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

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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.