The Circadian Clock Machinery During Early Development of Senegalese sole (Solea senegalensis): Effects of Constant Light and Dark Conditions

Circadian rhythms are established very early during vertebrate development. In fish, environmental cues can influence the initiation and synchronization of different rhythmic processes. Previous studies in zebrafish and rainbow trout have shown that circadian oscillation of clock genes represents one of the earliest detectable rhythms in the developing embryo, suggesting their significance in regulating the coordination of developmental processes. In this study, we analyzed the daily expression of the core clock components Per1, Per2, Per3, and Clock during the first several days of Senegalese sole development (0–4 d post fertilization or dpf) under different lighting regimes, with the aim of addressing when the molecular clock first emerges in this species and how it is affected by different photoperiods. Rhythmic expression of the above genes was detected from 0 to 1 dpf, being markedly affected in the next few days by both constant light (LL) and dark (DD) conditions. A gradual entrainment of the clock machinery was observed only under light-dark (LD) cycles, and robust rhythms with increased amplitudes were established by 4 dpf for all clock genes currently studied. Our results show the existence of an embryonic molecular clock from the 1st d of development in Senegalese sole and emphasize the significance of cycling LD conditions when raising embryos and early larvae. (Author correspondence: munoz.cueto@uca.es; carlos.pendon@uca.es)     

Cryptochrome restores dampened circadian rhythms and promotes healthspan in aging Drosophila

Circadian clocks generate daily rhythms in molecular, cellular, and physiological functions providing temporal dimension to organismal homeostasis. Recent evidence suggests two‐way relationship between circadian clocks and aging. While disruption of the circadian clock leads to premature aging in animals, there is also age‐related dampening of output rhythms such as sleep/wake cycles and hormonal fluctuations. Decay in the oscillations of several clock genes was recently reported in aged fruit flies, but mechanisms underlying these age‐related changes are not understood. We report that the circadian light–sensitive protein CRYPTOCHROME (CRY) is significantly reduced at both mRNA and protein levels in heads of old Drosophila melanogaster. Restoration of CRY using the binary GAL4/UAS system in old flies significantly enhanced the mRNA oscillatory amplitude of several genes involved in the clock mechanism. Flies with CRY overexpressed in all clock cells maintained strong rest/activity rhythms in constant darkness late in life when rhythms were disrupted in most control flies. We also observed a remarkable extension of healthspan in flies with elevated CRY. Conversely, CRY‐deficient mutants showed accelerated functional decline and accumulated greater oxidative damage. Interestingly, overexpression of CRY in central clock neurons alone was not sufficient to restore rest/activity rhythms or extend healthspan. Together, these data suggest novel anti‐aging functions of CRY and indicate that peripheral clocks play an active role in delaying behavioral and physiological aging.     

Association Study of a Variable-Number Tandem Repeat Polymorphism in the Clock Gene PERIOD3 and Chronotype in Norwegian University Students

Circadian rhythms are endogenously generated cycles involving physiological parameters, such as core body temperature, hormone levels, blood pressure, sleep, and metabolism, with a period length of around 24?h. The circadian clock in mammals is regulated by a set of clock genes that are functionally linked together, and polymorphisms in clock genes could be associated with differences in circadian rhythms. A variable-number tandem repeat (VNTR) in the human clock gene PERIOD3 (PER3) has been suggested to correlate with a morning (lark) versus evening (owl) chronotype as well as with the circadian rhythm sleep disorder “delayed sleep phase disorder” (DSPD). The authors examined 432 healthy Norwegian university students in search of further support for an association between the PER3 polymorphism and diurnal preference. The Horne-Östberg Morningness-Eveningness Questionnaire (MEQ) and Preferences Scale (PS) were used to evaluate subjective chronotype. DNA samples were genotyped with respect to the 4-repeat and 5-repeat alleles of the VNTR PER3 polymorphism, and the genotype distribution was 192 (4-4), 191 (4-5), and 49 (5-5). The authors estimated that the power to detect an association of the 4-allele with preference for morningness or eveningness was 75%. The authors found no association between the PER3 clock gene and chronotype, indicating that the proposed role of PER3 needs further clarification. (Author correspondence: teresa.osland@med.uib.no)     

Circadian Oscillations of Molecular Clock Components in the Cerebellar Cortex of the Rat

The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum. (Author correspondence: mrath@sund.ku.dk)     

DIURNAL EXPRESSION OF CLOCK GENES IN PINEAL GLAND AND BRAIN AND PLASMA LEVELS OF MELATONIN AND CORTISOL IN ATLANTIC SALMON PARR AND SMOLTS

In Atlantic salmon, the preadaptation to a marine life, i.e., parr-smolt transformation, and melatonin production in the pineal gland are regulated by the photoperiod. However, the clock genes have never been studied in the pineal gland of this species. The aim of the present study was to describe the diurnal expression of clock genes (Per1-like, Cry2, and Clock) in the pineal gland and brain of Atlantic salmon parr and smolts in freshwater, as well as plasma levels of melatonin and cortisol. By employing an out-of-season smolt production model, the parr-smolt transformation was induced by subjecting triplicate groups of parr to 6 wks (wks 0 to 6) under a 12?h:12?h light-dark (LD) regime followed by 6 wks (wks 6 to 12) of continuous light (LL). The measured clock genes in both pineal gland and brain and the plasma levels of melatonin and cortisol showed significant daily variations in parr under LD in wk 6, whereas these rhythms were abolished in smolts under LL in wk 12. In parr, the pineal Per1-like and Cry2 expression peaked in the dark phase, whereas the pineal Clock expression was elevated during the light phase. Although this study presents novel findings on the clock gene system in the teleost pineal gland, the role of this system in the regulation of smoltification needs to be studied in more detail. (Author correspondence: ti.hu@hotmail.com)     

Low-Carbohydrate,High-Protein Diet Affects Rhythmic Expression of Gluconeogenic Regulatory and Circadian Clock Genes in Mouse Peripheral Tissues

Recent studies have demonstrated that metabolic changes in mammals induce feedback regulation of the circadian clock. The present study evaluates the effects of a low-carbohydrate high-protein diet (HPD) on circadian behavior and peripheral circadian clocks in mice. Circadian rhythms of locomotor activity and core body temperature remained normal in mice fed with the HPD diet (HPD mice), suggesting that it did not affect the central clock in the hypothalamus. Two weeks of HPD feeding induced mild hypoglycemia without affecting body weight, although these mice consumed more calories than mice fed with a normal diet (ND mice). Plasma insulin levels were increased during the inactive phase in HPD mice, but increased twice, beginning and end of the active phase, in ND mice. Expression levels of the key gluconeogenic regulatory genes PEPCK and G6Pase were significantly induced in the liver and kidneys of HPD mice. The HPD appeared to induce peroxisome proliferator-activated receptor α (PPARα) activation, since mRNA expression levels of PPARα and its typical target genes, such as PDK4 and Cyp4A10, were significantly increased in the liver and kidneys. Circadian mRNA expression of clock genes, such as BMAL1, Cry1, NPAS2, and Rev-erbα, but not Per2, was significantly phase-advanced, and mean expression levels of BMAL1 and Cry1 mRNAs were significantly elevated, in the liver and kidneys of HPD mice. These findings suggest that a HPD not only affects glucose homeostasis, but that it also advances the molecular circadian clock in peripheral tissues. (Author correspondence: k-ooishi@aist.go.jp)     

Reduced Histone H3K9 Acetylation of Clock Genes and Abnormal Glucose Metabolism in ob/ob Mice

Recent chronobiological studies found significant correlation between lack of clock function and metabolic abnormalities. We previously showed that clock gene expressions were dampened in the peripheral tissues of obese and diabetic ob/ob mice. However, the molecular mechanism of the disturbance remained to be determined. In this study, we demonstrated for the first time that acetylation levels of histone H3 lysine 9 (H3K9) at the promoter regions of clock genes, such as Dbp, Per2, and Bmal1, in the adipose tissue of ob/ob mice were significantly reduced compared with those of its control C57BL/6J mice. Treatment with histone deacetylase (HDAC) inhibitors increased Dbp, but not Per2 or Bmal1, mRNA expression in adipose tissue, and it decreased blood glucose in these animals. In addition, 2-deoxyglucose uptake activity was significantly suppressed by silencing Dbp expression in cultured adipocytes. These results suggest that reduced H3K9 acetylation and subsequent decreased mRNA expression of the Dbp gene in adipose tissue are involved in the mechanism of development of abnormal glucose metabolism in ob/ob mice. (Author correspondence: akiofuji@jichi.ac.jp)     

Cyclic Presence and Absence of Conspecifics Alters Circadian Clock Phase But Does Not Entrain the Locomotor Activity Rhythm of the Fruit Fly Drosophila melanogaster

Circadian clocks use a wide range of environmental cues, including cycles of light, temperature, food, and social interactions, to fine-tune rhythms in behavior and physiology. Although social cues have been shown to influence circadian clocks of a variety of organisms including the fruit fly Drosophila melanogaster, their mechanism of action is still unclear. Here, the authors report the results of their study aimed at investigating if daily cycles of presence and absence (PA) of conspecific male visitors are able to entrain the circadian locomotor activity rhythm of male hosts living under constant darkness (DD). The results suggest that PA cycles may not be able to entrain circadian locomotor activity rhythms of Drosophila. The outcome does not change when male hosts are presented with female visitors, suggesting that PA cycles of either sex may not be effective in bringing about stable entrainment of circadian clocks in D. melanogaster. However, in hosts whose clock phase has already been set by light/dark (LD) cycles, daily PA cycles of visitors can cause measurable change in the phase of subsequent free-running rhythms, provided that their circadian clocks are labile. Thus, the findings of this study suggest that D. melanogaster males may not be using cyclic social cues as their primary zeitgeber (time cue) for entrainment of circadian clocks, although social cues are capable of altering the phase of their circadian rhythms. (Author correspondence: vsharma@jncasr.ac.in, vksharmas@gmail.com)     

ASSOCIATIONS OF METABOLIC PARAMETERS AND ETHANOL CONSUMPTION WITH MESSENGER RNA EXPRESSION OF CLOCK GENES IN HEALTHY MEN

Recent studies suggest that the impairment of circadian clock function causes various pathological conditions, such as obesity, diabetes, and alcoholism, and an altered mRNA expression of clock genes was found under these conditions. However, it remains to be determined whether clock gene expression varies depending on metabolic conditions even in healthy people. To address this issue, we investigated the associations of metabolic parameters and alcohol consumption with mRNA expression of clock genes (CLOCK, BMAL1, PER1, PER2, and PER3) in peripheral blood cells obtained from 29 healthy non-obese elderly men (age 51–78 yrs) who adhered to a regular sleep-wake routine, through a single time-of-day venous blood sampling at ～09:00?h. There were significant correlations between (1) waist circumference and mRNA level of PER1 (r?=?0.43), (2) plasma glucose concentration and PER2 (r?=?0.50), (3) ethanol consumption and BMAL1 (r?=?0.43), and (4) serum γ-GTP concentration (a sensitive marker of alcohol consumption) and PER2 (r?=?0.40). These results suggest mRNA expression of clock genes is associated with obesity, glucose tolerance, and ethanol consumption even in healthy people. (Author correspondence: akiofuji@jichi.ac.jp)     

Comparison of β-Adrenergic and Glucocorticoid Signaling on Clock Gene and Osteoblast-Related Gene Expressions in Human Osteoblast

Most living organisms exhibit circadian rhythms that are generated by endogenous circadian clocks, the master one being present in the suprachiasmatic nuclei (SCN). Output signals from the SCN are believed to transmit standard circadian time to peripheral tissue through sympathetic nervous system and humoral routes. Therefore, the authors examined the expression of clock genes following treatment with the β-adrenergic receptor agonist, isoprenaline, or the synthetic glucocorticoid, dexamethasone, in cultured human osteoblast SaM-1 cells. Cells were treated with 10^?6 M isoprenaline or 10^?7 M dexamethasone for 2?h and gene expressions were determined using real-time polymerase chain reaction (PCR) analysis. Treatment with isoprenaline or dexamethasone induced the circadian expression of clock genes human period 1 (hPer1), hPer2, hPer3, and human brain and muscle Arnt-like protein 1 (hBMAL1). Isoprenaline or dexamethasone treatment immediately increased hPer1 and hPer2 and caused circadian oscillation of hPer1 and hPer2 with three peaks within 48?h. hPer3 expression had one peak after isoprenaline or dexamethasone treatment. hBMAL expression had two peaks after isoprenaline or dexamethasone treatment, the temporal pattern being in antiphase to that of the other clock genes. Dexamethasone treatment delayed the oscillation of all clock genes for 2–6?h compared with isoprenaline treatment. The authors also examined the expression of osteoblast-related genes hα-1 type I collagen (hCol1a1), halkaline phosphatase (hALP), and hosteocalcin (hOC). Isoprenaline induced oscillation of hCol1a1, but not hALP and hOC. On the other hand, dexamethasone induced oscillation of hCol1a1 and hALP, but not hOC. Isoprenaline up-regulated hCol1a1 expression, but dexamethasone down-regulated hCol1a1 and hALP expression in the first phase. (Author correspondence: togariaf@dpc.agu.ac.jp)     

Circadian Modulation of Acute Alcohol Sensitivity But Not Acute Tolerance in Drosophila

An increased understanding of the factors affecting behavioral and neurological responses to alcohol and alcohol physiology is necessary given the tremendous toll alcohol abuse and alcoholism exert on individuals and society. At the behavioral and molecular levels, the response to alcohol appears remarkably conserved from Drosophila to humans, suggesting that investigations across model species can provide insight into the identification of common modulatory factors. We investigated the interaction between the circadian clock and alcohol sensitivity, alcohol tolerance, and alcohol absorbance in Drosophila melanogaster. Using a loss-of-righting reflex (LoRR) assay, we found that flies exhibit a circadian rhythm in the LoRR, with the greatest sensitivity to alcohol occurring from mid to late night, corresponding to the flies' inactive phase. As predicted, a circadian rhythm in the LoRR was absent in circadian mutant flies and under conditions in which the circadian clock was nonfunctional. Circadian modulation of the response to alcohol was not due to circadian regulation of alcohol absorbance. Similar to other animals, Drosophila develop acute and chronic tolerance to alcohol upon repeat exposures. We found that the circadian clock did not modulate the development of acute alcohol tolerance measured as the difference in sensitivity to alcohol between naïve and pre-exposed flies. Thus, the circadian clock modulates some, but not all, of the behavioral responses to alcohol exposure, suggesting that specific mechanisms underlie the observed circadian modulation of LoRR rather than global cellular circadian regulation. This study provides valuable new insights in our understanding of the circadian modulation of alcohol-induced behaviors that ultimately could facilitate preventative measures in combating alcohol abuse and alcoholism. (Author correspondence: lyons@bio.fsu.edu)     

Expression of Clock Genes in Human Subcutaneous and Visceral Adipose Tissues

Disrupted circadian rhythms are associated with obesity and metabolic alterations, but little is known about the participation of peripheral circadian clock machinery in these processes. The aim of the present study was to analyze RNA expression of clock genes in subcutaneous (SAT) and visceral (VAT) adipose tissues of male and female subjects in AM (morning) and PM (afternoon) periods, and its interactions with body mass index (BMI). Ninety-one subjects (41?±?11 yrs of age) presenting a wide range of BMI (21.4 to 48.6?kg/m²) were included. SAT and VAT biopsies were obtained from patients undergoing abdominal surgeries. Clock genes expressions were evaluated by qRT-PCR. The only clock gene that showed higher expression (p?<?.0001) in SAT in comparison to VAT was PER1 of female (372%) and male (326%) subjects. Different patterns of expression between the AM and PM periods were observed, in particular REV-ERBα, which was reduced (p?<?.05) at the PM period in SAT and VAT of both women and men (women: ～53% lower; men: ～78% lower), whereas CLOCK expression was not altered. Relationships between clock genes were different in SAT vs. VAT. BMI was negatively correlated with SATPER1 (r?=??.549; p?=?.001) and SATPER2 (r?=??.613; p?=?.0001) and positively with VATCLOCK (r?=?.541; p?=?.001) and VATBMAL1 (r?=?.468; p?=?.007) only in women. These data suggest that the circadian clock machinery of adipose tissue depots differs between female and male subjects, with a sex-specific effect observed for some genes. BMI correlated with clock genes, but at this moment it is not possible to establish the cause-effect relationship. (Author correspondence: mzanquetta@gmail.com)