R21: Entrainment of Peripheral Circadian Rhythms

描述（由申请人提供）：内源性每日（昼夜）节律支配许多行为和生理功能。下丘脑视交叉上核（SCN）中的主起搏器控制这些振荡，但产生它们的转录-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，如果不是无限期地，在孤立的器官后，他们被放置在文化。SCN产生神经和内分泌信号以调节外周组织中的昼夜节律振荡，但这些信号作为夹带线索（zeitgebers）的方式以及外周振荡器对主起搏器所采取的相位的决定因素知之甚少。虽然强大的工具，可在转基因小鼠的形式，需要新的方法来确定的行为，生理作用，和外围振荡器的重要性。在该R21申请中描述的实验将利用小鼠荧光素酶报告基因构建体，以确定SCN是否引起肝振荡，并确定哪些特定信号用作Zeitgeber。将检查神经递质肾上腺素和乙酰胆碱以及包括糖皮质激素、胰岛素和胰高血糖素在内的激素的作用。还将测试葡萄糖浓度波动的影响。将使用一种新的流通系统，其中将生理信号（激素或神经递质）添加到含有从转基因Per 2：：luc小鼠获取的肝脏重复切片的器官培养室中。这些信号调节周期和相位的能力将通过光度测定法建立，我们将确定是否符合夹带的标准。首次对外围振荡器的夹带范围进行了研究，并绘制了相位响应曲线。将评价昼夜节律组织的健康相关性，因为通过灌流流出物的测定来确定肝脏生物钟对VLDL-胆固醇分泌的不同时期的影响。一旦建立了这种光度测定系统，它不仅可以用于分析其他肝脏昼夜节律的夹带，而且还可以用于分析其他器官的时钟样行为。这些研究将为分析多振荡器系统提供重要的新工具，该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.