BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis

Circadian timing is generated through a unique series of autoregulatory interactions termed the molecular clock. Behavioral rhythms subject to the molecular clock are well characterized. We demonstrate a role for Bmal1 and Clock in the regulation of glucose homeostasis. Inactivation of the known clock components Bmal1 (Mop3) and Clock suppress the diurnal variation in glucose and triglycerides. Gluconeogenesis is abolished by deletion of Bmal1 and is depressed in Clock mutants, but the counterregulatory response of corticosterone and glucagon to insulin-induced hypoglycaemia is retained. Furthermore, a high-fat diet modulates carbohydrate metabolism by amplifying circadian variation in glucose tolerance and insulin sensitivity, and mutation of Clock restores the chow-fed phenotype. Bmal1 and Clock, genes that function in the core molecular clock, exert profound control over recovery from insulin-induced hypoglycaemia. Furthermore, asynchronous dietary cues may modify glucose homeostasis via their interactions with peripheral molecular clocks.     

Role of Sleep Restriction in Daily Rhythms of Expression of Hypothalamic Core Clock Genes in Mice

Lack of sleep time is a menace to modern people, and it leads to chronic diseases and mental illnesses. Circadian processes control sleep, but little is known about how sleep affects the circadian system. Therefore, we performed a 28-day sleep restriction (SR) treatment in mice. Sleep restriction disrupted the clock genes’ circadian rhythm. The circadian rhythms of the Cry1 and Per1/2/3 genes disappeared. The acrophase of the clock genes (Bmal1, Clock, Rev-erbα, and Rorβ) that still had a circadian rhythm was advanced, while the acrophase of negative clock gene Cry2 was delayed. Clock genes’ upstream signals ERK and EIFs also had circadian rhythm disorders. Accompanied by changes in the central oscillator, the plasma output signal (melatonin, corticosterone, IL-6, and TNF-α) had an advanced acrophase. While the melatonin mesor was decreased, the corticosterone, IL-6, and TNF-α mesor was increased. Our results indicated that chronic sleep loss could disrupt the circadian rhythm of the central clock through ERK and EIFs and affect the output signal downstream of the core biological clock.     

The choroid plexus harbors a circadian oscillator modulated by estrogens

The suprachiasmatic nucleus (SCN) of the hypothalamus is considered the master circadian oscillator in mammals. However, extra-SCN structures in the brain also display daily rhythms. Recently, we have demonstrated that the choroid plexus (CP) expresses core clock genes that are subjected to circadian regulation in a sex-dependent manner. By using CP explants cultured from female knock-in mice carrying the Period-luciferase transgene, we show that CP exhibits endogenous circadian rhythms of PERIOD2::LUCIFERASE expression. Furthermore, we demonstrate that estrogen declines following ovariectomy modulates the daily rhythm expression of Bmal1, Per1 and Per2 in female rat CP, corroborating data obtained in experiments where rat CP epithelial cell (CPEC) cultures were incubated with 17β-estradiol (E2). The molecular mechanism underlying these effects was also investigated, and we provide evidence that the estrogen receptor (ER) mediates the response of clock genes to E2.

In conclusion, our study proves that the CP harbors a circadian oscillator that is modulated by estrogens and demonstrates that E2 regulation occurs through an estrogen-receptor-dependent mechanism.     

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.     

Timed high-fat diet in the evening affects the hepatic circadian clock and PPARα-mediated lipogenic gene expressions in mice

A long-term high-fat diet may result in a fatty liver. However, whether or not high-fat diets affect the hepatic circadian clock is controversial. The objective of this study is to investigate the effects of timed high-fat diet on the hepatic circadian clock and clock-controlled peroxisome proliferator-activated receptor (PPAR) α-mediated lipogenic gene expressions. Mice were orally administered high-fat milk in the evening for 4 weeks. The results showed that some hepatic clock genes, such as Clock, brain-muscle-Arnt-like 1 (Bmal1), Period 2 (Per2), and Cryptochrome 2 (Cry2) exhibited obvious changes in rhythms and/or amplitudes. Alterations in the expression of clock genes, in turn, further altered the circadian rhythm of PPARα expression. Among the PPARα target genes, cholesterol 7α-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase, low-density lipoprotein receptor, lipoprotein lipase, and diacylglycerol acyltransferase (DGAT) showed marked changes in rhythms and/or amplitudes. In particular, significant changes in the expressions of DGAT and CYP7A1 were observed. The effects of a high-fat diet on the expression of lipogenic genes in the liver were accompanied by increased hepatic cholesterol and triglyceride levels. These results suggest that timed high-fat diets at night could change the hepatic circadian expressions of clock genes Clock, Bmal1, Per2, and Cry2 and subsequently alter the circadian expression of PPARα-mediated lipogenic genes, resulting in hepatic lipid accumulation.     

Clock genes regulate neurogenic transcription factors,including NeuroD1, and the neuronal differentiation of adult neural stem/progenitor cells

The circadian clock system plays multiple roles in our bodies, and clock genes are expressed in various brain regions, including the lateral subventricular zone (SVZ) where neural stem/progenitor cells (NSPCs) persist and postnatal neurogenesis continues. However, the functions of clock genes in adult NSPCs are not well understood. Here, we first investigated the expression patterns of Clock and Bmal1 in the SVZ by immunohistochemistry and then verified how the expression levels of 17 clock and clock-related genes changed during differentiation of cultured adult NSPCs using quantitative RT-PCR. Finally, we used RNAi to observe the effects of Clock and Bmal1 on neuronal differentiation. Our results revealed that Clock and Bmal1 were expressed in the SVZ and double-stained with the neural progenitor marker Nestin and neural stem marker GFAP. In cultured adult NSPCs, the clock genes changed their expression patterns during differentiation, and interestingly, Bmal1 started endogenous oscillation. Moreover, gene silencing of Clock or Bmal1 by RNAi decreased the percentages of neuronal marker Map2-positive cells and expression levels of NeuroD1 mRNA. These findings suggest that clock genes are involved in the neuronal differentiation of adult NSPCs and may extend our understanding of various neurological/psychological disorders linked to adult neurogenesis and circadian rhythm.     

Facilitated physiological adaptation to prolonged circadian disruption through dietary supplementation with essence of chicken

Synchrony between circadian and metabolic processes is critical to the maintenance of energy homeostasis. Studies on essence of chicken (EC), a chicken meat extract rich in proteins, amino acids and peptides, showed its effectiveness in alleviating fatigue and promoting metabolism. A recent study revealed that it facilitated the re-entrainment of clock genes (Bmal1, Cry1, Dec1, Per1 and Per2) in the pineal gland and liver in a rat model of circadian disruption. Here, we investigated the role of EC-facilitated circadian synchrony in the maintenance of the energy homeostasis using a mouse model of prolonged circadian disruption. Prolonged circadian disruption (12 weeks) resulted in hepatic maladaptation, manifested by a mild but significant (p?<?0.05) hepatomegaly, accompanied by disturbed hepatic lipid metabolism and liver injury (indicated by increased circulating hepatic enzymes). Evidently, there was marked elevations of hepatic inflammatory mediators (interleukin-1beta and interleukin-6), suggesting an underlying inflammation leading to the hepatic injury and functional impairment. Importantly, the disruption paradigm caused the decoupling between key metabolic regulators (e.g. mTOR and AMPK) and hepatic clock genes (Per1, Cry1, Dec1, Bmal1). Further, we showed that the loss of circadian synchrony between the master and hepatic clock genes (Per1, Cry1, Dec1, Bmal1) could be the underlying cause of the maladaptation. When supplemented with EC, the functional impairment and inflammation were abolished. The protective effects could be linked to its effectiveness in maintaining the synchrony between the master and hepatic clocks, and the resultant improved coupling of the circadian oscillators (Per1, Cry1, Dec1, Bmal1) and metabolic regulators (mTOR, AMPK). Overall, EC supplementation promoted the physiological adaptation to the prolonged circadian disruption through facilitation of endogenous circadian synchrony and the coupling of circadian oscillators and metabolic regulators. This forms an important basis for further elucidation of the physiological benefits of EC-facilitated circadian synchrony.     

Circadian rhythm gene expression and daily melatonin levels vary in athletes and sedentary males

The circadian rhythm is a 24-h cycle in which cells control metabolic and physiological processes throughout the day. In this study, we compared the expression patterns of major circadian rhythm-related genes: from blood of Bmal1, Ror-α, Cry1, Per2, Per1, and Nr1d1. In addition, changes in patterns of melatonin levels were observed in 16 subjects, eight males rugby players and eight males who did not exercise regularly. Blood was collected at 6:00, 10:00, 18:00, and 22:00. Bmal1, Ror-α, Cry1, Per2 (p < 0.001), Per1 (p < 0.01), and Nr1d1 (p < 0.05) genes related to circadian rhythm was higher in rugby players than in sedentary males. However, melatonin levels were higher in sedentary males than in rugby players (p < 0.05). These results indicate that long-term exercise in athletes can increase the expression of genes related to circadian rhythm and these may have an effect on daily melatonin levels as well.     

The Luteinizing Hormone Surge Regulates Circadian Clock Gene Expression in the Chicken Ovary

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors’ previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary. (Author correspondence: stischkau@siumed.edu)