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)     

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)     

Peripheral circadian clocks are diversely affected by adrenalectomy

Glucocorticoids are considered to synchronize the rhythmicity of clock genes in peripheral tissues; however, the role of circadian variations of endogenous glucocorticoids is not well defined. In the present study, we examined whether peripheral circadian clocks were impaired by adrenalectomy. To achieve this, we tested the circadian rhythmicity of core clock genes (Bmal1, Per1-3, Cry1, RevErbα, Rora), clock-output genes (Dbp, E4bp4) and a glucocorticoid- and clock-controlled gene (Gilz) in liver, jejunum, kidney cortex, splenocytes and visceral adipose tissue (VAT). Adrenalectomy did not affect the phase of clock gene rhythms but distinctly modulated clock gene mRNA levels, and this effect was partially tissue-dependent. Adrenalectomy had a significant inhibitory effect on the level of Per1 mRNA in VAT, liver and jejunum, but not in kidney and splenocytes. Similarly, adrenalectomy down-regulated mRNA levels of Per2 in splenocytes and VAT, Per3 in jejunum, RevErbα in VAT and Dbp in VAT, kidney and splenocytes, whereas the mRNA amounts of Per1 and Per2 in kidney and Per3 in VAT and splenocytes were up-regulated. On the other hand, adrenalectomy had minimal effects on Rora and E4bp4 mRNAs. Adrenalectomy also resulted in decreased level of Gilz mRNA but did not alter the phase of its diurnal rhythm. Collectively, these findings suggest that adrenalectomy alters the mRNA levels of core clock genes and clock-output genes in peripheral organs and may cause tissue-specific modulations of their circadian profiles, which are reflected in changes of the amplitudes but not phases. Thus, the circulating corticosteroids are necessary for maintaining the high-amplitude rhythmicity of the peripheral clocks in a tissue-specific manner.     

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)     

Differential Roles of Breakfast and Supper in Rats of a Daily Three-Meal Schedule Upon Circadian Regulation and Physiology

The timing of meals has been suggested to play an important role in circadian regulation and metabolic health. Three meals a day is a well-established human feeding habit, which in today's lifestyle may or may not be followed. The aim of this study was to test whether the absence of breakfast or supper significantly affects the circadian system and physiological function. The authors developed a rat model for their daily three meals study, whereby animals were divided into three groups (three meals, TM; no first meal, NF; no last meal, NL) all fed with the same amount of food every day. Rats in the NF group displayed significantly decreased levels of plasma triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and glucose in the activity phase, accompanied by delayed circadian phases of hepatic peripheral clock and downstream metabolic genes. Rats in the NL group showed lower concentration of plasma TC, HDL-C, and glucose in the rest phase, plus reduced adipose tissue accumulation and body weight gain. Real-time polymerase chain reaction (PCR) analysis indicated an attenuated rhythm in the food-entraining pathway, including down-regulated expression of the clock genes Per2, Bmal1, and Rev-erbα, which may further contribute to the delayed and decreased expression of FAS in lipogenesis in this group. Our findings are consistent with the conclusion that the daily first meal determines the circadian phasing of peripheral clocks, such as in the liver, whereas the daily last meal tightly couples to lipid metabolism and adipose tissue accumulation, which suggests differential physiological effects and function of the respective meal timings. (Author correspondence: azwfu2003@yahoo.com.cn)     

Indirect Effects of Glucagon-Like Peptide-1 Receptor Agonist Exendin-4 on the Peripheral Circadian Clocks in Mice

Circadian clocks in peripheral tissues are powerfully entrained by feeding. The mechanisms underlying this food entrainment remain unclear, although various humoral and neural factors have been reported to affect peripheral clocks. Because glucagon-like peptide-1 (GLP-1), which is rapidly secreted in response to food ingestion, influences multiple humoral and neural signaling pathways, we suggest that GLP-1 plays a role in the food entrainment of peripheral clocks. To test this, we compared the effects of exendin-4, a GLP-1 receptor agonist, on mRNA expression of the clock genes (Clock, Bmal1, Nr1d1, Per1, Per2, and Cry1) with those of refeeding. In addition, we investigated whether exendin-4 could affect the rhythms of the peripheral clocks. In male C57BL/6J mice, although refeeding rapidly (within 2 h) altered mRNA levels of Per1 and Per2 in the liver and that of Per1 in adipose tissue, a single i.p. injection of exendin-4 did not cause such changes. However, unlike the GLP-1 receptor antagonist exendin-(9–39), exendin-4 significantly influenced Per1 mRNA levels in the liver at 12 h after injection. Moreover, pretreatment with exendin-4 affected the rapid-feeding-induced change in Per1 not only in the liver, but also in adipose tissue, without effect on food intake. Furthermore, during light-phase restricted feeding, repeated dosing of exendin-4 at the beginning of the dark phase profoundly influenced both the food intake and daily rhythms of clock gene expression in peripheral tissues. Thus, these results suggest that exendin-4 modulates peripheral clocks via multiple mechanisms different from those of refeeding.     

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 ).     

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)     

Free Access to a Running-Wheel Advances the Phase of Behavioral and Physiological Circadian Rhythms and Peripheral Molecular Clocks in Mice

Behavioral and physiological circadian rhythms are controlled by endogenous oscillators in animals. Voluntary wheel-running in rodents is thought to be an appropriate model of aerobic exercise in humans. We evaluated the effects of chronic voluntary exercise on the circadian system by analyzing temporal profiles of feeding, core body temperature, plasma hormone concentrations and peripheral expression of clock and clock-controlled genes in mice housed under sedentary (SED) conditions or given free access to a running-wheel (RW) for four weeks. Voluntary wheel-running activity advanced the circadian phases of increases in body temperature, food intake and corticosterone secretion in the mice. The circadian expression of clock and clock-controlled genes was tissue- and gene-specifically affected in the RW mice. The temporal expression of E-box-dependent circadian clock genes such as Per1, Per2, Nr1d1 and Dbp were slightly, but significantly phase-advanced in the liver and white adipose tissue, but not in brown adipose tissue and skeletal muscle. Peak levels of Per1, Per2 and Nr1d1 expression were significantly increased in the skeletal muscle of RW mice. The circadian phase and levels of hepatic mRNA expression of the clock-controlled genes that are involved in cholesterol and fatty acid metabolism significantly differed between SED and RW mice. These findings indicated that endogenous clock-governed voluntary wheel-running activity provides feedback to the central circadian clock that systemically governs behavioral and physiological rhythms.     

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)