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The role of mitochondrial fission in beta cell function

线粒体裂变在 β 细胞功能中的作用

基本信息

批准号：
10538551
负责人：
Gregory Michael Ku
金额：
$ 37.77万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-22 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10538551
关键词：
Acute Address Affect Animal Model Apoptosis Apoptotic Behavior Beta Cell Biochemical Bioenergetics CASP3 gene CRISPR interference CRISPR/Cas technology Calcium Cell Line Cell Survival Cell physiology Cells Cellular Morphology Chemicals Chronic Cytoprotection Data Defect Development Diabetes Mellitus Disease Progression Down-Regulation Equilibrium Failure Foundations Future Genetic Glucose Goals Grant Human Insulin Insulin Resistance Kidney Bean Knock-out Knockout Mice Link Measures Metabolic Mitochondria Mitochondrial Swelling Monitor Morphology Mus NADP Non-Insulin-Dependent Diabetes Mellitus Optical reporter Organelles Outcome Outer Mitochondrial Membrane Oxygen Consumption Pathway interactions Patients Phase Protein Dynamics Quality Control Reactive Oxygen Species Reporter Respiration Reticulum Rodent Role Shapes Stains Structure of beta Cell of islet Testing Therapeutic diabetes pathogenesis diabetic human model improved in vivo inhibitor innovation insulin secretion islet knock-down mRNA Expression member mitochondrial dysfunction morphometry mouse model non-diabetic novel pandemic disease response therapeutic target

项目摘要

Project Summary/Abstract Nearly 10% of the world has diabetes and this number is projected to increase. Type 2 diabetes occurs when beta cells fail to produce sufficient insulin in the face of insulin resistance. One important aspect of beta cell failure is mitochondrial dysfunction. While mitochondrial have long been known to be critical for glucose stimulated insulin secretion, it is now clear that mitochondria are not simply static metabolic organelles. They are in a constant state of fission and fusion and disruption of this balance affects bioenergetics, mitophagy and cell survival. We propose that mitochondrial morphology could be a novel target for diabetes therapeutics. However, very little is known about how changes in mitochondrial morphology affect beta cell function and survival in vivo. The central objectives of this proposal are to understand how loss of mitochondrial fission affects insulin secretion, beta cell survival and mitophagy. Our specific aims are as follows. Aim 1: Define how mitochondrial fission regulates the metabolic amplifying pathway of insulin secretion. Based on our preliminary data, we hypothesize that mitochondrial fission is required for the NADPH amplifying pathway. We will use optical reporters to ascertain the oscillatory dynamics of NADPH in response to glucose in the setting of acute and chronic Drp1 knockout. Since Drp1 has roles outside of mitochondrial fission, we will use CRISPR interference to silence a pure regulator of mitochondrial fission, Mief1, for comparison with the Drp1 knockout. Finally, we will restore mitochondrial morphology in Drp1 deficient beta cells with concomitant knockdown of the mitochondrial fusion regulator mitofusin-2 (Mfn2) to rescue insulin secretion. Aim 2: Determine the role of mitochondrial fission in beta cell survival and mitochondrial quality control. In non-beta cells, mitochondrial fission is thought to be important for mitophagy but be required for apoptosis. Based on our preliminary data, we hypothesize that loss of Drp1 in the beta cell may protect cells from apoptosis but maytrigger mitophagy and mitochondrial quality control. Aim 3: Establish the relevance of Drp1 in human islets with and without type 2 diabetes. We will knockdown Drp1 in primary human beta cells from non-diabetic patients and measure their oxygen consumption, insulin secretion, and calcium dynamics. To test a role of mitochondrial dynamics in type 2 diabetes pathogenesis, we will measure levels of mitochondrial dynamics proteins in beta cells from type 2 diabetes patients and examine the mitochondrial morphology in these cells. Finally, to test a role of Drp1 downregulation in type 2 diabetes, we will re-express Drp1 in human diabetic beta cells and ask if glucose stimulated insulin secretion can be improved. These studies will lay the foundation for future diabetes therapeutics based on improving mitochondrial dynamics.

项目总结/摘要全世界近10%的人患有糖尿病，预计这一数字还会增加。2型糖尿病是什么？ β细胞在面对胰岛素抗性时不能产生足够的胰岛素。β细胞的一个重要方面失败是线粒体功能障碍。虽然线粒体长期以来一直被认为是葡萄糖的关键通过刺激胰岛素分泌，现在清楚的是线粒体不仅仅是静态的代谢细胞器。他们处于裂变和融合的恒定状态，这种平衡的破坏会影响生物能量学、线粒体自噬和细胞存活我们认为线粒体形态学可能成为糖尿病治疗的新靶点。然而，很少有人知道线粒体形态的变化如何影响β细胞功能，体内存活率。这项建议的中心目标是了解线粒体分裂的损失是如何发生的。影响胰岛素分泌、β细胞存活和线粒体自噬。我们的具体目标如下。目标1：如何定义线粒体分裂调节胰岛素分泌的代谢放大途径。根据我们初步的数据，我们假设线粒体分裂是必需的NADPH放大途径。我们将使用光学报告，以确定振荡动力学的NADPH响应葡萄糖在设置急性和慢性Drp 1敲除。由于Drp 1在线粒体分裂之外发挥作用，我们将使用CRISPR 干扰沉默线粒体分裂，Mief 1的纯调节，与Drp 1敲除比较。最后，我们将在Drp 1缺陷β细胞中恢复线粒体形态，同时敲低线粒体融合调节因子mitofusin-2（Mfn 2），以拯救胰岛素分泌。目标2：确定在β细胞存活和线粒体质量控制中的线粒体分裂。在非β细胞中，线粒体分裂被认为对于线粒体自噬是重要的，但对于细胞凋亡是必需的。根据我们的初步数据，我们假设β细胞中Drp 1的缺失可能保护细胞免于凋亡，但可能不可避免地触发线粒体自噬和线粒体质量控制。目的3：确定Drp 1在人类胰岛中的相关性， 2糖尿病.我们将在来自非糖尿病患者的原代人β细胞中敲除Drp 1，并测量它们的表达。氧消耗、胰岛素分泌和钙动力学。为了测试线粒体动力学在 2型糖尿病发病机制，我们将测量2型糖尿病患者β细胞中线粒体动力学蛋白的水平，糖尿病患者，并检查这些细胞中的线粒体形态。最后，测试Drp 1的作用我们将在人类糖尿病β细胞中重新表达Drp 1，并询问葡萄糖是否可以改善刺激的胰岛素分泌。这些研究将为未来的糖尿病奠定基础基于改善线粒体动力学的疗法。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A Superfolder Green Fluorescent Protein-Based Biosensor Allows Monitoring of Chloride in the Endoplasmic Reticulum.

DOI：
10.1021/acssensors.2c00626
发表时间：
2022-08-26
期刊：
ACS SENSORS
影响因子：
8.9
作者：
Shariati, Kaavian;Zhang, Yaohuan;Giubbolini, Simone;Parra, Riccardo;Liang, Steven;Edwards, Austin;Hejtmancik, J. Fielding;Ratto, Gian Michele;Arosio, Daniele;Ku, Gregory
通讯作者：
Ku, Gregory

Selective monitoring of insulin secretion after CRISPR interference in intact pancreatic islets despite submaximal infection.