Metabolic Profiling of Pancreatic Beta Cells

产生胰岛素的β细胞对葡萄糖刺激的反应以代谢活动增加为特征，导致信号分子的产生，如ATP。对葡萄糖的健康反应包括启动燃料消耗、氧化磷酸化和ATP产生的振荡，这导致胰岛素分泌的脉动模式。大量研究表明，糖尿病和肥胖会干扰这种代谢级联反应。因此，开发治疗这些代谢紊乱的新方法需要有能力监测这一级联反应中的关键步骤。测试版 然而，细胞并不是由同质的细胞群体组成的，因此，研究代谢参数的方法应该在单个细胞的分辨率上应用。我们建议开发一种可以应用于单个细胞分辨率的分析系统，并描述对葡萄糖的代谢反应，重点是振荡的ATP产生。我们将开发一种新的ATP探针，它将基于荧光共振能量转移(FRET)，可以在亚细胞分辨率下成像。该探针将是DNA编码的蛋白质，可以通过病毒载体以转基因的形式传递给原代贝塔细胞。该探测器将不需要外源性辅助因子，其活性也不会消耗ATP。使用适当的靶向序列将该探针定向到特定的亚细胞间隔，例如线粒体，将允许准确地监测首选感兴趣位置的ATP的可用性。我们将进一步将ATP成像与目前监测膜电位和NADH的方法相结合，允许同时监测与燃料消耗有关的代谢级联中的三个关键步骤。我们将使用这些参数将β细胞群体细分为不同的代谢表型。我们的长期目标是创建一个涵盖各种药物、激素和因子的代谢影响的数据库。这样的代谢谱数据库将用于表征和预测新化合物和培养条件对胰岛细胞的影响，并将有助于建立组织来源的胰岛细胞代谢功能质量标准化的平台。

The response of insulin producing beta cells to glucose stimulation is characterized by increased metabolic activity, leading to the production of signaling molecules such as ATP. A healthy response to glucose involves the initiation of oscillations in fuel consumption, oxidative phosphorylation, and ATP production, which lead to a pulsatile pattern of insulin secretion. Numerous investigations have demonstrated that diabetes and obesity interfere with this metabolic cascade. Accordingly, development of new approaches to the treatment of these metabolic disorders requires the ability to monitor key steps in this cascade. Beta cells, however, do not consist of a homogenous population of cells and thus, methodologies that study metabolic parameters should be applied at the resolution of the individual cell. We propose here to develop a system of assays that can be applied at the resolution of a single cell and profile the metabolic response to glucose, focusing on oscillatory ATP production. We will develop a new probe for ATP, which will be based on Fluorescence Resonance Energy Transfer (FRET) and can be imaged at sub-cellular resolution. The probe will be DNA encoded protein that can be delivered as a transgene to primary beta cells using viral vectors. The probe will not require exogenous co-factors and its activity will not consume ATP. The targeting of this probe to specific sub-cellular compartments, such as the mitochondria, using appropriate targeting sequences will allow for accurate monitoring of ATP availability at preferred sites of interest. We will further combine the imaging of ATP with current methodologies for the monitoring of membrane potential and NADH, allowing for simultaneous monitoring of three key steps in the metabolic cascade involved in fuel consumption. We will use these parameters to subdivide the population of beta cells into different metabolic phenotypes. Our long-term goal is to create a database covering the metabolic effect of a w|de spectrum of pharmalogical agents, hormones and factors. Such a database of metabolic profiles will be used to characterize and predict the effect of new compounds and culturing conditions on beta cells and will be useful in establishing a platform for quality standardization of tissue derived islet cells in terms of metabolic function.