R21: Entrainment of Peripheral Circadian Rhythms

描述(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-翻译反馈环分布在全身的细胞中。这些外周昼夜节律振荡即使不是无限期的，也会持续许多个周期，在孤立的器官中放置在培养中。SCN产生神经和内分泌信号以调节周围组织的昼夜振荡，但这些信号作为诱导性线索(斑点)的方式以及外围振荡器对主起搏器采取的相位的决定因素尚不清楚。虽然以转基因小鼠的形式提供了强大的工具，但需要新的方法来确定外周振荡器的行为、生理作用和重要性。本R21申请中描述的实验将利用小鼠荧光素酶报告结构，以确定SCN是否引起肝脏振荡，并确定哪些特定信号作为斑点。神经递质肾上腺素和乙酰胆碱以及包括糖皮质激素、胰岛素和高血糖素在内的激素的作用将被研究。还将测试葡萄糖浓度波动的影响。将使用一种新的流动系统，在该系统中，生理信号(激素或神经递质)被添加到包含从转基因PER2：：Luc小鼠身上获得的复制肝脏切片的器官培养室内。这些信号调节周期和相位的能力将通过光度学来确定，我们将确定是否满足夹带的标准。对于外围振荡器，将首次考察夹带的范围，并绘制相位响应曲线。昼夜节律组织的健康相关性将被评估，因为通过对灌流流出物的分析，确定不同时期肝钟夹带对极低密度脂蛋白-胆固醇分泌的影响。一旦建立了这个发光测量系统，它不仅可以用来分析其他肝脏昼夜节律的夹带，还可以用来分析其他器官的钟状行为。这些研究将为分析多振子系统提供重要的新工具，多振子系统控制正常的生理功能，其故障可能导致疾病。

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.