The Role of Circadian Clocks in Metabolic Disease

The circadian clock is a highly conserved timing system, resonating physiological processes to 24-hour environmental cycles. Circadian misalignment is emerging as a risk factor of metabolic disease. The molecular clock resides in all metabolic tissues, the dysfunction of which is associated with perturbed energy metabolism. In this article, we will review current knowledge about molecular mechanisms of the circadian clock and the role of clocks in the physiology and pathophysiology of metabolic tissues.     

Circadian fluctuations in circulating plasminogen activator inhibitor-1 are independent of feeding cycles in mice

To evaluate the involvement of the day-night feeding cycle in the circadian regulation of circulating plasminogen activator inhibitor-1 (PAI-1) concentrations, mice were fed with a diet for eight hours during either daytime (DF) or nighttime (NF) for one week. The reversed feeding cycle did not affect the circadian phases of plasma PAI-1 levels as well as the nocturnal wheel-running activity, although the phase of Pai-1 mRNA expression was significantly advanced for 8.6 hours in the livers of DF, compared with NF mice. The day-night feeding cycle is not a critical Zeitgeber for circadian rhythm of circulating PAI-1.     

Circadian clocks in changing weather and seasons: Lessons from the picoalga Ostreococcus tauri

Daylight is the primary cue used by circadian clocks to entrain to the day/night cycle so as to synchronize physiological processes with periodic environmental changes induced by Earth rotation. However, the temporal daylight pattern is not the same every day due to erratic weather fluctuations or regular seasonal changes. Then, how do circadian clocks operate properly in varying weather and seasons? In this paper, we discuss the strategy unveiled by recent studies of the circadian clock of Ostreococcus tauri, the smallest free‐living eukaryotic organism. It combines mechanisms controlling light inputs and clock sensitivity, shaping both the dynamics of the core circadian oscillator and its forcing by light so as to ensure stable and precise synchronization in all weather and seasons. Editor's suggested further reading in BioEssays: Another place, another timer: Marine species and the rhythms of life Abstract     

Circadian clock gene expression regulates cancer cell growth through glutaminase

Glutamine is an essential amino acid for malignant tumor cells. Glutaminase that metabolizes glutamine reaches a maximum expression in tumors immediately before the maximum proliferation rate. Tumor cells grow at different rates during the day. We postulated that the activity of glutaminase in tumor cells is subject to the regulation of circadian clock gene. We measured glutaminase by western blot analysis and circadian clock gene expression by real-time polymerase chain reaction in the liver and tumor cells at six equispaced time points of the day in individual mice of a 12/12 h light/dark schedule. The results showed that the tumor-bearing mice, under normal diurnal conditions, are circadianly entrained, as reflected by the normal host locomotor activity rhythms and rhythmic liver clock gene expression. The tumors within these mice are also circadianly organized, as reflected by circadian clock gene （Bmall） expression. What is most remarkable is that kidney-type glutaminase also showed circadian rhythms in the same pattern with tumor circadian clock gene expression in liver cancer xenograft model, indicating that conditionally inhibiting glutaminase activity may provide a new target for cancer therapy.     

Protein Synthesis in Euglena gracilis is Light-and Temperature-Dependent,Oscillating in a Circadian,Temperature-Compensated Manner

Abstract: Incorporation of radiolabelled amino acids into proteins of Euglena gracilis revealed that the amount of labelled protein depends on the conditions of illumination and temperature of cultivation. Protein synthesis was generally lower under dark conditions except at 37 °C. The largest amounts of labelled protein were measured at 21 °C and decreased at higher and lower temperatures. By separating the labelled proteins of the membraneous cell fraction from subcultures under a range of culture conditions, the synthesis of some specific proteins was found to be light- and/or temperature-dependent. On incubating cells taken at different times during a light/dark cycle and under constant conditions, a circadian rhythm of ³⁵S-methionine- as well as ³⁵S-cysteine-incorporation was detected. Thereby the cells incorporated ten-times less cysteine than methionine. Protein synthesis always peaked during the last quarter of the daily light phase, confirming the rhythmic rise in total protein. The length of the rhythm period, approximately 24 h, was nearly independent of the applied temperature in the range of 16 to 27 °C.     

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.     

光敏色素与转录因子结合直接调控植物基因表达和发育

植物的光控发育一直是植物学中一个非常活跃的研究领域,是近该领域又取得重大突破性进展,人们通过酵母双杂交技术克隆到在体内与光敏色素相互作用的转录因子,并证实被光活化的光敏色素直接进入细胞核与转录因子结构合启动基因表达,本文就此研究进展作简要介绍。     

大鼠视交叉上核与松果体中Clock基因转录的昼夜节律性及不同光反应性

本研究旨在观察和比较视交叉上核（suprachiasmatic nucleus,SCN）与松果体（pineal gland,pG）中Clock基因内源性昼夜转录变化规律以及光照对其的影响。Sprague-Dawley大鼠在持续黑暗（constant darkness,DD）和12h光照：12h黑暗交替（12hourlight：12hour-darkcycle,LD）光制下分别被饲养8周（n=36）和4周n=36）后,在一昼夜内每隔4h采集一组SCN和PG组织（n=6）,提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点（circadian times．CT or zeitgeber times．ZT）各样品中Clock基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律性转录变化。结果如下：（1）SCN中Clock基因mRNA的转录在DD光制下呈现昼低夜高节律性振荡变化（P〈0．05）,PG中Clock基因的转录也显示相似的内源性节律外观,即峰值出现于主观夜晚（SCN为CTl5,PG为CT18）,谷值位于主观白天（SCN为CT3,PG为CT6）（P〉0．05）。（2）LD光制下SCN中Clock基因的转录也具有昼夜节律性振荡（P〈0．05）,但与其DD光制下节律外观相比,呈现反时相节律变化（P〈0．05）,且其表达的振幅及峰值的mRNA水平均增加（P〈0．05）,而PG中Clock基因在LD光制下转录的相应节律参数变化却恰恰相反（P〈0．05）。（3）在LD光制下,光照使PG中Clock基因转录的节律外观反时相于SCN（P〈0．05）,即在SCN和PG的峰值分别出现于光照期ZT10和黑暗期ZT17,谷值分别位于黑暗期ZT22和光照期ZT5。结果表明,Clock基因的昼夜转录在SCN和PG中存在同步的内源性节律本质,而光导引在这两个中枢核团调节Clock基因昼夜节律性转录方面有着不同的作用。     

The chondrocyte-intrinsic circadian clock is disrupted in human osteoarthritis

Peripheral clocks are essential for driving cell differentiation. In osteoarthritis, loss of the normal differentiated chondrocyte (cartilage cell) phenotype is causative of disease. We investigated whether clock gene expression differed in osteoarthritic compared to “healthy” chondrocytes and used RNAi to determine whether the differences observed could affect chondrocyte phenotype. Following serum shock, PER2 expression was significantly higher, whereas BMAL1 expression was significantly lower, in osteoarthritic chondrocytes. Knockdown of BMAL1 in “healthy” chondrocytes was associated with higher cell proliferation and MMP13 expression, features characteristic of the osteoarthritic chondrocyte phenotype. Chondrocyte-intrinsic clock disruption may be a critical early step in osteoarthritis development.     

Isoform switching facilitates period control in the Neurospora crassa circadian clock

A striking and defining feature of circadian clocks is the small variation in period over a physiological range of temperatures. This is referred to as temperature compensation, although recent work has suggested that the variation observed is a specific, adaptive control of period. Moreover, given that many biological rate constants have a Q₁₀ of around 2, it is remarkable that such clocks remain rhythmic under significant temperature changes. We introduce a new mathematical model for the Neurospora crassa circadian network incorporating experimental work showing that temperature alters the balance of translation between a short and long form of the FREQUENCY (FRQ) protein. This is used to discuss period control and functionality for the Neurospora system. The model reproduces a broad range of key experimental data on temperature dependence and rhythmicity, both in wild‐type and mutant strains. We present a simple mechanism utilising the presence of the FRQ isoforms (isoform switching) by which period control could have evolved, and argue that this regulatory structure may also increase the temperature range where the clock is robustly rhythmic.     

Clock gene Per2 modulates epidermal tissue repair in vivo

Wound healing can be influenced by genes that control the circadian cycle, including Per2 and BMAL1, which coordinate the functions of several organs, including the skin. The aim of the study was to evaluate the role of PER2 during experimental skin wound healing. Two groups (control and Per2-KO), consisting of 14 male mice each, were anesthetized by inhalation, and two 6 mm wounds were created on their dorsal skin using a punch biopsy. A silicone ring was sutured around the wound perimeter to restrict contraction. The wound healing process was clinically measured daily (closure index) until complete wound repair. On Day 6, histomorphometric analysis was performed using the length and thickness of the epithelial migration tongue, in addition to counting vessels underlying the lesion by immunofluorescence assay and maturation of collagen fibers through picrosirius staining. Bromodeoxyuridine (BrdU) incorporation and quantification were performed using the subcutaneous injection technique 2 h before euthanasia and through immunohistochemical analysis of the proliferative index. In addition, the qualitative analysis of myofibroblasts and periostin distribution in connective tissue was performed by immunofluorescence. Statistically significant differences were observed in the healing time between the experimental groups (means: 15.5 days for control mice and 13.5 days for Per2-KO; p = 0.001). The accelerated healing observed in the Per2-KO group (p < 0.05) was accompanied by statistical differences in wound diameter and length of the migrating epithelial tongue (p = 0.01) compared to the control group. Regarding BrdU immunoreactivity, higher expression was observed in the intact epithelium of Per2-KO animals (p = 0.01), and this difference compared to control was also present, to a lesser extent, at the wound site (p = 0.03). Immunofluorescence in the connective tissue underlying the wound showed a higher angiogenic potential in the Per2-KO group in the intact tissue area and the wound region (p < 0.01), where increased expression of myofibroblasts was also observed. Qualitative analysis revealed the distribution of periostin protein and collagen fibers in the connective tissue underlying the wound, with greater organization and maturation during the analyzed period. Our research showed that the absence of the Per2 gene positively impacts the healing time of the skin in vivo. This acceleration depends on the increase of epithelial proliferative and angiogenic capacity of cells carrying the Per2 deletion.     

斑马鱼生物钟研究进展

斑马鱼是生物钟研究领域中一种新兴的脊椎动物模型。文章总结了斑马鱼生物钟研究的一些进展, 以及利用斑马鱼研究生物钟的特点及优势。由于光照和温度作为重要的外部信号在斑马鱼生物钟调节中发挥重要作用, 文章主要就近期光和温度对斑马鱼钟基因及调节通路的研究进行了概述, 最后对斑马鱼生物钟研究的未来提出了展望。     

Temperature Dependency of Circadian Period in a Single Cell (Acetabularia)

The circadian rhythm in the oxygen production of 30 individual Acetabularia cells has been studied at different temperatures. The temperature induced period variation was continuously evaluated over the whole data record of each individual cell with an advanced spectral analysis technique. The observed circadian periods of O₂ production displayed a well established region of temperature compensation between 25 °C and 30 °C with a Q₁₀, value of 0.9, whereas between 15°C and 22°C a positive temperature coefficient was measured (Q₁₀ at 22 °C 0.9, Q₁₀ at 20°C 0.8, Q₁₀at 17°C 0.7).     

Circadian expression of connexins in the mouse heart

Significant circadian variations exist in the frequency of cardiac arrhythmia; however, the underlying mechanism is largely unknown. Connexins are essential in the propagation of electrical activity throughout the heart and are an important determinant of conduction velocity. Their dysfunction is related to the genesis of cardiac arrhythmia. In this study, we investigated if cx40 and cx43 expressed circadianly in the mouse heart using suprachiasmatic nuclei (SCN) lesion and pharmacological autonomic nervous system block mouse. Significant 24-h variations were observed in the expression of cx40 and cx43 in the sham-operated mice. In the SCNX mouse, all genes examined lost circadian rhythm. In the ANSB mouse, the expressions of Bmal1 was dampened significantly but still had circadian rhythm, whereas the two connexin gene expressions lost rhythm. These results suggest that cx40 and cx43 gene expressions have clear circadian rhythm and might be regulated by the central clock in the SCN through the ANS.