage by anticipating the transitions between day and night. The circadian rhythms of sleep, melatonin secretion and body core temperature are thought to be generated by the suprachiasmatic nucleus of the hypothalamus, the anatomic locus of the mammalian circadian clock. Autosomal semi-dominant mutations in rodents with fast or slow biological clocks (that is, short or long endogenous period lengths; tau) are associated with phase-advanced or delayed sleep-wake rhythms, respectively. These models predict the existence of familial human circadian rhythm variants but none of the human circadian rhythm disorders are known to have a familial tendency. Although a slight 'morning lark' tendency is common, individuals with a large and disabling sleep phase-advance are rare. This disorder, advanced sleep-phase syndrome, is characterized by very early sleep onset and offset; only two cases are reported in young adults. Here we describe three kindreds with a profound phase advance of the sleep-wake, melatonin and temperature rhythms associated with a very short tau. The trait segregates as an autosomal dominant with high penetrance. These kindreds represent a well-characterized familial circadian rhythm variant in humans and provide a unique opportunity for genetic analysis of human circadian physiology.     

Expression of clock genes in human peripheral blood mononuclear cells throughout the sleep/wake and circadian cycles

The rhythmic expression of circadian clock genes in the neurons of the suprachiasmatic nucleus (SCN) underlies the manifestation of endogenous circadian rhythmicity in behavior and physiology. Recent evidence demonstrating rhythmic clock gene expression in non-SCN tissues suggests that functional clocks exist outside the central circadian pacemaker of the brain. In this investigation, the nature of an oscillator in peripheral blood mononuclear cells (PBMCs) is evaluated by assessing clock gene expression throughout both a typical sleep/wake cycle (LD) and during a constant routine (CR). Six healthy men and women aged (mean±SEM) 23.7±1.6 yrs participated in this five-day investigation in temporal isolation. Core body temperature and plasma melatonin concentration were measured as markers of the central circadian pacemaker. The expression of HPER1, HPER2, and HBMAL1 was quantified in PBMCs sampled throughout an uninterrupted 72 h period. The core body temperature minimum and the midpoint of melatonin concentration measured during the CR occurred 2:17±0:20 and 3:24 ±0:09 h before habitual awakening, respectively, and were well aligned to the sleep/wake cycle. HPER1 and HPER2 expression in PBMCs demonstrated significant circadian rhythmicity that peaked early after wake-time and was comparable under LD and CR conditions. HBMAL1 expression was more variable, and peaked in the middle of the wake period under LD conditions and during the habitual sleep period under CR conditions. For the first time, bi-hourly sampling over three consecutive days is used to compare clock gene expression in a human peripheral oscillator under different sleep/wake conditions.     

Understanding circadian rhythmicity in Neurospora crassa: from behavior to genes and back again

Circadian clocks have been described in organisms ranging in complexity from unicells to mammals, in which they function to control daily rhythms in cellular activities and behavior. The significance of a detailed understanding of the clock can be appreciated by its ubiquity and its established involvement in human physiology, including endocrine function, sleep/wake cycles, psychiatric illness, and drug tolerances and effectiveness. Because the clock in all organisms is assembled within the cell and clock mechanisms are evolutionarily conserved, simple eukaryotes provide appropriate experimental systems for dissecting the clock. Significant progress has been made in deciphering the circadian system in Neurospora crassa using both genetic and molecular approaches, and Neurospora has contributed greatly to our understanding of (1) the feedback cycle that comprises a circadian oscillator, (2) the mechanisms by which the clock is kept in synchrony with the environment, and (3) the genes that reside in rhythmic output pathways. Importantly, the lessons learned in Neurospora are relevant to our understanding of clocks in higher eukaryotes.     

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed^1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere^1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms^3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous^5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects^5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms^5,8-13.Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals^14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection^13,16,17 or stable transduction^5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells²⁰. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.     

Ticking for Metabolic Health: The Skeletal‐Muscle Clocks

To be prepared for alternating metabolic demands occurring over the 24‐hour day, the body preserves information on time in skeletal muscle, and in all cells, through a circadian‐clock mechanism. Skeletal muscle can be considered the largest collection of peripheral clocks in the body, with a major contribution to whole‐body energy metabolism. Comparison of circadian‐clock gene expression between skeletal muscle of nocturnal rodents and diurnal humans reveals very common patterns based on rest/active cycles rather than light/dark cycles. Rodent studies in which the circadian clock is disrupted in skeletal muscle demonstrate impaired glucose handling and insulin resistance. Experimental circadian misalignment in humans modifies the skeletal‐muscle clocks and leads to disturbed energy metabolism and insulin resistance. Preclinical studies have revealed that timing of exercise over the day can influence the beneficial effects of exercise on skeletal‐muscle metabolism, and studies suggest similar applicability in humans. Current strategies to improve metabolic health (e.g., exercise) should be reinvestigated in their capability to modify the skeletal‐muscle clocks by taking timing of the intervention into account.