Metabolic Profiling of Pancreatic Beta Cells

胰腺β细胞的代谢分析

• 批准号: 6879325
• 负责人: Orian S Shirihai
• 金额: $ 15.84万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879325
• 关键词: adenosine triphosphate; biotechnology; fluorescence resonance energy transfer; fluorescent dye /probe; glucose metabolism; laboratory mouse; membrane potentials; metabolomics; molecular probes; nicotinamide adenine dinucleotide; pancreatic islets; technology /technique development

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.

产生胰岛素的 β 细胞对葡萄糖刺激的反应的特点是代谢活动增加，从而导致 ATP 等信号分子的产生。对葡萄糖的健康反应涉及启动燃料消耗、氧化磷酸化和 ATP 产生的振荡，从而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能够监测该级联中的关键步骤。贝塔 然而，细胞并不由同质细胞群组成，因此，研究代谢参数的方法应应用于单个细胞的分辨率。我们在此建议开发一种可应用于单细胞分辨率的检测系统，并分析对葡萄糖的代谢反应，重点关注振荡 ATP 的产生。我们将开发一种新的 ATP 探针，该探针基于荧光共振能量转移 (FRET)，并且可以以亚细胞分辨率成像。探针将是 DNA 编码的蛋白质，可以使用病毒载体作为转基因传递至原代 β 细胞。该探针不需要外源辅因子，其活性也不会消耗 ATP。使用适当的靶向序列将该探针靶向特定的亚细胞区室（例如线粒体）将允许准确监测首选感兴趣位点的 ATP 可用性。我们将进一步将 ATP 成像与当前监测膜电位和 NADH 的方法相结合，从而能够同时监测涉及燃料消耗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剂、激素和因子的代谢效应的数据库。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β细胞的影响，并将有助于建立组织来源的胰岛细胞在代谢功能方面的质量标准化平台。