Sleep and circadian rhythms: Key components in the regulation ... - Core

FEBS Letters 582 (2008) 142–151

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Sleep and circadian rhythms: Key components in the regulation of energy metabolism Aaron D. Laposkya,b,*, Joseph Bassb,c, Akira Kohsakac, Fred W. Tureka,b a

Northwestern University, Department of Neurobiology and Physiology, 2205 Tech Drive, Hogan 2-160, Evanston, IL 60208-3520, United States b Northwestern University, Center for Sleep and Circadian Biology, 2205 Tech Drive, Hogan 2-160, Evanston, IL 60208-3520, United States c Evanston Northwestern Healthcare and Research Institute, Department of Medicine, United States Received 15 May 2007; accepted 16 June 2007 Available online 14 August 2007 Edited by Peter Tontonoz and Laszlo Nagy

Abstract In this review, we present evidence from human and animal studies to evaluate the hypothesis that sleep and circadian rhythms have direct impacts on energy metabolism, and represent important mechanisms underlying the major health epidemics of obesity and diabetes. The first part of this review will focus on studies that support the idea that sleep loss and obesity are ‘‘interacting epidemics.’’ The second part will discuss recent evidence that the circadian clock system plays a fundamental role in energy metabolism at both the behavioral and molecular levels. These lines of research must be seen as in their infancy, but nevertheless, have provided a conceptual and experimental framework that potentially has great importance for understanding metabolic health and disease. Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Sleep; Circadian rhythms; Circadian clock genes; Obesity; Metabolic syndrome; Sleep deprivation

0. Overview A major aspect of virtually all behavioral and physiological processes is daily variation mediated by the circadian timing system. Diurnal rhythms are generated by an internal biological clock that is synchronized to the 24-h day by environmental cues, primarily the light:dark cycle. When external timing signals are absent, the 24-h rhythms ‘‘free run’’ with a period close to 24 h, and are called ‘‘circadian’’ (about a day) rhythms. Many rhythms are overt and easy to recognize, such as the sleep–wake cycle, locomotor activity, and feeding behavior. The sleep–wake cycle is arguably the master output rhythm of the circadian clock, because the regulation of most behaviors and physiological activities depend on whether the organism is asleep or awake. A wide-range of less-easily observed rhythms occurs internally and includes the daily regulation of body temperature, adrenal corticosterone and pituitary hormone release, neuropeptide and neurotransmitter levels, * Corresponding author. Address: Northwestern University, Department of Neurobiology and Physiology, Center for Sleep and Circadian Biology, 2205 Tech Drive, Hogan 2-160, Evanston, IL 60208-3520, United States. Fax: +1 847 467 4065. E-mail address: [email protected] (A.D. Laposky).

sympathetic activation, energy metabolism (e.g., lipolysis, gluconeogenesis, insulin sensitivity, basal metabolic rate), as well as gene transcription. The evolution of a circadian system suggests that the ability of an organism to coordinate itself with the environment (external synchronization) and to maintain temporal organization of endogenous processes (internal synchronization) confers optimal health and survival potential. As we begin to unravel the many links between sleep, circadian rhythms, and metabolism, the survival benefits of temporal organization are beginning to emerge. The first mammalian circadian clock gene was discovered in 1997 by researchers at Northwestern University, and was given the name Clock (Circadian locomotor output cycles kaput), to represent the altered circadian phenotype in the Clock mutant mouse [1,2]. Since that time, there have been major advances in understanding the molecular basis of the circadian clock, including the discovery of many other circadian clock genes and the ongoing elucidation of transcriptional–translational feedback loops through which these clock genes interact [3]. Two important studies in the Clock mutant mouse provided evidence that circadian clock genes may be involved in more than just keeping time. First, it was demonstrated that mice homozygous for the Clock mutation (on a C57Bl/6J background) exhibit significant increases in daily wake time (+2 h), and a smaller amount of rapid eye movement (REM) sleep rebound following sleep deprivation compared to littermate controls [4]. The high-wake phenotype observed in Clock/Clock mice entrained to a 12L:12D cycle persisted when they were free-running in constant darkness. Therefore, the regulatory effect of the Clock gene on sleep extends beyond temporal consolidation and includes a component of sleep homeostasis, the amount of daily sleep time and the compensatory response to sleep deprivation. More recently, we discovered that Clock/Clock mice on a C57Bl/6J background develop alterations in energy metabolism, including greatly attenuated diurnal feeding and locomotor activity rhythms, hyperphagia, and obesity with accompanying symptoms of the metabolic syndrome: hyperleptinemia, hyperlipidemia, hepatic steatosis, and hyperglycemia with insufficient compensatory insulin production, a hallmark of type 2 diabetes mellitus [5]. These remarkable data indicate that the molecular circadian clock has an important and direct role in maintaining energy homeostasis in mice. It is possible that the effects of clock genes on multiple physiological systems are mediated through central, as well as peripheral pathways. Recent landmark discoveries

0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.06.079

A.D. Laposky et al. / FEBS Letters 582 (2008) 142–151

have shown that circadian clock genes exist not only in the brain, but in many peripheral tissues important in energy metabolism and cardiovascular function, including the heart, liver, adipose tissue, and pancreas [6,7]. Thus, clock genes are positioned to interact with or to represent a core component of many different molecular pathways, thereby, potentially having many different functional roles. In this review, we will present evidence from human and animal studies to evaluate the hypothesis that sleep and circadian rhythms have direct impacts on energy metabolism, and may represent important mechanisms underlying the explosion of obesity and the metabolic syndrome that has occurred in the United States, that is now becoming recognized in many developing countries as well. The first part of this review will focus on evidence that has led to the idea that sleep loss and obesity are ‘‘interacting epidemics.’’ The second part will discuss recent evidence that the circadian system may play a fundamental role in energy metabolism at both the behavioral and molecular levels. It should be noted that much of the provocative evidence that sleep and the circadian clock are involved in energy metabolism has appeared only in recent years. Therefore, this line of research must be seen as in its infancy, but nevertheless, one that provides a framework for exciting future research and discussion, and one with potentially great importance for understanding metabolic health and disease.

1. Obesity and sleep loss: ‘‘interacting epidemics?’’ Two concurrent health trends have emerged over the past few decades that are having a significant effect on society and the health care system: (1) a continuous increase in the incidence and severity of obesity and the metabolic syndrome, and (2) a reduction in the average daily amount of sleep time resulting in chronic sleep debt. Data from the Center for Disease Control and Prevention (National Health and Nutrition Examination Surveys, www.cdc.gov) have shown that since the 1980’s, the amount of overweight and obesity in the United States has more than doubled in adults and nearly tripled in children and adolescents, affecting over 60% of the population in some states [8]. Obesity is not limited to the United States and is increasing around the world [9]. There are numerous secondary complications resulting from obesity, particularly symptomatology of the metabolic syndrome, including insulin resistance, hyperglycemia, dyslipidemia, hypertension, inflammation, and coagulation disorders. Obesity and the metabolic syndrome have been linked to heart attack, stroke, diabetes, cancer, obstructive sleep apnea, and arthritis, making them responsible, at least in part, for some of the major health problems facing the industrialized world today. Over the same time period that obesity has reached epidemic levels, the amount of daily sleep time achieved by American adults has decreased by 1–2 h and recent data show that adolescents are even more severely sleep deprived than adults [10– 13]. As of 2006, there were at least 35 epidemiological studies linking alterations in sleep time with adverse health outcomes, including obesity, diabetes, metabolic syndrome, and cardiovascular disease [14]. The important question is whether these parallel outcomes are indeed ‘‘interacting’’ or simply correlated with no cause and effect relationship. The following sections will overview evidence from clinical research and animal studies that support a direct role of sleep loss as an underlying

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cause or pre-disposing factor for alterations in energy homeostasis and disease states, such as obesity and diabetes. 1.1. Human laboratory studies Van Cauter and colleagues were the first to report, in wellcontrolled, experimentally rigorous clinical studies, changes in glucose regulation and appetite following just a few days of partial sleep restriction. For example, healthy male subjects restricted to a 4-h sleep opportunity each night for six consecutive nights exhibited elevated glucose levels and decreased insulin sensitivity following ingestion of breakfast on the last day of sleep restriction [15]. Sleep restricted subjects also had a significant decrease in the rate of glucose disposal and a reduction in insulin secretion in response to an intravenous glucose tolerance test. The extent to which glucose and insulin responses were impaired is comparable to what occurs in aging or in the early stages of diabetes, suggesting that even longer periods of sleep restriction in healthy young adults may indeed represent a risk factor for impaired glucose homeostasis and diabetes. In a second study by Van Cauter and colleagues, 4 h of sleep restriction for only two consecutive nights resulted in notably reduced circulating levels of the anorectic hormone, leptin, and increased levels of the orexigenic hormone, ghrelin, along with self-reports of increased appetite [16]. These findings are in line with epidemiological data from the Wisconsin Sleep Cohort Study that included over 1000 subjects, which showed reduced leptin and increased ghrelin levels in self-reported short sleepers [17]. These clinical studies controlled for daily caloric intake and activity levels, but it was estimated that if the self-reported appetite ratings in sleep restricted subjects were translated into actual caloric intake, it would represent an extra 350–500 k/cal per day, enough to result in excess weight gain if the pattern were to be maintained on a chronic basis. Other physiological changes detected following experimental sleep restriction include elevated sympathovagal balance, hypercortisolemia, increased thyroid hormone turnover, elevated C-reactive protein levels, and increased secretion of pro-inflammatory cytokines [15,18,19]. An important question to consider is whether the neuroendocrine and metabolic effects of sleep curtailment result directly from the sleep loss itself, or through changes in other physiological pathways affected by sleep loss, such as the sympathetic nervous system or the hypothalamo-pitiutary-adrenal (HPA) axis; the specific mechanisms underlying the effects of sleep loss on energy metabolism remain to be elucidated. The major impact of the initial clinical studies is that they provide laboratory support for the epidemiological based links between sleep and energy metabolism. The experimental studies also show that sleep loss can precede and lead to significant alterations in energy homeostasis. 1.2. Animal laboratory studies In rodent studies, chronic total sleep deprivation paradigms have been utilized to demonstrate effects of sleep loss on energy metabolism. A series of studies using the classic disk-over water (DOW) technique of sleep deprivation in rodents revealed that chronic total sleep deprivation over a 2-week period resulted in hyperphagia, increased energy expenditure (i.e. weight loss), initial hyperthermia followed by hypothermia, elevated sympathetic tone (increased norepinephrine

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levels and adrenal hypertrophy) and an increased thyroid hormone turnover [20]. Other important neuroendocrine changes following sleep deprivation using the DOW included a decrease in the satiety hormone, leptin, as well as reduced serum levels of insulin-like growth factor 1 (IGF-1), growth hormone, and prolactin [21]. Interestingly, if sleep deprivation was stopped, many of these symptoms were largely reversible following a period of sleep recovery, indicating that some property inherent to the sleep state mediated these changes. We have performed intraperitoneal glucose tolerance tests in rats following 1 day of sleep restriction (20 h of sleep deprivation followed by 4 h of sleep) by sleep depriving rats in a slowly rotating wheel (unpublished data). These preliminary findings show that sleep loss results in decreased insulin secretion and impaired glucose disposal following a single injection of dextrose (2 g/kg i.p.). Interestingly, chemical sympathectomy using 6-hydroxydopamine significantly reduced, but did not eliminate, the extent of insulin and glucose impairment following sleep loss, indicating that sympathetic tone may be an important mediator of sleep loss on insulin and glucose homeostasis in rodents. We are currently examining whether repeated sleep restriction can exacerbate the diabetic state in Zucker Diabetic Fatty (ZDF) rats. Other sleep deprivation methods in rats have been used to show metabolic consequences of sleep deprivation of varying durations. During a 20-day period of REM sleep loss, rats developed hyperphagia, increased metabolic rate (i.e. elevated uncoupling protein-1 expression in brown adipose tissue), and decreased leptin levels accompanied by upregulated mRNA expression of neuropeptide-Y (NPY), an orexigenic neuropeptide, and downregulated mRNA expression of pro-opiomelanocortin (POMC), an anorectic neuropeptide, in the hypothalamus [22,23]. Even a short period of sleep deprivation for 5 h by gentle handling increased feeding while decreasing leptin and increasing ghrelin concentrations in rats [24]. In combination, these studies show that chronic total sleep deprivation in rats and chronic partial sleep restriction in humans both result in notable changes in components of energy metabolism that could predispose or directly result in altered metabolic function. Many consequences of sleep loss are consistent between human and animal studies including: (1) increased food intake and appetite, (2) decreased leptin and increased ghrelin levels, (3) activation of the sympathetic nervous system, (4) elevation of corticosterone levels, (5) alterations in glucose utilization and, (6) an increase in cardiovascular risk factors. The major difference between human and animal studies is that short sleep time in humans is associated with increased BMI, whereas chronic total sleep deprivation in animals usually leads to decreased body weight, or negative energy balance. One reason for this difference could involve methodological issues. The majority of animal studies have used prolonged total sleep deprivation, rather than chronic partial sleep restriction to assess changes in food intake, body weight or other measures of energy homeostasis. Second, all current animal methods of sleep deprivation involve some degree of forced locomotion or physical manipulation of the animals to keep them awake, whereas human studies have controlled for activity by keeping subjects under bed rest conditions. Third, human studies of chronic partial sleep restriction have controlled nutrient intake by giving control and experimental subjects meals with identical caloric intake or by using constant glucose infusion, therefore, these


experiments do not assess ad lib food intake and body weight changes as has been the case in animal sleep deprivation studies. Another factor that could lead to differences in the body weight response of humans and rodents could involve physiological differences. In rodents, brown adipose tissue (BAT) contributes to heat loss through non-shivering thermogenesis and could substantially contribute to negative energy balance following sleep deprivation, particularly in response to increased sympathetic tone; BAT is not present in adult humans. In summary, except for the effects on body weight, the majority of neuroendocrine changes following sleep loss are similar between humans and animals, indicating that such animal models will continue to be valuable in determining the mechanistic links and common physiological and molecular components between sleep regulatory and energy metabolism regulating systems.

1.3. Effects of altered metabolism on sleep–wake patterns While it is clear that sleep is important in maintaining energy balance, there are a variety of examples of how metabolic pathways reciprocally interact to influence sleep–wake regulation. A major finding that set the stage for investigating neural interconnections linking sleep and metabolic homeostasis was the discovery that the appetite regulating neuropeptides, hypocretin-1 (Hcrt-1) and hypocretin-2 (Hcrt-2) (also termed orexin-1 and orexin-2) are involved in sleep–wake regulation [25]. The hcrt system has an important wake-promoting function and is involved in the control of motor activity. A defect in the hcrt-2 receptor, as well as destruction of hcrt-containing neurons in the hypothalamus, have been determined as causative factors in the canine and human forms of the sleep disorder, narcolepsy [26]. Interestingly, survey investigations have reported that BMI may be elevated in narcoleptic patients [27], and animal studies indicate that feeding and energy expenditure problems in hcrt deficient mice are directly related to the disruption in sleep–wake patterns [28]. Therefore, hcrt represents an important example of a molecule involved in the co-regulation of sleep and energy metabolism, as it may play a role in coordinating periods of sleep, food consumption and energy storage. Other metabolic hormones and perturbations influence sleep–wake patterns. Diet induced obesity in mice leads to an increase in non-rapid eye movement (NREM) sleep time and increased sleep fragmentation, whereas in rats, food deprivation results in decreased sleep time [29,30]. Appetite regulating hormones and neuropeptides, such as insulin, ghrelin, and neuropeptide Y (NPY) exert effects on sleep time when administered centrally or peripherally [31]. Acute administration of the adiposity-signalling hormone, leptin, decreases REM sleep and increases NREM sleep time in rats [32]. Furthermore, substances such as histamine and serotonin have important roles in the control of both feeding and sleep–wake patterns [31]. We have recently shown that leptin deficient, ob/ob, mice exhibit high amounts of sleep time, a severely fragmented sleep– wake pattern, and a disrupted circadian distribution of REM sleep time and NREM EEG delta power, a quantitative measure of sleep homeostasis [33]. We have similar findings in sleep–wake patterns in db/db mice and ZDF rats, which produce leptin, but harbor a mutation in the leptin receptor (unpublished observations). Cytokines, such as tumor necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1), are produced in


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recruited macrophages in adipocyte tissue, are elevated in obesity, and have notable effects on sleep [31]. Thus, many of the primary neuropeptides and neurotransmitters involved in the regulation of energy homeostasis also mediate sleep– wake states, providing further evidence for overlap at the mechanistic level between sleep and metabolic regulatory systems. A common thread in animal models of obesity (whether genetic or diet induced) is that total sleep time is increased, however, sleep is less consolidated as evidenced by frequent arousals from sleep and increased sleep during the normal wake period (i.e. during the dark phase for nocturnal rodents).

of a variety of physiological processes, but that also feeds back to affect the sleep–wake regulatory process itself. In turn, the allostatic load resulting from sleep loss could then feed into other physiological systems to exert a wide range of effects, including adaptations in energy metabolism. The biological model of allostasis may prove valuable in establishing a framework around which to investigate and understand the many physiological consequences of chronic partial sleep loss.

1.4. A novel model of chronic partial sleep loss: allostasis One of the major factors governing sleep–wake regulation is sleep homeostasis. Following short-term sleep deprivation, humans and animals compensate by increasing sleep intensity, as defined by NREM EEG delta (1–4 Hz) power, and usually NREM and REM sleep time. We have recently developed a model of chronic partial sleep restriction in rats and have made the novel discovery that following repeated days of sleep loss, rats lose the ability to generate a homeostatic sleep response, as depicted in Fig. 1 [34]. We propose that this pattern is indicative of a change in the recovery strategy from homeostasis to allostasis [35]. It is conceivable that sleep loss creates an allostatic load on the organism that not only alters the regulation

The discovery of the molecular basis of the circadian clock (i.e. circadian clock genes) and the development of animal models (genetic and non-genetic) of circadian disruption have played a crucial role in understanding how the circadian system controls a wide-range of output rhythms, and has elucidated other potentially important roles of the circadian system that extend well beyond its role as a time keeper. One of the most exciting recent discoveries is that circadian clock genes exist throughout the brain, as well as in numerous peripheral tissues throughout the body [3,6,7]. These discoveries have resulted in novel ways of thinking about how the circadian system is involved in regulating multiple physiological and molecular processes, including those relating to energy

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Fig. 1. Chronic partial sleep restriction in rats: change from homeostatic to allostatic sleep response. (A) Schematic diagram of experimental design. A 24-h baseline (BL) sleep–wake recording was collected beginning 4 h after light onset (zeitgeber time, ZT 4). Over the next five consecutive days animals were sleep deprived for 20 h (SD1–SD5) followed by a restricted 4-h sleep opportunity (SR1-SR5). Thereafter, animals had a 3-day ad libitum sleep recovery (R1-R3) opportunity. The 12:12 light:dark cycle is indicated at the bottom of the figure (open bar = light phase, dark bar = dark phase). (B) NREM delta power during the daily sleep opportunities. Sleep intensity was quantified by determining the amount of slow wave activity (i.e. NREM delta [1.0–4.0 Hz] power) in the EEG during NREM sleep. Following the first day of sleep deprivation in the wheel, there was a robust increase in sleep intensity during the 4-h sleep opportunity (grey bar on SD1), compared to baseline levels, indicating strong homeostatic drive for sleep. Surprisingly, rats were not able to exhibit a homeostatic drive for sleep on subsequent days of sleep restriction (i.e. SD2–SD5, NREM delta power regressed to baseline levels). This lack of sleep homeostasis even persisted throughout the full 3-day recovery period. (C) Representation of an EEG trace during NREM sleep (10 s) during normal sleep conditions (top) and following acute sleep deprivation (bottom). The bottom trace depicts an increase in the degree of slow wave activity in response to acute sleep loss. The ability to generate this amount of slow wave activity is greatly diminished when animals are repeatedly sleep restricted. We interpret this pattern as a strategic change from homeostasis to allostasis during conditions of chronic partial sleep loss.

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metabolism, and the role that the circadian clock may play as an underlying factor in many diseases, such as obesity and the metabolic syndrome, and associated cardiovascular disease. This section will provide an overview of the anatomical and molecular components of the circadian clock system and will discuss recent findings pointing to an important role of circadian regulation and circadian clock genes in energy metabolism.

2.1. The circadian clock: external and internal synchronization A primary role of the circadian clock is to entrain the organism to environmental cues, such as the light:dark cycle (external synchronization). Entrainment allows the animal to predict many fluctuations in the environment, such as dusk and dawn, food availability, predator risk, prey availability, and temperature changes that require survival-dependent changes in behavior. Another critical role of the circadian system is to maintain internal synchronization between multiple behaviors, physiological systems, and molecular pathways to insure that each of these processes, ranging from the sleep–wake cycle, to hormonal rhythms, to gene transcription, are properly aligned. Different rhythms exhibit peaks and troughs of activity at different times of day, but an important aspect of circadian regulation is that these rhythms are expressed in a particular phase relationship to one another. For example, the initial period of nocturnal sleep in humans is characterized by slow wave sleep, elevated growth hormone (GH) levels and increased blood glucose concentration. In combination, reduced glucose utilization by non-insulin dependent and insulin dependent tissues during slow wave sleep and the ability of GH to increase hepatic gluconeogenesis and reduce insulin release from pancreatic beta cells, allows glucose levels to be maintained throughout the prolonged fasting state of sleep. On the other hand, cortisol levels increase near the end of the sleep period and contribute to enhanced cardiovascular tone and glucose utilization, two physiological processes that contribute to optimal waking behavior. While these represent only two examples of internal synchronization related to sleep and metabolism, there are many scenarios of temporal coordination between physiological systems that are involved in a wide range of functions, including not only energy metabolism, but also cognitive performance, immune function, autonomic nervous system homeostasis, cell growth, and possibly drug treatment efficacy (i.e. chronopharmacology). A common example of circadian desynchronization occurs in shift-work, where an individual is forced to be awake, to eat, and to perform physical and cognitive tasks at times of day that may not be aligned with endogenous internal rhythms. Indeed, shift work has been linked to increased BMI and higher rates of gastrointestinal disorders, cardiovascular disease, as well as diabetes [36]. Few animal models of ‘‘shift work’’ have been developed, but one provocative study demonstrated that reversing the light:dark cycle on a weekly basis hastened death in cardiomyopathic hamsters, suggesting that circadian desynchronization may have exacerbated and complicated the pre-disposed disease state in these animals [37]. Body weight, diabetes, and cardiovascular disease increase with age, and there is evidence of anatomical changes in circadian pacemaker neurons in the brain, as well as changes in the timing of circadian gene expression in the paraventricular nu-


cleus (PVN), a region of the hypothalamus important for the integration of metabolic signals, as well as output of the autonomic nervous system [38]. Interestingly, Buijs and colleagues have hypothesized that a predisposing factor for type 2 diabetes may be an imbalance in the temporal regulation of sympathetic and parasympathetic inputs to target endocrine organs, such as the liver, adrenal gland and adipose tissue [39,40]. It is well-known that adverse cardiovascular events, such as hypertension, congestive hear failure, arrhythmias, and myocardial infarction are more likely to occur at certain times of day [41]. Until recently, the hypothesis that internal synchronization may play an important role in physiology has been inferred based on easily observable rhythms without direct measurements of phase relationships between internal rhythms. The possibility of internal desynchronization as an underlying factor in disease states is particularly attractive in view of the recent and accumulating evidence that many tissues throughout the body contain the molecular circadian clock gene machinery. It is conceivable that in situations such as shift-work, travel across time zones, and even conditions of sleep deprivation, individual oscillators can become desynchronized from one other, whereas, under routine conditions, these separate oscillators are entrained in an organized phase relationship. 2.2. The suprachiamatic nucleus: master circadian pacemaker The suprachiasmatic nuclei (SCN) consist of two bilaterally paired groups of neurons situated just above the optic chiasm in the anterior hypothalamus [3,42]. This structure represents the master circadian pacemaker in the brain and its activity underlies the maintenance and coordination of most behavioral and physiological rhythms (i.e., internal synchronization) of the body. The pacemaker function of the SCN was initially suggested in rodents with the demonstration that SCN lesions eliminate a wide range of rhythms, including the sleep–wake cycle, locomotor activity, corticosteroid and leptin levels, glucose tolerance, as well as feeding and drinking behavior [42]. Neurons in the SCN also contain hormones and neurotransmitters that are synthesized and released in a circadian manner, including transforming growth factor-a (TGF-a), prokineticin-2 (PK2), gamma-aminobutyric acid (GABA), and vasopressin [7]. Both TGF-a and PK2 suppress locomotor activity, raising the possibility that their endogenous pattern of release in the hypothalamus contributes to the locomotor activity rhythm. While it has remained unclear exactly how SCN activity is translated into synchronized effector signals to drive and coordinate multiple behavioral and physiological rhythms, progress has been made in identifying some of the key anatomical pathways comprising the SCN input and output system. 2.3. Anatomical model of SCN out signaling The SCN receives light input from the eye via the retinohypothalamic tract (RHT), which originates in non-visual photoreceptive retino-ganglion cells [7]. Interestingly, these cells are distinct from rods and cones, and contain the photopigment, melanopsin. In the absence of light:dark cues, the circadian clock in the SCN ‘‘free-runs’’ with a period close to, but not exactly, 24 h. Signal transduction through the RHT represents the major pathway by which the SCN is entrained to precisely match the environmental light:dark cycle.


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Fig. 2. Schematic of SCN input and output pathways in the hypothalamus. Environmental light:dark cues are transduced to the suprachiasmatic nuclei (SCN) via the retinohypothalamic tract (RHT). The SCN sends a major projection to the ventral and dorsal subparaventricular zone (vSPZ and dSPZ), which then transmits SCN signals to a variety of nuclei involved in sleep–wake, feeding, energy expenditure, and autonomic regulation (details provided in text). ARC, arcuate nucleus; DMH, dorsomedial nucleus of the hypothalamus; LHA, lateral hypothalamic area (including hypocretin and melanin concentrating hormone – containing neurons); PVN, paraventricular nucleus; MPOA, medial preoptic area; VLPO, ventrolateral preoptic area. Coronal sections of the anterior and posterior hypothalamic areas – note that the SCN, SPZ and ACR are located in the mediobasal hypothalamus surrounding the third ventricle. (Adapted from Saper [43]).

Saper and colleagues have performed eloquent neuronal tracing and lesion studies to define the major hypothalamic targets of SCN efferent projections [43]. They have proposed a model by which the major SCN projection leads to the ventral and dorsal regions of the subparaventricular zone (SPZ) of the hypothalamus (Fig. 2). The SPZ is a relay between the SCN and the dorsomedial nucleus of the hypothalamus (DMH), which, in turn, sends projections to areas such as: (1) the ventrolateral preoptic area (VLPO), important in sleep–wake control, (2) the paraventricular nucleus (PVN), which contains neurons that synthesize corticotrophin releasing hormone (CRH) and neurons that mediate pre-ganglionic outputs of the autonomic nervous system, and (3) the lateral hypothalamic area, where the hunger-stimulating neuropeptides, hypocretin and melanin-concentrating hormone, are produced. Discrete neurotoxic lesions of the SPZ and DMH impair sleep and metabolic rhythms, indicating that these nuclei are important anatomical sites where the SCN is integrated with sleep and metabolic regulatory signals [44]. The DMH receives input from the arcuate nucleus, an area of the mediobasal hypothalamus that has been well characterized with respect to the regulation of food intake and energy expenditure, as well as for the ability to monitor peripheral energy status, particularly through leptin receptor signaling [43]. In nocturnal rodents, DMH activity, as measured by c-fos expression, is highest during the dark phase, in conjunction with the major period of wakefulness, activity, and food intake [43]. If food availability is restricted to a specific block of time during the light phase over multiple consecutive days, rhythms in wakefulness, locomotor activity, body temperature, cortico-

sterone release, and DMH c-fos expression shift to anticipate (by a few hours) the next block of food availability [45,46]. This pattern of food entrainment still occurs in SCN-lesioned animals, indicating that a separate food entrainable oscillator (FEO) exists, independent of the SCN [47]. There has been great interest in identifying the anatomical location of the FEO, however, despite several investigations, it has remained elusive. While a recent study provided convincing data that the FEO may reside within the DMH [44], contradictory evidence to this hypothesis has also been reported [48]. The ability of the organism to entrain to a cue, other than the light:dark cycle, has broad implications for understanding circadian organization. The existence of the FEO opens up the possibility that peripheral oscillators may be controlled by a range of factors, including particular behaviors, neuro-hormonal signals, or local peripheral processes. Therefore, not only may circadian disruption predispose an animal or human to alterations in metabolic function (e.g. obesity, diabetes, and cardiovascular disease), it is conceivable that circadian clocks are reciprocally modulated by inputs relaying information about adiposity, glucose homeostasis, sympathetic and parasympathetic balance, or behavioral status (e.g. feeding, sleep deprivation). For example, sleep deprivation has been shown to shift the circadian clock, even when stress and overall activity levels have been controlled [49]. In murine models of obesity and diabetes, it is common to find attenuated rhythms of food intake, locomotor activity, and body temperature [50– 53]. Intriguingly, when db/db mice, Zucker obese rats, and H1 receptor deficient mice are provided with food only during the dark phase, their increase in weight gain is attenuated,

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Fig. 3. Transcriptional–translational feedback loop of core circadian clock genes. This diagram depicts the positive (CLOCK:BMAL1) and negative (PER:CRY) limbs of the circadian clock gene feedback loop that is present in virtually all cells throughout the body. REV-ERBa and RORa represent components of a secondary feedback loop that either activate (solid line with arrow) or inhibit (dashed line) Bmal1 activity. The CLOCK:BMAL1 transcription complex has the ability to directly mediate the activity of genes (clock controlled genes) critically involved in energy metabolism, including Dbp and Ppara. In turn, PPARa can directly bind to Bmal1 and influence activation of the positive limb of the feedback loop. BMAL1, brain muscle arnt like factor; CLOCK, circadian locmotor output cycles kaput; CRY, cryptochrome; DBP, albumin D-element binding protein; PER, period; PPARa, peroxisome proliferator-activated factor alpha; REV-ERBa, reverse erythroblastosis virus alpha; RORa, retinoic acid receptor-related orphan receptor alpha; RXRa, retinoid X receptor alpha. (Adapted from Kohsaka [6]).

locomotor rhythms are improved, and other altered metabolic parameters show improvement [50–52]. The ability of a high fat diet to induce weight gain is prevented when rats are fed only during the primary eating phase, the dark period [54]. A novel study showed that in response to restricted feeding, Zucker obese rats exhibited more robust food anticipatory behavior compared to lean rats, indicating a stronger response of the FEO [55]. Additionally, our laboratory has performed a variety of studies that demonstrate caloric restriction and experimental manipulations of glucose availability and metabolism alter the response of the circadian system to the phase shifting and entraining effects of light [56–58]. All of these studies provide evidence for intersections between circadian and metabolic control systems. An understanding of the fundamental basis for this interplay is beginning to emerge due to advances in the field of molecular biology and the discovery/characterization of circadian clock genes. 2.4. The molecular basis of circadian rhythms: circadian clock genes The ability of the SCN to entrain to the L:D cycle and to generate endogenous rhythms in the absence of environmental cues is based on molecular events involving an integrated circuit of circadian clock genes that exists in individual neurons of the SCN. This circuit, which is highly conserved across spe-

cies, involves transcriptional–translational feedback loop(s) of core circadian clock genes, and cycles naturally with a near 24-h periodicity [3]. In mammals, Clock and Bmal1 (also called Mop3) encode the bHLH-PAS domain-containing transcription factors, CLOCK and BMAL1, which dimerize to form the positive (i.e. active) limb of the feedback loop (Fig. 3). The CLOCK:BMAL1 complex drives transcription of the negative limb (i.e. repressor) genes, Period (Per1,Per2, and Per3) and Cryptochrome (Cry1 and Cry2), by binding to E-box cisregulatory enhancer elements in their promoter region. In the cytoplasm, PER and CRY proteins dimerize, translocation to the nucleus, and inhibit further Per and Cry transcription by suppressing activity of CLOCK:BMAL1, thus, completing the negative loop. Post-translational events, such as the cytoplasmic phosphorylation of PER:CRY by casein kinase Ie (CKIe), can slow the translocation of PER:CRY into the nucleus and influence the timing of the molecular clock. In addition, CLOCK:BMAL1 activates transcription of the orphan nuclear receptors, reverse erythroblastosis virus a (Rev-erba), which inhibits Bmal1 expression, and retinoic acid receptor-related orphan receptor a (Rora), which activates Bmal1 expression, to form a secondary feedback loop. The significance of REV-ERBa and RORa as components of the clock machinery for understanding how the circadian clock and energy regulating systems may be tied together at the molecular level is that


these proteins are directly involved in other molecular pathways related to energy metabolism [59], as we will discuss below.

2.5. Clock controlled genes and clock-metabolic gene interplay The ability of non-SCN tissue to generate self-sustaining oscillations in clock gene expression was first clearly demonstrated in cultures of the rat retina and rat-1 fibroblast cells [60]. Subsequently, the Per1 gene was shown to exhibit circadian oscillations in liver explants using the bioluminescent reporter gene, luciferase [61]. Several core clock genes in the liver can entrain to scheduled feeding (i.e. the FEO), independently of clock gene rhythms in the SCN [45–47]. The activity of the core circadian clock genes must somehow be translated into downstream signals in order to affect cellular processes and to translate these cellular events into physiological and behavioral responses. DNA microarray analysis has revealed that around 5–10% of the transcriptome displays oscillations in mRNA expression levels in the SCN, liver, heart, vasculature, and adipose tissue [62–66]. There is little overlap in the genes that cycle between different tissues, indicating that the downstream targets of the core clock machinery are unique to particular tissues, which diversifies the role of the circadian clock in the regulation of individual tissue/organ systems. Furthermore, the same clock gene can exhibit different expression profiles between tissues, indicating that its role may not be identical throughout the body [67]. Many clock controlled genes (CCGs) are components of cellular metabolic pathways raising the possibility that metabolic and circadian gene networks may be co-regulated [6,68]. This also means that a disrupted molecular circadian clock may underlie a wide-range of disorders and disease processes, including obesity, diabetes, and other components of the metabolic syndrome. Recent reviews have described detailed pathways by which the circadian clock may directly mediate lipid and glucose metabolism, as well as cardiovascular function [41,67,69,70]. Of particular interest are CCGs that contain binding sites in their promoter regions for core clock genes, which include albumin D-element binding protein (Dbp), orphan nuclear receptors (NRs) (i.e. Rev-erba and Rora), NRs (i.e. peroxisome proliferator-activated receptor a [PPARa]), and plasminogen activator inhibitor type 1 (PAI-1). The family of orphan NRs consisting of Rev-erba and Rora have received significant attention as a potential molecular link between the circadian, metabolic, and cardiovascular systems. Recent studies have shown reciprocal binding between the CLOCK:BMAL1 transcription factor and Rev-erba and Rora, that forms the basis of a circadian clock gene feedback loop (described above)[3]. Nuclear receptors serve important roles in lipid and carbohydrate metabolism, as well as in mediating vascular inflammatory responses [59]. For example, Reverba mRNA expression promotes adipocyte differentiation and REV-ERBa is phosphorylated by glycogen synthase kinase 3, an enzyme involved in carbohydrate metabolism [59]. Using mouse fibroblast cells, it was shown that Bmal1 is a mediating factor in adipocyte differentiation, as well as downstream effects of PPARc [71]. RORa, which activates Bmal1 transcription, also mediates lipogenesis and lipid storage in skeletal muscle [59]. Rev-erba has been implicated in the regulation of cytokine-mediated inflammatory responses in vascular smooth muscle cells, as well as in the regulation of the

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expression of PAI-1 [59]. Increased levels of PAI-1 inhibit tissue plasminogen activator (tPA), resulting in reduced fibrinolysis and an increased risk for thrombolytic events. Another nuclear receptor, PPARa, is a lipid responsive protein involved in lipid and glucose metabolism. In Clock mutant mice on a mixed background, the circadian expression of Ppara mRNA in the liver and of Pai-1 mRNA in the heart was significantly attenuated compared to control animals [72,73]. Another study found that Clock mutant mice (mixed background) exhibited impaired gluconeogenesis in response to an insulin tolerance test and were resistant to the hyperglycemic effects of a high fat diet [74]. Taken together, these findings indicate that NRs may represent an important molecular link between the circadian clock and components of the metabolic syndrome, including adiposity, hypertension, and hyperlipidemia. Type II diabetes has also been associated with the metabolic syndrome and a number of intriguing links between diabetes and circadian rhythmicity have been identified. Core clock genes, as well as Dbp and Rev-erb-a, are expressed in a circadian manner in insulin-releasing cells of the pancreas [75]. Streptozotocin-induced diabetes in rats results in notable phase advances in the rhythmic expression of circadian clock genes and CCGs in the heart [76]. In a mouse model of diabetes, mild STZ-induced beta cell damage resulted in exaggerated circadian responses in locomotor activity in response to light (i.e. phase shifts), whereas, subsequent treatment with insulin in these same animals corrected the phase response to the light pulse [58]. Oppositely, reduced glucose availability induced by 2-deoxy-D -glusose injections attenuated the phase shifting effects of light on activity in mice [57]. Finally, there are indications that Clock/Clock mutant and Bmal1 / deficient mice exhibit deficits in insulin regulation and beta cell function [74]. Thus, clock gene disruption in pancreatic beta cells could underlie the loss of rhythmicity in insulin secretion and glucose tolerance exhibited by patients with type II diabetes.

3. Summary This review has presented some of the accumulating evidence indicating that chronic partial sleep loss, circadian desynchronization, and/or alterations in clock gene function can lead to significant changes in energy homeostasis and cardiovascular function. The discovery that circadian clock genes are directly involved in molecular pathways of lipid and glucose regulation, inflammatory responses, and sleep–wake regulation, has provided a new framework in which to examine the role of the circadian clock system in multiple disease processes. The fields of sleep research and circadian biology are in a unique position to make major contributions to the understanding of behavioral, physiological, and molecular mechanisms that are responsible for epidemic diseases, such as obesity, diabetes, and metabolic syndrome. Acknowledgement: This work was supported by NIH Grant P01 AG11412.

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