Screening of Molecules for entrainment of circadian rhythms in mammalian central and peripheral clock cells

许多生物体的许多生化、生理和行为过程都表现出昼夜节律。哺乳动物的中央昼夜节律振荡器位于大脑的视交叉上核（SCN）。由SCN产生的电生理活动和/或体液因子的昼夜波动调节外周节律。昼夜节律的产生机制被认为是一个涉及复杂分子相互作用的网络结构。因此，阐明这一分子网络的结构需要基于综合数据库的建模方法，该数据库是由主时钟细胞中的转录本、蛋白质和代谢物的全面定量构建的。在这项研究中，我们首先从转基因大鼠中建立了大鼠SCN衍生细胞系，其中小鼠Per1基因启动子与荧光素酶报告基因相连。在静态培养中，SCN衍生细胞系的发光具有很强的节律性。在用福斯克林处理细胞系后，完成了昼夜节律的时间依赖性相位提前或延迟。此外，我们已经建立了一个高效的代谢组系统，利用极其敏感的毛细管电泳-质谱（CE-MS）技术来估计细胞代谢物。然后，我们利用这些方法分析了SCN衍生细胞系中大约800种代谢物，并构建了一个分子数据库，收集了在细胞水平上具有昼夜波动和福斯克林处理后快速增加的代谢物。细胞能量电荷标志物ATP、ADP、NADP和NADPH的显著振荡表明能量代谢受昼夜节律控制。此外，相位提前后的能量电荷与相位延迟后的能量电荷相比处于极低的水平。结果反映了一个事实，即相推进所需的转变时间比相延迟所需的转变时间更长。

Many biochemical, physiological and behavioral processes in many organisms exhibit circadian rhythms. The central circadian oscillator of mammals is located at the suprachiasmatic nucleus(SCN) of the brain. The circadian fluctuation of electrophysiological activities and/or humoral factors produced by the SCN regulates the peripheral rhythms. The mechanism for the generating carcadian rhythms is regarded as a network structure involving complicated molecular interactions. Thus, the elucidation of the structure of this molecular network requires modeling methods based on an integrated database constructed with the comprehensive quantafcation of transcripts, proteins, and metabolite in the master clock cells. In this study, we first established a rat SCN derived cell line from a transgenic rat in which the mouse Per1 gene promoter is linked to a luciferase reporter. Light emission from the SCN derived cell line was robustly rhythmic in static culture. The time dependent phase advance or delay of the circadian rhythm was accomplished after the treatment of the cell line with forskolin. In addition, we have established an efficient metabolome system to estimate cellular metabolites utilizing an extremely sensitive Capillary electrophoresis-Mass spectrometry(CE-MS) technique. Then we analyzed approximately 800 metabolites in the SCN derived cell line using these methods, and constructed a molecular database that collected metabolites with a circadian fluctuation and a rapid increase after forskolin treatment in the cellular level. A prominent oscillation of the markers of the cellular energy charge, ATP, ADP, NADP, and NADPH, indicated that the energy metabolism was under the control of circadian rhythms. In addition, the energy charge after phase advance felt into an extremely low level in comparison with that after phase delay. The results reflected a fact that a longer transition time was necesary for phase advance than that for phase delay.