It is well established that circadian rhythms play an important role in maintaining homeostasis and normal body function. Therefore it follows that disruption of circadian regulation can affect normal physiological and biochemical function and can lead to diseases. The effects of disruption in circadian rhythms can range from minor and easily reversible conditions to severe damage to the system. Jet lag is a very simple and common case where a disruption of circadian rhythms is observed [37
]. Changes in time zones often cause sleep disorders (night-time insominia or abnormal day-time sleeping) and temporary cognitive defects. Similar observations were made when rats were subjected to an abrupt shift in the light/dark cycle, in which case the animals displayed an increase in night time (active period) rest [38
]. A series of studies performed by Cho et al showed that flight attendants who frequently fly across different time zones show cognitive defects associated with the reduction in the temporal lobe volume [39
]. Another classic example which illustrates that disruption of circadian regulation can cause disorder comes from studies done in shift workers [40
]. These studies suggest that the incidence of cancer, psychological disorders, metabolic syndrome, diabetes, and cardio-vascular diseases is higher in these individuals. Studies on the role of molecular clock components in regulating physiological processes have shown that clock plays a critical role in coordinating many of important processes including metabolism, cell cycle progression and immune regulation among many others. Furthermore, several valuable studies performed with genetic knockouts for the clock components have given an insight into the processes that are regulated by circadian rhythms. provides some of the important functions, physiological target genes and characteristics of genetic knockouts for these clock components. In this section, we focus on selected disorders including metabolic syndrome, obesity, diabetes, inflammatory disorders and cancer that can be the cause or the result of disruption in circadian rhythms and discuss processes at the transcription level that are involved. In addition we discuss how understanding circadian rhythms in gene expression can be useful in optimizing dose timing to maximize the efficacy of drug action and at the same time reduce adverse effects and in the identification of new therapeutic targets.
2.1. Metabolic syndrome, obesity and diabetes
Maintaining a proper glucose concentration in plasma is very important, as neuronal cells in the brain require a constant supply of glucose as their primary energy source since they are not capable of oxidizing amino acids or fatty acids. An extensive systemic network of organs which includes liver, muscle, adipose, pancreas, and the brain itself functions in maintaining blood glucose homeostasis [44
]. When the animal is fed, the elevated glucose level in the blood stimulates the pancreas to secrete insulin. Insulin promotes glucose uptake by peripheral tissues, glycogen synthesis in liver and muscle, and inhibits glucose production via gluconeogenesis in the liver and kidney. It also stimulates the production of fatty acids and lipids in the liver. Excess glucose taken up by the adipose tissue and not used for glycolysis is used for lipogenesis. In the fasting state, appropriate blood glucose concentrations are maintained by glycogen breakdown in the liver along with gluconeogenesis in the liver and the kidney so that there is a constant glucose supply for the brain. In addition, lipids stored in the adipose tissue can be used as an energy fuel by other tissues. Any imbalance in the timely coordination between these organs can affect glucose and energy homeostasis and may lead to disorders such as metabolic syndrome, obesity, and diabetes.
Several lines of evidence suggest that metabolic processes are under the control of circadian regulation. First, microarray studies in various organs demonstrate that genes involved in controlling metabolic processes show circadian rhythms [2
]. Many of the genes involved in regulating key reactions in pathways like glycolysis, gluconeogenesis, lipid biosynthesis and metabolism, and oxidative phosphorylation are either directly or indirectly under the control of core clock genes. Studies using Clock
gene mutants demonstrate that these transgenic rodents show characteristics of metabolic syndrome such as hyperleptinemia, hyperlipidemia, hepatic steatosis, hyperglycemia, and hypoinsulinemia [47
]. In addition, Clock
mutant rodents shows dysregulation in the circadian pattern of peptide hormones like ghrelin, orexin, and CART (Cocaine and amphetamine regulated transcript) which play important roles in regulating food intake and metabolic processes. These observations are consistent with the fact that Clock
mutant rodents are hyperphagic and obese with excessive accumulation of adipose tissue. PPAR alpha is an important nuclear receptor transcription factor that is involved in regulating lipid metabolism. Studies suggest that PPAR alpha expression is regulated by CLOCK via an E-box rich region present in the Ppar
alpha gene. The rhythmic expression of PPAR alpha is lost in the liver of Clock
mutant rodents [48
]. These results suggest that CLOCK plays an important role in regulating metabolic processes in peripheral tissues. Similarly, BMAL 1 which is the heterodimeric partner of CLOCK is also important in regulating metabolic processes. Lamia et al. 2008 performed a liver specific deletion of the Bmal1
gene in rodents and found that these animals exhibited hypoglycemia restricted to the fasting period, increased glucose clearance and loss of rhythmic expression in the hepatic genes involved in glucose metabolism [49
]. The orphan nuclear receptor NR1D1 (REV-ERB alpha) which is an important core clock transcription factor is found to play an important role in regulating hepatic gluconeogenesis [50
]. These results suggests that core clock genes present in peripheral tissues play an important role in regulating metabolic processes, and disruption of these genes results in metabolic disorder.
Evidence to support the fact that circadian rhythms in gene expression are affected in diabetes and metabolic syndrome comes from studies conducted using animal models of obesity and/or diabetes. Studies in the severely obese diabetic model, KK-Ay
mice showed that the circadian expression of certain core clock genes and genes involved in metabolic processes were disrupted [51
]. Also studies suggest that high fat fed animals show disruption in circadian rhythms in mRNA expression of clock genes [52
]. Evidence for disruption of circadian rhythms is not just limited to obese animals, but is also found in non-obese diabetic animal models. The Goto-Kakizaki (GK) rat is a polygenic model of non-obese diabetes which was developed by multiple generational breeding of Wistar-Kyoto (WKY) rats selected for high glucose intolerance [53
]. Studies suggest that serum melatonin (the hormone involved in circadian entrainment) concentrations are reduced in the GK diabetic rats because of the disruption in the expression of melatonin synthesizing enzymes present in the pineal gland of these animals [54
]. It was further observed that expression of melatonin receptor in beta islet cells was up-regulated in the GK rats which might be a compensatory response to the decrease in melatonin. In another study Frese et al. 2007 found that the rhythmic expression of both GLUT2 and glucokinase is altered in the beta cells of these rats [56
]. It was also reported that the circadian rhythm of glucose uptake by cultured muscle cells and adipocytes from GK rats were altered [57
]. These results suggest that the circadian rhythm in gene expression in diabetic animals is disrupted and this dysregulation can be causally related to the development of diabetes.
Although it has been well established that the core circadian mechanism regulates the expression of genes involved in metabolic processes, recent evidence suggests that the converse is also true. Zang et al. 2009 performed a genome wide siRNA screen in order to identify the genes that can potentially regulate the internal clock mechanism [58
]. This is a high throughput plate-based in vitro screen where U2OS (human osteosarcoma) cells were treated with 90,000 different individual siRNA and the effect of knock down on the circadian clock was studied by analyzing the changes in rhythm amplitude, phase, or period length of the luciferase reporter gene expression which was under the control of Bmal1
gene promoter. The result from this study showed that many genes that are involved in metabolic pathways such as insulin signaling, folate metabolism and others were found to regulate the core clock components.
The significance of understanding the circadian regulation of gene expression and how it is affected in disease states may be useful in optimizing dosing time for drugs in order to get maximum therapeutic efficacy and minimum toxicity. From microarray studies in liver tissue of Wistar rats we found that HMG-CoA reductase (3-Hydroxy-3-methylglutarylcoenzyme A reductase), Sqle (Squalene epoxidase) and Cyp7a1 (Cytochrome p450 (cholesterol hydrolase 7 alpha)) all had circadian rhythms with peak expression occurring at the middle of the active (dark) period [22
]. All of these proteins are involved in the biosynthesis of cholesterol and are the therapeutic targets for cholesterol-lowering drugs. For example statins are potent inhibitors of HMG-CoA reductase and are prescribed to patients with hypercholesterolemia. The current practice is that patients take the drug in the night before going to bed (inactive period) which would seem inappropriate as the HMG-CoA reductase peaks during the active period (which is day time in humans). Human studies have also confirmed the observation that HMG-CoA reductase peaks during the active day time period [59
]. This result suggests that for humans taking statins in the morning may either increase efficacy or decrease muscle toxicity.
Therapeutic targets for two important drugs (metformin and thiazolidinediones) used to treat diabetes regulate and/or show circadian rhythms. Metformin, a biguanide class drug used to treat T2DM, reduces blood glucose by activating AMPK (AMP- activated protein kinase) which in turn inhibits the expression of PEPCK, an important enzyme in regulating gluconeogenesis. Um et al. 2007 showed that the administration of metformin can lead to a phase advancement in the circadian expression of clock genes in rodents through AMPK by targeting the CKξ protein which phosphorylates and destabilizes PER proteins [60
]. Furthermore, the gluconeogenic enzyme PEPCK which is inhibited by AMPK also shows circadian rhythms in gene expression in peripheral tissues [61
]. The other class of drugs, thiazolidinediones, which are PPAR gamma agonists, bind to the nuclear receptor PPAR gamma and activate the PPAR gammamediated transcription of a number of specific genes that regulate adipogenesis and energy metabolism. Thiazolidinediones act as insulin sensitizers by increasing the uptake of glucose by organs and reduce plasma free fatty acids by increasing their uptake by adipose tissue. PPAR gamma is found to be rhythmically expressed in adipose tissue [63
]. Studies reported by Wang et al. 2008 show that vascular PPAR gamma also exhibits rhythmic expression and is important in controlling the circadian regulation of blood pressure and heart rate [64
]. In this study thiazolidinedione treatment resulted in the up-regulation of aortic BMAL1, while CHIP and promoter assays showed that Bmal1 is a direct PPAR gamma target gene.
2.2. Inflammatory diseases and autoimmune disorders
Immune responses and inflammation are very important biological processes which serve to protect the body from invading pathogens and cancer cells. Such processes are not confined to a single organ or cell type. Rather, they are regulated by a plethora of cell types present in different organs along with small molecule or protein mediators that are secreted by these cells. Some examples include white blood cells (monocytes and lymphocytes) in circulation, macrophages infiltrated into adipose tissues and lungs, Kupffer cells in liver, mast cells and macrophages in muscle, and microglial cells in the brain. Circadian regulation of these processes in these cell types occurs at different levels of organization. Studies in both humans and rodents demonstrate that the core clock genes are rhythmically expressed in cells regulating inflammation and immune response processes [65
]. Additionally, these studies indicate that inflammatory mediators also show circadian rhythms. The mRNA expression profiles and plasma protein profiles or cytokines such as IL-6, IL-2, IL-12, TNF alpha, GM-CSF, Interferon gamma, and others show circadian rhythmicity in both humans and rodents. For example, plasma concentrations of IL-6 and TNF alpha which are important pro-inflammatory cytokines peak at the beginning of the active cycle (early morning in humans and just after lights off in rodents) [68
]. Rhythms are also observed in cell counts and trafficking of monocytes, neutrophils, natural killer (NK) cells, T-lymphocytes and B-lympohocytes [69
]. Numbers of monocytes and T and B lymphocytes in blood peak during the inactive period of the organism, whereas neutrophils and natural killer cells peak during the active period. Hormones like melatonin, glucocorticoids and prolactin which show robust circadian rhythms in their secretion are also found to regulate immune response processes [29
Evidence from recent studies suggests that the core molecular clock components can directly modulate the expression of genes regulating immune response and inflammation. Liu et al. 2006 showed that in rodents PER2 is a very important regulator of interferon gamma production, and hence plays an important role in the normal functioning of NK cells [69
mutant mice showed defects in interferon gamma production and impaired NK cell function. Migita et al. 2004 showed that orphan nuclear receptor REV-ERB alpha, which is also a component of the molecular clock, regulates the expression of IL-6 and cyclooxygenase-2 genes in vascular smooth muscle cells [70
]. It was also reported that REV-ERB alpha increased the transactivation of nuclear factor kappa B (NFκB) which is an important transcription factor involved in regulating the expression of a large number of immune response genes. A study by Wang et al. 2006 demonstrated that in liver, Rev-erb
alpha and beta regulate the expression of plasminogen activator inhibitor type 1, a critical regulator of the fibrinolytic system and of the inflammatory response in the vascular wall [71
]. Furthermore, the orphan receptors RORα and RORγ were also found to play an important role in the development and functioning of immune cells both under normal conditions and in inflammatory or autoimmune disorders [17
]. Mutation of RORγ in rodent models resulted in abnormalities in lymphoid organ development and these animals failed to develop lymphoid nodes and Payer’s patches [16
]. These mutants also showed reduced survival rates of thymocytes, the precursor cells of T lymphocytes and NK cells. Animal models for ROR
α or ROR
γ mutants show reduced susceptibility to autoimmune and inflammatory disorders suggesting that both RORα and RORγ might be attractive pharmacological targets for both inflammatory and autoimmune disorders including asthma, chronic colitis, and multiple sclerosis [17
]. For example, both ROR
α and ROR
γ mutant rodents are less susceptible to ovalbumin-induced lung inflammation and showed reduced easinophil and lymphocytes levels, attenuated pulmonary inflammation, and lower levels of protein and mRNA expression of Th2 cytokines in lungs [72
]. Both these orphan receptors are critical for the differentiation and functioning of TH17 cells which plays a key role in the above mentioned inflammatory and autoimmune diseases. Together, these studies suggest that core molecular clock components can regulate inflammatory and immune responses and can be potential therapeutic targets for inflammation and autoimmune-related disorders.
Circadian rhythms in gene expression and/or protein production/secretion of immune modulating agents are relevant to clinical observations found in chronic inflammatory conditions. For example in rheumatoid arthritis (RA), a systemic chronic inflammatory disorder, symptoms like joint pain, morning stiffness and functional disability peak in the early active period (morning in the case of humans) [68
]. This is consistent with gene expression and plasma protein profiles of pro-inflammatory cytokines. Both IL-6 and TNF alpha are elevated in the disease and peak early in the morning (active period) in RA patients. It should be noted that circadian rhythms in cytokine expression are preserved even in the disease state, although the mean level of expression is elevated when compared to healthy controls.
Synthetic glucocorticoids (corticosteroids) are potent anti-inflammatory agents used to treat symptoms of RA. Although they are widely used for the treatment of a plethora of inflammatory and autoimmune disorders, long-term therapy can cause severe metabolic disorders leading to Cushing’s syndrome, hypertension, hyperglycemia, muscle atrophy, and dyslipidemia [35
]. Optimizing dose and timing of administration are necessary to obtain maximum therapeutic use and minimize adverse effects. It is a general practice for RA patients to take immediate-release corticosteroids in the morning at around 6 to 8 AM [74
]. However, it has been suggested that this dose schedule is inappropriate as the pro-inflammatory stimulus that causes the irritation and swelling of synovial tissue has already been triggered by the increase in the pro-inflammatory cytokines which peak at early morning. Studies performed in RA patients showed that administration of immediate-release corticosteroids during the night (2 AM) before the rise in the pro-inflammatory cytokines improved the efficacy of the drug by inhibiting proinflammatory cytokine production [75
]. It was also observed that the blood concentration of the drug resembled the normal circadian rhythm of the endogenous corticosteroid. This treatment regimen requires lower doses of the drug and hence reduces the magnitude of adverse effects. A problem with this treatment schedule is that it is impractical to wake the patients in the middle of the night to give the drugs regularly. This problem is circumvented by the development of a modified release oral tablet that contains a coating that can burst open 4 h after intake because of the penetration of water [74
Another inflammatory disease that shows rhythmicity in clinical symptoms is asthma. It is a common observation that asthma in humans worsens during the night. The increased occurrence of night-time asthmatic attacks is related to a number of factors that show circadian rhythms. The first and foremost factor is lung functioning. In normal humans, lung function has a modest circadian rhythm, but in asthmatic patients the amplitude of the circadian rhythm is very high with minimum functioning during the night [76
]. In humans, levels of cortisol, epinephrine, and beta-adrenergic receptors are low during the night [77
]. Cortisol (endogenous glucocorticoid) is a potent anti-inflammatory agent and epinephrine helps in bronchodilation through the beta-adrenergic receptor. That all these factors are low at night results in the worsening of symptoms. Further worsening the condition, both cellular markers of inflammation like alveolar easinophils, neutrophils, lymphocytes, and chemical mediators like histamine and their responsiveness to inhaled bronchoconstrictors are also found to be high during the night period [77
]. This circadian rhythm in the complex interaction between these factors results in the worsening of nocturnal asthma and drugs like theophylline and β2
-adrenergic receptor agonists that are used for the treatment of asthma are recommended to be given to patients at higher dose in the evening than in the morning [79