R21: Entrainment of Peripheral Circadian Rhythms

描述（由申请人提供）：内源性每日（昼夜）节律控制许多行为和生理功能。下丘脑视交叉上核（SCN）中的主起搏器控制着这些振荡，但产生这些振荡的转录-翻译反馈回路分布在全身的细胞中。这些外周昼夜节律振荡持续许多周期，如果不是无限期的，在孤立的器官放置在培养后。SCN产生神经和内分泌信号，以调节外周组织的昼夜节律振荡，但这些信号作为牵引线索（授时因子）的方式以及外周振荡器向主起搏器所采取的阶段的决定因素尚不清楚。虽然有强大的工具以转基因小鼠的形式可用，但需要新的方法来确定外周振荡的行为，生理作用和重要性。本R21应用程序中描述的实验将利用小鼠荧光素酶报告基因构建，以确定SCN是否携带肝脏振荡，并确定哪些特定信号作为zeitgeber。神经递质肾上腺素和乙酰胆碱的作用，以及激素包括糖皮质激素，胰岛素和胰高血糖素，将被检查。葡萄糖浓度波动的影响也将被测试。将使用一种新颖的流式系统，将生理信号（激素或神经递质）添加到含有从转基因Per2::luc小鼠的肝脏复制部分的器官培养室中。这些信号调节周期和相位的能力将通过光度法确定，我们将确定是否满足夹带标准。第一次对于外围振荡器，将检查夹带范围和相位响应曲线将被构造。随着肝时钟在不同时期对vldl -胆固醇分泌的夹带影响通过渗滤出水测定，昼夜节律组织的健康相关性将被评估。一旦这个光度测定系统建立起来，它不仅可以用来分析其他肝脏昼夜节律的干扰，还可以用来分析其他器官的类似时钟的行为。这些研究将为分析控制正常生理功能和其功能障碍可能导致疾病的多振荡系统提供重要的新工具。

DESCRIPTION (provided by applicant): Endogenous daily (circadian) rhythms govern many behavioral and physiological functions. A master pacemaker in the suprachiasmatic nucleus of the hypothalamus (SCN) controls these oscillations, but the transcriptional-translational feedback loops that generate them are distributed in cells throughout the body. These peripheral circadian oscillations persist for many cycles, if not indefinitely, in isolated organs after they are placed in culture. The SCN generates neural and endocrine signals in order to regulate circadian oscillations in peripheral tissues, but the way in which these signals act as entraining cues (zeitgebers) and the determinants of the phase taken by peripheral oscillators to the master pacemaker are poorly understood. Although powerful tools are available in the form of transgenic mice, novel approaches are required to determine the behavior, the physiological role, and the importance of the peripheral oscillators. The experiments described in this R21 application will utilize a mouse luciferase reporter construct in order to establish whether the SCN entrains hepatic oscillations, and to determine which specific signals serve as zeitgebers. The roles of the neurotransmitters epinephrine and acetylcholine, as well as hormones including glucocorticoids, insulin and glucagon, will be examined. The influence of fluctuations in glucose concentration will also be tested. A novel-flow through system will be used in which physiological signals (hormones or neurotransmitters) are added to organ culture chambers containing replicate sections of liver taken from transgenic Per2::luc mice. The ability of these signals to regulate period and phase will be established by luminometry, and we will determine whether the criteria of entrainment are met. For the first time for a peripheral oscillator, the range of entrainment will be examined and phase response curves wil be constructed. The health relevance of circadian organization will be evaluated as the impact of entrainment of the hepatic clock to various periods upon secretion of VLDL-cholesterol is determined through assay of the perifusion effluent. Once this luminometry system is established, it may be used to analyze not only entrainment of other hepatic circadian rhythms, but also the clock-like behavior of other organs. These studies will provide important new tools for analysis of the multi-oscillator system that governs normal physiological function and whose malfunction may contribute to disease.