Acute and chronic jet lag schedules, constant light conditions and counter-phase forced activity, which may be considered model paradigms for shift- and night-work conditions in humans, as well as rodent circadian mutants, have been employed to study circadian disruption and its consequences on physiology, health, and disease development (see Fig. 15.1).

In addition, we have developed a chronic jet lag schedule consisting of a 6 h LD-advance each two days, which generated a stable desynchronization of two activity components in mice (Casiraghi et al. 2012) and exerted severe metabolic alterations (unpublished results). Moreover, animals under similar chronic jet lag protocols showed biphasic corticosterone rhythms (Filipski et al. 2004), attenuated AVP and Pk2 expression in the SCN (Yan 2011), and alterations in PER2 expression rhythms in the SCN, thymus, and liver (Castañon-Cervantes et al. 2010). These persistent alterations due to chronic desynchronization have important consequences in health (see below) and might reflect the risks to which individuals working in frequent rotating shifts are subjected.

In humans, light-pulses at night induced acute melatonin suppression and subjective sleepiness in a dose-dependent response to light exposure duration (Chang et al. 2012).

Although it has been known for several decades that many metabolic processes, such as glucose and cholesterol metabolism, are regulated by the circadian clock, it is only in the past few years that the severe metabolic consequences of circadian disruption have emerged. The SCN exerts a hierarchical daily control on metabolic rhythms by neurohumoral efferents to other brain regions (mainly several hypothalamic nuclei) and to autonomic centers, which drive hormones (e.g., glucocorticoids) and behavior (e.g., feeding) producing a set of physiological signals that synchronize peripheral tissues (reviewed in Delezie and Challet 2011; Huang et al. 2011). In addition, food-entrainable oscillators exert a secondary daily control on peripheral clocks (reviewed in Delezie and Challet 2011) and fine-tune the balance between energy intake and expenditure. Pancreatic (Marcheva et al. 2010) and thyroid hormones regulating carbohydrate and lipid metabolism (and thus basal metabolic rate) are under circadian control, as well as rate-limiting enzymes of metabolic pathways (Panda et al. 2002). Several molecular clock components are found to be regulators of cell metabolism through the so-called nuclear receptor transcription factors (as Retinoic acid-related orphan receptor α, RORα, reverse viral erythroblastis oncogene products, REV-ERBα, and peroxisome proliferator-activated receptors, PPAR) which sense metabolites such as fat-soluble hormones, vitamins, lipids, oxysterols, bile acids, and xenobiotics. For instance, CLOCK and BMAL1 act as transcription factors of PPAR (α, γ, δ) and estrogen-related receptor (ERR) family members (α, β, γ). Conversely, REV-ERBα and PPARα directly regulate BMAL1 expression, and relevant cellular (nutrient-redox) metabolic sensors are able to interact with clock genes (Sahar and Sassone-Corsi 2012). For instance NADH/NAD + balance regulates CLOCK/BMAL1 and NPAS2/BMAL1 heterodimer activity (Rutter et al. 2001) and, in addition, the NAD + -deacetylase dependent SIRT1 induced by food restriction can directly bind to CLOCK/BMAL1 (Nemoto et al. 2004). This physiological and molecular cross-talk of both clockwork and metabolic networks provides a mechanistic framework to understand how systematic circadian disruption will lead to metabolic alterations (Eckel-Mahan and Sassone-Corsi 2009). For example, circadian Clock mutant mice develop obesity (Turek et al. 2005), and diabetes was found in Clock-Bmal1 double-mutants due to impaired insulin secretion (Marcheva et al. 2010) and gluconeogenesis suppression (Rudic et al. 2004). Moreover, liver-specific deletion of Bmal1 in mice confirmed its strong involvement in hepatic glucose metabolism (Lamia et al. 2008).

Impaired entrainment in the laboratory also generates metabolic alterations, as when exposing mice to T cycles of 20 h, leading to larger body mass gain and increased insulin/glucose ratio (Karatsoreos et al. 2011). In human subjects exposed to controlled 28 h sleep–wake cycles under dim light, misalignment between the free-running central circadian pacemaker and the behavioral sleep–wake and fasting/feeding rhythms, resulted in increased plasma glucose and insulin concentrations, increased blood pressure, and decreased plasma leptin concentrations (Scheer et al. 2009). In addition, constant exposure of mice to bright or dim light led to increased body mass and reduced glucose tolerance as compared to mice housed under a normal LD cycle, despite equivalent levels of caloric intake and total daily activity output (Fonken et al. 2010). There is a well-documented increased risk for metabolic syndrome in humans subjected to shift work (reviewed in Zimberg et al. 2012) perhaps as the result of pathological adaptation to chronically sleeping and eating at abnormal circadian times. Disrupted sleep must be taken as an important factor in glucose metabolism alterations increasing the risk for obesity (Gangwisch 2009; Nedeltcheva et al. 2009; Spiegel et al. 2009) as show in a recent study with humans provided with 5 days with insufficient sleep, showing weight gain due to increased food intake exceeding energy needs (Markwald et al. 2013). On the other hand, a “night-eating” syndrome was detected in women, with a delayed circadian pattern of melatonin and food intake, presenting evening hyperphagia and frequent awakenings accompanied by food intake, while retaining a normal sleep–wake cycle. This alteration in feeding behavior is accompanied by delayed and low-amplitude rhythms in cortisol and several nutrients intake, and altered rhythms of metabolic hormones as insulin, ghrelin, and leptin (Goel et al. 2009). In addition, a failure to manage cholesterol in shift workers could lead to cardiovascular alterations (Ha and Park 2005) such as the ones discussed in the following section.

Autonomic nuclei that regulate the cardiovascular system are directly controlled by neural efferents from SCN and/or by circadian hypothalamic–pituitary factors acting on corticoid stress hormones, as well as by neurohumoral effectors on vascular tone. Cardiovascular risk markers as cortisol, nonlinear dynamic heart rate control, and hemostatic factors are higher during the morning, when adverse cardiovascular events are most likely to occur (reviewed in Morris et al. 2012b; Ruger and Scheer 2009). These circadian acute cardiovascular events are also suggested to be dependent on specific phase-relationship between peripheral oscillators at cardiovascular tissues (Davidson et al. 2005). Epidemiological studies show a consistent association for shift work as a factor for cardiovascular disease (reviewed in Knutsson and Boggild 2000), such as coronary failure (Nakamura et al. 1997; Tenkanen et al. 1997) or increased blood pressure in night workers (Lo et al. 2010; Su et al. 2008). A meta-analysis study of almost 2,000,000 shift workers detected higher risk for coronary events (especially myocardial infarct) in the nocturnal shift (Vyas et al. 2012). It is suggested that cardiovascular risk markers might respond unfavorably to exogenous stressors such as increased nocturnal activity (Morris et al. 2012b), promoting alterations in shift workers with misalignment between the circadian and the activity–rest cycles.

Disruption of circadian regulation in the laboratory increases several risk factors of cardiovascular disease. Chronic LD-shifts increased plasmatic plasminogen activator inhibitor-1 (PAI-1) levels (Oishi and Ohkura 2013), which promotes hypofibrinolysis. The aforementioned study of Scheer et al. (2009) in humans forced to follow a 28 h sleep–wake cycle indicated increased blood pressure during wakefulness. Altered cardiovascular functions are found in the 22 h short-period hamster τ, with a point mutation in Ck1ε circadian regulatory gene, which exhibits abnormal 24 h LD behavioral entrainment and disrupted rhythms. These animals had cardiomyopathy, extensive fibrosis, and severely impaired contractility with shortened life-expectancy (Martino et al. 2008), while under T22 cycles (i.e., their normal endogenous period) behavioral rhythms and cardiac phenotype are normalized. Several vascular dysfunctions were found in clock gene-deficient mice. In Bmal1 knock-out mice, abnormal thrombosis and vascular remodeling responses were found, while Clock mutants showed impeded responses to vascular injury; moreover, in Bmal1, Clock, and Per2 mutant mice, vascular endothelial dysfunction was found (Anea et al. 2009; Viswambharan et al. 2007). Also, blood pressure was altered in Cry 1–2 null mice, showing hypertension due to deregulation of salt homeostasis caused by abnormal high synthesis of adrenal mineralocorticoid aldosterone (Doi et al. 2010).

The gastrointestinal tract (GT) displays circadian rhythms in several functions and variables, such as motility, maintenance and replacement of the protective barrier, absorption rates, and enzyme secretion rhythms (reviewed in Konturek et al. 2011). Furthermore, rhythmic clock gene expression has been found in peripheral oscillators throughout the GT, which can be shifted, or entrained, by timed meals (Hoogerwerf et al. 2007; Mazzoccoli et al. 2012; Polidarova et al. 2011). The correct phase-relationship between feeding time and the activity–rest cycle led to normal entrainment of the GT, by a myriad of neural and humoral signals related with the interplay between SCN activity, food-entrainable oscillators, and metabolism (Stenvers et al. 2012). There are evidences showing that circadian disruption leads to alterations in gastrointestinal physiology. Increased general symptoms for gastrointestinal disorders were more prevalent among shift workers as compared with day workers (Koller 1983), and aircrew members, especially those on long-distance flights, had more upper-GT symptoms than ground staff (Enck et al. 1995). The consequences on gastrointestinal functions of shifting working schedules might be related to an increase in food consumption during the rest cycle, since time of feed has been shown to be the strongest Zeitgeber in gastrointestinal oscillators (Malloy et al. 2012; Polidarova et al. 2011), resulting in a misalignment between circadian clocks among the body. There is a higher prevalence on peptic ulcer among shift workers (Higashi et al. 1988; Segawa et al. 1987), which might be related to higher incidences in Helicobacter pylori infections in shift workers (Pietroiusti et al. 2006; Zober et al. 1998). Irritable bowel disease prevalence was also higher in nurses working rotating shifts (Nojkov et al. 2010).

Circadian disruption by chronic lighting in rats fed ad libitum abolished rhythmic clock gene expression in the liver and colon, while a restricted feeding schedule restored these rhythms (Polidarova et al. 2011). Similar results were found in SCN-ablated mice, where restricted feeding re-established locomotor, feeding, and stool output rhythms (Malloy et al. 2012). Per1–2 double knockout mice exhibited abolished rhythms in colonic functions, such as motility, intracolonic pressure changes, stool output, and circular muscle contraction (Hoogerwerf 2010).

The circadian system exerts a tight control over the immune system and its function, which in turn feedbacks over the circadian clock (reviewed in Cermakian et al. 2013; Scheiermann et al. 2013). Robust rhythms in the number of immune cells, plasmatic levels of proinflammatory cytokines, and immune cell-produced peptide hormones, as well as in major humoral responses, have been described vastly, in humans and animal models (Fortier et al. 2011; Haus and Smolensky 1999; Silver et al. 2012). In addition, circadian disruption has profound effects on the immune system, which has been evidenced both in animal models and in human studies. Female nurses working in a three-shift rotating system showed a progressive decrease in natural killer (NK) cell activity and CD16⁺ CD56⁺ cells number (Nagai et al. 2011). Shift working has also been linked to a higher risk for common infections, with the highest risk in workers going through three different shifts (Mohren et al. 2002). Impeding normal LD-entrainment by chronic jet lag under weekly 6 h phase-advances altered the inflammatory response of mice to an experimental challenge with lipopolysaccharide (LPS). After four consecutive shifts, jet lagged mice displayed a fivefold increase in mortality and severe hypothermia due to LPS, as compared to LD-entrained controls. In this chronic jet lag the expression of the cytokines IL-1β, IL-10, IL-12, IL-13, GM-CSF, and TNF-α was significantly increased 24 h after LPS injection, and in vitro macrophage stimulation with LPS produced higher levels of IL-6 (Castañon-Cervantes et al. 2010). Chronic jet lag also impaired NK cells cytolytic activity, as well as altered rhythms in clock gene and cytokine expression (Logan et al. 2012b). Deleterious effects of abnormal entrainment were also reported in a mice model of experimental colitis, where chronic inversions of the LD cycle every 5 days led to increased symptoms as reduced body mass, abnormal intestinal histopathology, and potentiated inflammatory response (Preuss et al. 2008).

Changes in the sleep–wake rhythm, which are frequently concomitant with circadian disruption also have profound effects on the immune system. An altered sleep–wake pattern changed the HPA axis activity increasing cortisol, and consequently affecting the expression of proinflammatory cytokines, such as IL-6 or TNF-α (Vgontzas and Chrousos 2002). Additionally, NK cell activity, antigen uptake, circulating immune complexes, and secondary antibody responses were altered by sleep loss (Krueger et al. 2003), while IL-1β and TNF-α levels increased during sleep deprivation (Majde and Krueger 2005). In addition, sleep deprivation leads to the suppression of immune surveillance, which may permit the establishment and/or growth of malignant clones.

Alterations of clock genes, especially those of the period family, disrupt immune functions. Per3 clock gene polymorphisms have also been associated with circadian disruption and with increased cancer risk together with elevated IL-6 concentrations (Guess et al. 2009). Homozygous Per1 mutant mice exhibited altered cytokine rhythms (e.g., interferon-γ) and cytolytic factors (e.g., perforin and granzyme B) in splenic NK cells (Logan et al. 2013). On the other hand, Per2-deficient mice were more resistant to LPS-induced endotoxic shock than control wild-type mice. Moreover, serum levels of IFN-α and IL-1β were dramatically decreased in Per2−/− mice following an LPS challenge, attributable to defective NK and NKT cell function (Liu et al. 2006). In addition mice carrying hematopoietic Cry1−/− Cry2−/− mutant cells presented a potentiated immune response to LPS administration, with higher levels of circulating proinflammatory cytokines (Narasimamurthy et al. 2012).

Chronodisruption may result in altered immune responses, such as aberrant immune cell trafficking and abnormal cell proliferation cycles, which can lead to cancer (Mormont and Levi 1997). Per2 mutant mice have been shown to present enhanced cancer susceptibility (Fu et al. 2002; Wood et al. 2008), along with disrupted circadian rhythms of NK cell gene expression and function.

There is an intimate connection between the circadian clock and multiple endocrine axes, which involve several aspects of the daily regulation of hormonal release. A highly specialized organization configures the SCN neural output to both hypothalamic neuroendocrine neurons (i.e., cells containing or releasing corticotrophin-releasing hormone, CRH, tyrotrophin-releasing hormone, TRH, or gonadotrophin-releasing hormone GnRH) and pre-autonomic neurons, as well as to other brain structures (Kalsbeek and Fliers 2013). Such output ensures the coordination between behavioral, endocrine, and metabolic rhythms needed for the circadian adjustment of the organism. For instance, glucose homeostasis is tightly regulated in order to predict the energy needs for the circadian onset of behavioral activity, by increasing hypothalamic CRH and adrenal cortisol release, and by activating hypothalamic orexins at the right time of day (Kalsbeek et al. 2011).

One of the best known examples of endocrine circadian regulation is represented by the synthesis and release of pineal melatonin, which is under the control of sympathetic autonomic efferents commanded by the SCN (Moore 1996). Blood melatonin increases during the night, it is suppressed by light (Zawilska et al. 2009), and its amplitude and duration are proportional to photoperiod duration (Hazlerigg 2012). Indeed, melatonin can be considered a daily and calendar signal which communicates circadian and circannual cues to the body. In recent years, additional evidence suggested an antioxidant role for this hormone, which falls outside the scope of this chapter (Reiter et al. 2010).

It is well known that sleep disruption affects autonomic and endocrine responses. A representative example is the fact that cortisol rhythms are altered in night-working emergency physicians (Machi et al. 2012), shift-working nurses (Korompeli et al. 2009) and in police officers working night or afternoon shifts than day shifts, with maximal differences occurring after 5 days of shift work (Wirth et al. 2011). Moreover, flights involving 7 h of jet lag produced a decrease in cortisol levels that lasted for 9 days after the flight (Pierard et al. 2001). Furthermore, in laboratory conditions, in which subjects were kept in a 28 h forced desynchronization protocol, cortisol was more strongly coupled to the circadian endogenous component than to the environmental “day.” Indeed, an inverted pattern of cortisol secretion was observed when sleep–wake and circadian cycles were misaligned. On the other hand, leptin rhythm followed only the sleep–wake cycle component, with conflict phases of misalignment between the circadian and behavioral component resulting in suppression of circulating leptin levels (Scheer et al. 2009). More information is needed (and on the way) regarding the role of disturbed hormonal cycles on circadian synchronization, both at central and peripheral level.