Summary. Background: Most physiologic processes exhibit diurnal fluctuations controlled by the circadian regulation of sleep–wake behavior and feeding cycles. In addition, many cell types express endogenous circadian rhythms that affect cell‐specific processes. Independent reports support the hypothesis that thrombopoietin (TPO) is under circadian control.

Objectives: The current study tested the hypothesis that CLOCK, a circadian transcription factor, may regulate Thpo, the gene encoding TPO.

Methods: Circadian gene expression patterns were analyzed in mice and in human cell lines, Small interfering RNA was used to knock down CLOCK expression in cell lines, and gene expression was also examined in ClockΔ19/Δ19 mutant mice.

Results: It was found that there was a diurnal rhythm in the expression of Thpo in vivo in mice, and that this was associated with concomitant rhythms of protein abundance. Thpo was rhythmically expressed in human cell lines, consistent with the gene being directly or indirectly regulated by the circadian clock. Silencing of CLOCK in the Huh7 human hepatoma cell line led to a significant reduction in the rhythmicity of Thpo expression. The expression of Mpl in murine marrow also displayed diurnal rhythmicity in vivo. In ClockΔ19/Δ19 mutant mice, Thpo and Mpl expression was disrupted and there was an increase in the number of mature megakaryocytes, but no change in the ploidy distribution within the megakaryocyte population.

Conclusions: These findings establish that Clock regulates Thpo and Mpl expression in vivo, and demonstrate an important link between the body’s circadian timing mechanisms and megakaryopoiesis

Circadian (c. 24 h) rhythms of physiology are entrained to either the environmental light‐dark cycle or the timing of food intake. In the current work the hypothesis that rhythms of platelet turnover in mammals are circadian and entrained by food intake was explored in mice. Mice were entrained to 12 h light‐dark cycles and given either ad libitum (AL) or restricted access (RF) to food during the light phase. Blood and megakaryocytes were then collected from mice every 4 h for 24 h. It was found that total and reticulated platelet numbers, plasma thrombopoietin (TPO) concentration and the mean size of mature megakaryocytes were circadian but not entrained by food intake. In contrast, a circadian rhythm in the expression of Arnt1 in megakaryocytes was entrained by food. Although not circadian, the expression in megakaryocytes of Nfe2, Gata1, Itga2b and Tubb1 expression was downregulated by RF, whereas Ccnd1 was not significantly affected by the feeding protocol. It is concluded that circadian rhythms of total platelet number, reticulated platelet number and plasma TPO concentration are entrained by the light‐dark cycle rather than the timing of food intake. These findings imply that circadian clock gene expression regulates platelet turnover in mammals

Circadian rhythms control a vast array of biological processes in a broad spectrum of organisms. The contribution of circadian rhythms to the development of megakaryocytes and the regulation of platelet biology has not been defined. This study tested the hypothesis that murine megakaryocytes exhibit hallmarks of circadian control. Mice expressing a PER2::LUCIFERASE circadian reporter protein and C57BI/6 mice were used to establish if megakaryocytes expressed circadian genes in vitro and in vivo. Mice were also subjected to 3 weeks on a restricted feeding regime to separate food-entrained from light-entrained circadian rhythms. Quantitative real time polymerase chain reaction (PCR), flow cytometry and imunohistochemistry were employed to analyse gene expression, DNA content and cell-cycle behavior in megakaryocytes collected from mice over a 24-h period. Megakaryocytes exhibited rhythmic expression of the clock genes mPer2 and mBmal1 and circadian rhythms in megakaryopoiesis. mPer2 and mBmal1 expression phase advanced 8 h to coincide with the availability of food; however, food availability had a more complex effect on megakaryopoiesis, leading to a significant overall increase in megakaryocyte ploidy levels and cell-cycle activity. Normal megakaryopoiesis requires synchrony between food- and light-entrained circadian oscillators.

Circadian (∼24 hours) clocks are ubiquitous in nature and are important regulators of behaviour, physiology and metabolism. Circadian clocks can synchronise biological processes with environmental cycles, buffer biological systems to maintain homeostasis and partition mutually antagonistic processes to different temporal spaces within the daily cycle. Clocks act cell-autonomously (intrinsically) and systemically (extrinsically) to coordinate whole organism biology and there is epidemiological evidence indicating that chronic disruption of behavioural rhythms increases the risk of developing cancer and cardiovascular disease. Although the genetic mechanism of the mammalian clock has been largely deciphered, the physiological relevance of clocks often remains elusive. Findings from humans and animal models suggest that the circadian clock and diurnal rhythms have an important role in megakaryopoiesis and the risk of a cardiovascular event. This short review will introduce the mammalian circadian clock and discuss how circadian clocks and diurnal rhythms influence platelet production and function.

Background

A complex relationship exists between diet and sleep but despite its impact on human health, this relationship remains uncharacterized and poorly understood. Drosophila melanogaster is an important model for the study of metabolism and behaviour, however the effect of diet upon Drosophila sleep remains largely unaddressed.

Methodology/Principal Findings

Using automated behavioural monitoring, a capillary feeding assay and pharmacological treatments, we examined the effect of dietary yeast and sucrose upon Drosophila sleep-wake behaviour for three consecutive days. We found that dietary yeast deconsolidated the sleep-wake behaviour of flies by promoting arousal from sleep in males and shortening periods of locomotor activity in females. We also demonstrate that arousal from nocturnal sleep exhibits a significant ultradian rhythmicity with a periodicity of 85 minutes. Increasing the dietary sucrose concentration from 5% to 35% had no effect on total sucrose ingestion per day nor any affect on arousal, however it did lengthen the time that males and females remained active. Higher dietary sucrose led to reduced total sleep by male but not female flies. Locomotor activity was reduced by feeding flies Metformin, a drug that inhibits oxidative phosphorylation, however Metformin did not affect any aspects of sleep.

Conclusions

We conclude that arousal from sleep is under ultradian control and regulated in a sex-dependent manner by dietary yeast and that dietary sucrose regulates the length of time that flies sustain periods of wakefulness. These findings highlight Drosophila as an important model with which to understand how diet impacts upon sleep and wakefulness in mammals and humans.