The role of mitochondrial fission in beta cell function

项目摘要/摘要 世界上近10%的人患有糖尿病，这一数字预计还会增加。2型糖尿病在以下情况下发生 面对胰岛素抵抗，β细胞无法产生足够的胰岛素。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 刺激胰岛素的分泌，现在很明显，线粒体不是简单的静态代谢细胞器。他们 处于不断的裂变和融合状态，这种平衡的破坏会影响生物能量学、有丝分裂和 细胞存活。我们认为线粒体形态可能成为糖尿病治疗的新靶点。 然而，人们对线粒体形态的变化如何影响β细胞功能和 活体内存活。这项提议的中心目标是了解线粒体裂变的丢失是如何 影响胰岛素分泌、β细胞存活和有丝分裂。我们的具体目标如下。目标1：定义如何 线粒体分裂调节胰岛素分泌的代谢放大途径。根据我们的初步调查 数据，我们假设线粒体分裂是NADPH放大途径所必需的。我们将使用 光学记者确定急性脑缺血时NADPH对葡萄糖反应的振荡动力学 和慢性Drp1基因敲除。由于Drp1具有线粒体分裂以外的作用，我们将使用CRISPR 干扰以沉默线粒体分裂的纯调控因子Mief1，以与Drp1基因敲除进行比较。 最后，我们将恢复DRp1缺陷的β细胞的线粒体形态，并伴随着敲除 线粒体融合调节因子丝裂原蛋白-2(Mfn2)可挽救胰岛素分泌。目标2：确定 线粒体分裂与β细胞存活和线粒体质量控制。在非β细胞中，线粒体 分裂被认为是吞噬有丝分裂的重要因素，但也是细胞凋亡所必需的。根据我们的初步数据， 我们推测，在β细胞中丢失drp1可能会保护细胞免于凋亡，但可能是引发了吞噬核分裂。 和线粒体质量控制。目的3：确定DRp1在人类胰岛中的相关性 2糖尿病。我们将在非糖尿病患者的原代人类β细胞中敲除Drp1，并测量它们的 耗氧量、胰岛素分泌和钙动力学。测试线粒体动力学在类型性疾病中的作用 2糖尿病的发病机制，我们将测量2型糖尿病β细胞中线粒体动力学蛋白的水平。 并检查这些细胞中线粒体的形态。最后，测试Drp1的作用 在2型糖尿病中，我们将在人类糖尿病β细胞中重新表达Drp1，并询问葡萄糖 刺激的胰岛素分泌可以得到改善。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治疗。

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.