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Endocrine Systems Regulated by the Circadian Clock
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In addition to regulation of behavioral rhythms such as sleep/wake and fasting/feeding cycles, the circadian clock also regulates rhythms of the endocrine system. Cortisol rhythms are regulated through a feedback loop known as the hypothalamic-pituitary (HPA) axis (Chap. 379). Hypothalamic secretion of CRH and AVP promotes secretion of pituitary adrenocorticotropic hormone (ACTH), which in turn regulates rhythmic cortisol secretion from the adrenal cortex. Cortisol release increases towards the morning, and this increase is believed to prepare the brain and peripheral tissues for daytime activity and food intake. Daytime sleep can blunt circulating cortisol levels, presumably through the occurrence of non-REM sleep. AVP secretion in mice occurs prior to sleep to promote water intake, thereby preventing dehydration during the sleep period. Several hormonal systems are in fact influenced more by sleep than by circadian rhythms. For instance, secretion of growth hormone (GH) is profoundly blunted during acute overnight wakefulness. The secretion of this hormone is primarily dependent on the occurrence of slow-wave sleep, which is a homeostatically driven sleep stage that occurs primarily in the first part of the sleep period. Cortisol also exhibits a peak close after wakefulness: the cortisol awakening response (CAR). This peak seems to be independent of a circadian rhythm, as the CAR is severely blunted by acute overnight wakefulness. Curtailed sleep and overnight wakefulness increase the activity of the HPA axis and may increase diurnal cortisol levels. Sleep also influences melatonin amplitude, such that sleep deprivation can increase melatonin levels. In working environments, the effects of curtailed sleep are often confounded by mistimed exposure to light. Even low levels of light are able to potently suppress melatonin secretion. Together with altered timing in light exposure, perturbed hormonal levels may represent a mechanism through which altered timing and duration of sleep may impact central and peripheral circadian oscillators.
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Centrally controlled rhythms of melatonin and cortisol are considered key regulators of extra-SCN and peripheral oscillators. Glucocorticoid receptors exist in both the central nervous system and in peripheral tissues such as skeletal muscle, liver, and adipose tissue. Following acute shifts in light-dark or feeding cycles, 24-h rhythms of circulating cortisol appear to shift more slowly than other rhythms and may thus contribute to adverse effects of circadian misalignment by hampering proper realignment of peripheral clocks. Glucocorticoids shift clock gene expression in muscle, kidney, and lung, while the powerful synthetic glucocorticoid dexamethasone is able to synchronize (e.g., reset) circadian rhythms of cells in culture, including liver cells. Consistent with a role for glucocorticoid regulation of the clock, both adrenalectomy, which results in a lack of cortisol, and exogenous corticosteroid supplementation significantly disrupt the circadian clock system.
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Several peripherally produced hormones and peptides are not only produced rhythmically but can also feedback to central clocks, including the SCN. For instance, both cortisol and thyroid hormones regulate their own rhythmic synthesis by feedback to central brain regions, i.e., the hypothalamus (for cortisol) and pituitary (for both hormones). Several other peripherally produced factors have been proposed to influence the central clock, including fatty acids produced by the adipose tissue and fibroblast growth factor 21, a hormone primarily produced by the liver. Peripheral hormones that signal energy state and hunger also exhibit circadian rhythms. The most extensively studied hormones are leptin, which is released from white adipose tissue cells, and ghrelin, which is released from the upper fundus region of the stomach. Ghrelin also exhibits significant peaks related to anticipated meal timing, which persist for several days of fasting in humans. Circulating rhythms of leptin and ghrelin are disrupted in circadian mutant mice and are also perturbed in humans subjected to circadian misalignment. For instance, Per and Cry mutant mice exhibit severely blunted leptin rhythms, and wild-type mice exposed to jetlag—through repeatedly altered light-dark cycles—show a reduced wake-associated decrease in leptin. Similarly, humans forced to live 28-h days exhibit increased 24-h profiles of ghrelin, and conversely decreased levels of leptin. Ghrelin and leptin signal to several regions of the brain, including integrative appetitive regions of the hypothalamus such as the arcuate and paraventricular region. Through actions in several such central sites, these hormones influence rhythms of food intake and energy homeostasis in a nutrient-dependent manner.
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Role for the Clock in Metabolic Homeostasis
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Circadian control of glucose homeostasis has long been recognized, as early studies demonstrated variation in glucose tolerance and insulin action across the day. For example, oral glucose tolerance is impaired in the evening and afternoon compared with the morning due to a combination of circadian control of both peripheral insulin sensitivity and pancreatic β-cell insulin secretion. Another example is the “dawn phenomenon,” whereby glucose levels peak prior to the onset of activity. Further, destruction of the SCN has been shown to abolish circadian regulation of glucose metabolism in rats, and daily cycles of insulin secretion and glucose tolerance are often perturbed in patients with type 2 diabetes. Changes have also been observed in first-degree relatives of patients with type 2 diabetes, possibly highlighting a key hereditary component of the circadian clock in the pathogenesis of type 2 diabetes.
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Ablating clock genes in mice has revealed a key function for both central and peripheral clocks in regulating energy homeostasis. The circadian system has been shown to regulate rhythmic insulin secretion from the pancreas via both neural signals and hormonal levels (e.g., cortisol and norepinephrine), as well as via cell autonomous clock regulation within the pancreatic β-cell itself. An early observation was that whole-body mutant ClockΔ19/Δ19 mice developed obesity without displaying hyperinsulinemia, a phenomenon that indicated concurrent β-cell failure. This was later confirmed using pancreas- and β-cell–specific Bmal1-deficient mice, which exhibited glucose intolerance, hypoinsulinemia, and impaired glucose-stimulated insulin secretion. The molecular clock within other peripheral tissues such as liver, adipose tissue, and skeletal muscle also regulate circadian fluctuations in insulin sensitivity and glucose disposal, which are highest in the morning and decline towards the evening. Liver-specific Bmal1 mutant studies have revealed liver clock promotion of gluconeogenesis, glycogenolysis, and mitochondrial oxidative metabolism in the sleep/fasting period while promoting glycogen synthesis in the wake/feeding period. Muscle-specific Bmal1 deficient mice display reduced glucose tolerance, concomitant with lower levels of proteins involved in glucose uptake by muscle cells (e.g., the glucose transporter GLUT4). Ablation of the Cry1 and Cry2 repressors in the negative limb of the clock alters glucagon and glucocorticoid signaling in the liver, contributing to hyperglycemia and impaired glucose tolerance in these mutant mice. Together, these genetic studies in mice suggest a role for tissue-specific clocks in the partitioning of energy utilization across the sleep-wake cycle.
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Importantly, peripheral clocks also interact with other environmental factors such as diet and time of feeding. For example, high-fat feeding leads not only to obesity and metabolic syndrome in mice, but also to perturbed clock gene expression across multiple peripheral tissues and a disrupted sleep-wake/fasting-feeding cycle, as revealed by increased activity and feeding during the daytime. Furthermore, mice that are fed a high-fat diet exclusively during the light phase gain significantly more weight than mice that are fed the same diet during the dark period—the active period for mice. Additionally, the metabolic phenotypes arising from ad lib high-fat feeding can be significantly ameliorated by restricting the time of high-fat feeding exclusively to the dark period. Time-restricted feeding can also increase the activity of brown adipose tissue in mice and reduce hepatic glucose production to instead promote beta oxidation of fatty acids. These findings have been corroborated in human interventional studies, which have demonstrated that time-restricted feeding can improve metabolic homeostasis and promote weight loss. Time-restricted feeding may also modulate central regulation of sleep and hunger, as a study found that humans who restricted their food intake to a shorter than ad lib period also consumed less daily calories and reported improved sleep.
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Finally, animal studies have further shown that when the light-dark cycle is disrupted or animals are subjected to conditions mimicking “jetlag”—by artificially advancing or delaying the daily light period—there is desynchronization amongst circadian clocks and subsequent weight gain. Accumulating evidence in humans suggests that circadian misalignment both disrupts and desynchronizes circadian clocks across tissues. Prolonged circadian misalignment using forced desynchrony protocols reduces insulin sensitivity in the pre- and postprandial state. Under such conditions, insulin secretion fails to suppress glucose levels, suggesting inadequate β-cell compensation. Moreover, resting metabolic rate declines significantly both in the awake and sleeping state, altogether providing potential explanations why shift work can increase the risk of obesity, type 2 diabetes, and the metabolic syndrome.
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Human genetic association studies also support a role for clock genes in metabolic homeostasis and beta cell function. Carriers of a certain BMAL1 polymorphism have a greater risk of developing type 2 diabetes, while CLOCK variants have been found to interact with diet, such that variants can have a protective effect on insulin sensitivity in individuals with high monounsaturated fat intake or in individuals provided a low-fat diet. Instead, the minor allele of another CLOCK gene variant has been associated with increased waist circumference, but only in those with high saturated fat intake. Similarly, NPAS2 and BMAL1 variants have been associated with a greater risk of hypertension. Melatonin receptor MTNR1B gene variants, which result in increased expression of MTNR1B, have been associated with elevated fasting blood glucose levels and reduced insulin secretion irrespective of their level of glycemic control, consistent with the known effect of melatonin on insulin secretion and lower insulin secretion in the evening. These association studies highlight the role of the circadian system in metabolism, as well as potential for interactions of external perturbations—such as circadian misalignment—with a protective or adverse genetic profile.
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A large proportion of society recurrently shifts sleep-wake times between working/non-free days and free days. This social jetlag has been increasingly tied to metabolic disruptions, including a greater risk of obesity and type 2 diabetes. As this involves recurrent phase advances and phase delays—like shift work but of smaller magnitude—it is possible that social jetlag also results in perturbed energy expenditure, in combination with disruptions to the circadian rhythm of hunger drive, further increasing the risk of obesity. Repeated shifts in the food- and SCN-driven rhythm of insulin release may similarly over time increase the risk of type 2 diabetes. Shifted feeding rhythms in relation to the sleep-wake cycle and the timing of SCN activity may be causally involved in this pathogenesis. This is exemplified by the disorders known as night-eating syndrome and sleep-related eating disorder. In the former syndrome, a large part of daily calorie consumption occurs in the evening and nighttime hours, and this shifted meal pattern has been associated with a delayed timing of the internal clock. Some evidence exists that these syndromes are associated with obesity. Individuals who report sleeping fewer hours or who are subjected to restricted sleep for a few consecutive days have also been found to consume more calories later in the evening, perhaps explaining why sleep restriction increases the risk of obesity. These associations have also been observed in individuals with later onset of sleep, i.e., evening chronotypes. Night-eating syndrome and later chronotypes have also been linked to type 2 diabetes and may be more common than other eating disorders such as binge-eating disorder. Both conditions have also been found to be associated with impaired glycemic control—such as a greater likelihood of hemoglobin A1c values exceeding 7%—in patients already suffering from type 2 diabetes, emphasizing how proper alignment of internal circadian rhythms with external factors are key contributing factors for long-term metabolic homeostasis.
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Circadian Clocks in Relation to Brain Health and Cognition
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Molecular circadian clocks are present not only within the extra-SCN regions of the brain but also in neurons, astrocytes, and microglia. Emphasizing the functional significance of properly aligned clocks for brain health, shift workers have been found to have decreased grey matter in brain regions involved in memory and executive functions, with more notable effects in individuals who had shorter recovery periods between the onset of each shift work cycle. Adults performing rotating shift work for many years have also been shown to exhibit signs of accelerated cognitive aging. Notably, evidence suggests that these effects may be reversible, as those who have stopped carrying out shift work exhibit normal cognitive performance 5 or more years later.
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Recent studies have also uncovered an important role for perturbed circadian and sleep-wake rhythms in neurodegenerative conditions such as Alzheimer’s disease (AD), Huntington disease (HD), and Parkinson’s disease (PD). Amyloid beta, a key pathognomonic component of AD, normally exhibits circadian fluctuations in the extracellular space in the brain, as well as in the cerebrospinal fluid and plasma in humans, peaking during the active period and falling during sleep. Of note, these daily rhythms of amyloid beta accumulation are dampened in mice that are prone to develop AD; reduced fluctuations in plasma amyloid beta fluctuations have also been noted in older compared with younger individuals. It is believed that removal of amyloid beta (and other neurotoxic substances) during the nighttime sleep period is facilitated by a lymphatic-like system that relies on glial cells (the “glymphatic” system). Whereas the function of this system has been shown to be important in mice, its circadian components and relevance to humans remain to be determined. Consistent with a role for circadian rhythms in the pathogenesis of AD, ablation of core clock genes throughout the brain or within subregions of the brain increases oxidative stress and neuronal cell death, while promoting scarring of brain tissue (astrogliosis). Furthermore, perturbed light-dark cycles increased pathology associated with oxidative stress, and single-nucleotide polymorphisms in Clock and Bmal1 have been associated with increased risk of developing AD.
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Evidence also indicates that the relationship between the circadian/sleep-wake system and AD is bidirectional. For example, patients suffering from AD exhibit several signs of perturbed circadian rhythms, the most prominent of such phenomena being “sundowning,” whereby AD patients become more agitated and exhibit delirium-like symptoms in the afternoon or evening. Studies have furthermore indicated that in severe forms of AD, the circadian rhythm is phase delayed. Aged AD-prone mice also display perturbed sleep-wake patterns, which can be corrected by immunization against amyloid beta or by an orexin antagonist. Further research will help uncover the primary pathogenic contribution of the circadian system, and its independent contribution from perturbed sleep, in conditions like AD. Notably, evidence suggests that interventions that increase daytime light exposure and include melatonin supplementation are able to ameliorate symptoms of AD, presumably by counteracting disrupted circadian rhythms.
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While the relation between shift work and depression has not been extensively studied, disruption of sleep and circadian rhythms and the pathogenesis of depression are intimately interlocked. Clock genes have also been implicated in depression and mood both in animal and human studies. Polymorphisms of genes that regulate sleep and circadian rhythms—for instance, a long gene variant of PER3—have also been linked to bipolar disorder and schizophrenia, while CRY2 and CLOCK gene polymorphisms are associated with seasonal affective disorder, a type of depression arising in the fall and winter months when the levels of sunlight are lowest. Bipolar disorder is furthermore often triggered by circadian disruptions or curtailed sleep. Both bipolar disorder and schizophrenia have been linked to various forms of circadian disruption following disease onset, and a critical component of disease treatment often involves normalizing sleep and sleep-wake rhythms.
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Sleep deprivation by itself is known to reduce alertness, impair decision-making, and increase risk for accidents—after 18–24 h of continuous wakefulness, several skills exhibit the same degree of decline as following mild alcohol intoxication. However, cognitive abilities may suffer even further when sleep restriction is combined with circadian misalignment as in shift work. In one study, participants were subjected to ~33-h long days in parallel with reduced sleep (equivalent to 5.6 h sleep in a 24-h period), yielding a forced desynchrony protocol coupled with sleep loss. When subjects were tested at the nadir of their circadian period, the subjects’ reaction speed dropped almost by an order of magnitude compared with controls. In another study, researchers noted almost a 36% greater incidence of serious medical errors in resident interns who regularly worked 24-h or longer shifts compared with those who were randomly assigned to work up to 16-h long shifts. Furthermore, errors that resulted in patient death were three times more likely to occur in residents working extended hours compared with those who only worked up to 16-h long shifts.
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Circadian Regulation of Gastrointestinal Homeostasis and the Microbiota
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Physiologic aspects of the gastrointestinal (GI) tract exhibit day-night variations that anticipate and prepare for food intake and digestion during the active period. Gastric emptying, as well as colonic motility, are considerably greater during the active phase, as the phasic motor program supporting movement of digested material along the intestine is approximately twice as fast during the day compared with night. Bile acid secretion also exhibits circadian rhythmicity in the intestine, as does absorption and the expression of many nutrient uptake transporters in the intestinal wall, including the main glucose transporter protein SGLT1. The permeability of the intestinal wall also varies throughout the sleep-wake cycle, and mice exposed to chronic sleep fragmentation exhibit increased intestinal permeability, which may enable inflammatory molecules from bacteria to reach the systemic circulation.
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The composition and function of the microbe population living in the intestine (i.e., the gut microbiota) also display circadian rhythmicity, orchestrated by both host circadian clock gene expression and food intake rhythms. Accordingly, circadian disruption, either by environmental or genetic means, perturbs these microbial rhythms, disrupting both bacterial levels and the metabolic functions of the gut microbiota. For example, alterations in the expression and functions of the gut microbiota have been noted in humans exposed to acute jetlag, and evidence suggests that curtailing sleep, which often accompanies shift work and jetlag, can alter the gut microbiota. By increasing local and systemic inflammation, circadian disruption of the gut microbiota may be causally involved in the increased risk of inflammatory bowel disease (Crohn’s disease and ulcerative colitis) and colon cancer in shift workers. Gender-specific differences have also been reported, as female mice display more pronounced microbial rhythms. Interestingly, the gut microbiome has also been shown to influence the rhythms of host tissues, such as the intestine and liver, suggesting that a bidirectional relationship exists between tissues that regulate metabolic processes and the gut microbiome across the sleep-wake cycle. These findings may furthermore have clinical implications, given that the gut microbiome may both directly (in the gut lumen) and indirectly (through host-microbiota interactions) impact pharmacokinetic and pharmacodynamic properties of therapeutic drugs across the 24-h day-night cycle.
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Cardiovascular Health and the Circadian Clock
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An early epidemiologic observation was a greater incidence of myocardial infarction in the morning hours, with the lowest risk during the period preceding sleep. Other cardiovascular outcomes such as sudden cardiac death and syncope also exhibit a daily peak in the morning. Blood pressure (BP) typically peaks around 21:00 h and decreases later during sleep, partially due to a circadian nighttime dip of around 3–6 mmHg in systolic BP and 2–3 mmHg in diastolic BP. A dip in blood pressure of either less than 10% or greater than 20% during normal sleep has been associated with worse cardiovascular prognosis. Heart rate also typically decreases during sleep, while mistimed sleep leads to higher heart rate during their sleep time. Thus, a combination of reasons—which may also involve altered glucocorticoid levels and increased platelet aggregation—may contribute to a greater risk of cardiovascular disease in the morning. Subsequent epidemiologic studies also have demonstrated that shift work increases the risk of dyslipidemia and hypertension, as well as the risk of coronary heart disease, including myocardial infarction. These findings are in line with interventional findings in which circadian misalignment has been induced either by inverting the sleep-wake cycle or by imposing 28-h days on healthy human subjects. These studies have found that circadian misalignment elevates 24-h blood pressure, particularly during sleep. These changes may be causally related to how the autonomic system is regulated during sleep, as evidenced by reduced vagal cardiac control when the sleep-wake cycle is inverted.
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Circadian Disruption and Cancer
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In 2007, the International Agency for Research on Cancer declared that shift work that involves circadian disruption is likely carcinogenic to humans. While evidence for an association between shift work and general cancer incidence is mixed, accruing evidence supports a link between shift work and increased risk of developing colon and breast cancer, as well as having a poorer cancer prognosis. Telomere shortening, a phenomenon in aging that destabilizes the genome, has also been observed in shift workers as well as in individuals suffering from short sleep. This may reduce the ability of damaged or senescent cells to undergo apoptosis, and instead lead to uninhibited cell growth and cancer. An indirect role for the circadian clock has also come from retrospective studies on how cancer risk is related to food timing and duration of the nighttime fast in humans. These studies indicate that by portioning food intake to a restricted period of the day, circadian processes are optimized in a way that confers reduced risk of carcinogenic cell damage. Studies of recurring fasting have also been shown to lower the risk as well as to delay the onset of cancer.
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Experimental genetic evidence has also implicated clock disruption as a factor in tumorigenesis. Genetic loss of Per2 or Bmal1 has been shown to promote lung tumorigenesis, while studies in Per2 mutant mice have also revealed increased radiation-induced lymphoma associated with dysregulation of the cell cycle. However, disruption of the Cry gene in mice has also been implicated in tumor protection due to increased susceptibility to cell death. Thus, while both epidemiologic and experimental evidence suggests a link between circadian disruption and cancer, a full understanding of the role of circadian systems in tumorigenesis remains an area for investigation.
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Circadian Regulation of the Immune System
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Circadian misalignment and sleep restriction both alter population levels of immune cells and decrease the ability of immune cells to produce reactive radicals. Chronic circadian disruption may thereby impair the immune system’s ability to conduct immunosurveillance at the proper time of day and reduce the ability to mount an appropriate response upon exposure of pathogens during the recovery/sleep phase, when the immune system is typically more active. Instead, circadian misalignment increases a range of clinically used inflammatory markers (e.g., C-reactive protein, tumor necrosis factor α, and interleukin 6), and such changes have been noted even when the sleep-wake cycle is only prolonged to a slightly longer than normal 24.6-h day. While similar effects are also observed following acute total sleep deprivation or recurrent partial sleep restriction, circadian misalignment has been found to promote an even more pronounced elevation of such markers. Genetic clock disruption in peritoneal macrophages has also revealed clock control of Toll-like receptor 9, which is responsible for identifying molecules from foreign pathogens. Clock knockout mice also have reduced T cell antigen response, and mice immunized during the day had a stronger T cell response than mice immunized at night, supporting regulation of the immune system by the clock.
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Aging and the Circadian Clock
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Instability in the clock system is an often overlooked hallmark of aging. Aging is associated with a decline in the robustness of intrinsic rhythmic processes at the behavioral, physiologic, and molecular levels in both human and animal models. At the behavioral level, aging leads to reduced and fragmented sleep, dampened locomotor activity and feeding rhythms, and a reduced ability to entrain to light, as old rodents are 20 times less sensitive to the entraining effects of light relative to young animals. Even middle-aged individuals exposed to jetlag also exhibit more symptoms of circadian misalignment, such as more time awake and reduced alertness, compared with young individuals. On a physiologic level, some of the hallmarks of aging are a reduction in amplitude (e.g., flattening of circadian pattern) and a phase-advance (e.g., a shift in the timing of the peak or nadir) in rhythms of the endocrine and neuroendocrine systems, including sleep onset and offset. For example, cortisol, DHEA, and melatonin all have dampened rhythms and are phase-advanced in aging; the combination of such changes may, for instance, contribute to more fragmented sleep and lower levels of restorative slow-wave sleep in aged individuals. Aging also leads to alterations in peptide expression in the SCN (VIP and AVP) and reduced amplitude of rhythms of SCN electrical activity. Further, while the SCN-dependent body temperature rhythm—a generally accepted marker for the integrity of circadian rhythms—peaks in the evening and is lowest in the early morning in young individuals, aged healthy subjects display a phase advance and decrease in circadian amplitude in body temperature rhythms. Indeed, evidence suggests that internal desynchrony between core body temperature rhythms and the sleep-wake cycle may contribute to age-associated circadian alterations. On a molecular level, aging is associated with decreased expression and altered diurnal profiles of several of the core clock genes, including Clock and Bmal1, within both SCN and peripheral tissues such as heart and liver. Interestingly, the acute induction of Per1 in response to light was markedly reduced in the SCN of aged mice compared with young mice, potentially contributing to their delayed response to light entrainment. Mice lacking Bmal1 die prematurely compared with control mice, consistent with premature accumulation of reactive oxygen species. These mice have an accelerated onset of numerous age-related pathologies, including cataracts, sarcopenia, reduced organ size, and decreased hair growth. Instead, deficiency of Cryptochrome, a repressor of the internal clock repressor, has been associated with alterations in liver regeneration, while BMAL1 and PER2 may be important for proper neurogenesis in the hippocampus, a brain region in which adult mammals normally exhibit continuous cell division. Altogether, this suggests that the highly conserved circadian clock is important for regulating a wide range of homeostatic processes, including cell-cycle pathways, that when properly phased to each other promote organismal fitness.
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Measurements of altered circadian rhythms with age may serve as a useful biomarker for aging. An intriguing question is whether the decline in amplitude of rhythms correlates with a decline in function, and importantly whether restoration of these rhythms with age, through either behavioral or pharmacologic intervention, would delay the aging process. Of note, transplantation of the SCN from a young rat into an old rat “rescued” the rhythms of both locomotor activity and corticotropin hormone (CRH), suggesting that the SCN is an important target for age-related changes in clocks. Treatments targeting the SCN may therefore ameliorate some of the deterioration in aged individuals.