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β细胞对葡萄糖刺激的胰岛素反应的特征是代谢活性增加，导致信号分子（如ATP）产生。对葡萄糖的健康反应涉及在燃料消耗，氧化磷酸化和ATP产生中振荡的启动，这导致了胰岛素分泌的脉动模式。许多研究表明，糖尿病和肥胖症会干扰这种代谢级联。因此，开发新方法来治疗这些代谢疾病，就需要能够监视此级联的关键步骤。 beta 然而，细胞不包括同质细胞群，因此，研究代谢参数的方法应在单个细胞的分辨率上应用。我们在这里建议开发一种可以在单个细胞分辨率上应用的测定系统，并介绍了代谢反应对葡萄糖的反应，重点是振荡ATP的产生。我们将开发针对ATP的新探针，该探针将基于荧光共振能量转移（FRET），并可以在细胞下分辨率下成像。该探针将是DNA编码的蛋白质，可以使用病毒载体作为转基因传递到原代β细胞。该探针将不需要外源性副因素，其活性不会消耗ATP。使用适当的靶向序列将该探针靶向特定的亚细胞隔室，例如线粒体，将允许精确监视感兴趣的首选位点的ATP可用性。我们将进一步将ATP的成像与当前的方法结合起来，以监测膜电位和NADH，从而同时监测参与燃料消耗的代谢级联的三个关键步骤。我们将使用这些参数将β细胞的种群细分为不同的代谢表型。我们的长期目标是创建一个数据库，涵盖了药物，激素和因素的W | de spectrum的代谢效应。这种代谢概况的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，并将在建立质量标准化组织衍生胰岛细胞的平台方面有用。