Molecular Mechanisms Regulating Pancreatic Delta Cell Function and Dysfunction

项目摘要 在2型糖尿病（T2 D）患者和动物中，胰岛葡萄糖刺激的生长抑素（Sst）分泌丧失 该疾病的模型，这有助于破坏胰高血糖素和胰岛素分泌。人们普遍认为 Sst分泌从β-细胞发生在响应升高的细胞内Ca 2+，这主要是由于 内质网Ca ~（2+）释放。然而，控制胰岛细胞Ca 2 +ER处理的机制 以及它们在T2 D中是如何改变的，在很大程度上是未知的。我们实验室的数据发现，富含胰岛的两孔- 结构域K+通道（TALK-1）是一种ER定位的通道，为β细胞Ca ~（2+）ER释放提供逆流 Ca 2 +ER漏出。TALK-1介导的增强细胞Ca 2 +ER泄漏的电化学驱动力， 菌株Ca 2 +ER储存，限制葡萄糖刺激的Ca 2 +ER释放和Sst分泌。进一步的数据显示， 葡萄糖诱导的胰岛β细胞Ca 2 +-sens变构激活可增强胰岛β细胞Ca 2 +-ER释放和Sst分泌。 ing受体（CaSRs）。最后，我们的初步数据提供了第一个证据，表明糖尿病的条件减少， 胰岛细胞Ca 2 +ER储存，这有助于葡萄糖刺激的Ca 2+处理和Sst分泌的扰动 在糖尿病的情况下。根据这些令人兴奋的初步数据，本提案的总体目标是 阐明在糖尿病发病过程中，β细胞Ca 2 +ER是如何被控制和被破坏的。这个项目 将检验中心假设，即葡萄糖刺激的胰岛细胞Sst分泌被CaSR介导的Ca 2 +ER放大 释放，这是由TALK-1通道限制的Ca 2 +ER存储控制。这背后的基本原理是 本项目的目的是了解CaSR和TALK-1如何控制胰岛细胞Ca 2 +ER的处理和Sst的分泌， 用于恢复T2 D中葡萄糖刺激的Sst分泌和胰岛激素分泌的新治疗靶点。这 本课题的主要目的是：1）确定细胞内CaSR对Ca ~（2+）ER的调控机制 处理、Sst分泌和胰岛激素分泌;以及2）确定TALK-1通道如何控制Ca 2 +ER 释放调节β-细胞功能和功能障碍。在第一个目标下，对转基因小鼠进行β细胞消融， 将利用CaSR以及具有β细胞CaSR的shRNA敲低的人假胰岛来评估CaSR的作用。 在促分泌素调节β细胞Ca ~（2+）处理和Sst分泌过程中，Ca ~（2+）敏感受体的作用。aim 1将 还确定了糖尿病条件下β细胞Ca 2 +ER储存的耗竭如何影响CaSR信号传导和Sst 分泌物在第二个目标下，TALK-1通道在β细胞Ca 2 +ER处理和功能中的功能将是 在TALK-1的β细胞特异性消融的小鼠中和在含有β细胞的人假胰岛中测定 TALK-1敲低或表达显性负性TALK-1通道亚单位。此外，Aim 2将 确定TALK-1如何在应激条件下增强β细胞Ca 2 +ER耗竭， 糖尿病导致胰岛细胞功能障碍。该项目意义重大，因为它有望照亮机械- 在T2 D中改变胰岛细胞Ca 2 +ER处理和破坏胰岛激素分泌的细菌。此外，该项目将 鉴定用于使Sst分泌正常化和减少T2 D中胰岛功能障碍的药理学策略。

Project Summary Islet glucose-stimulated somatostatin (Sst) secretion is lost in patients with type-2 diabetes (T2D) and in animal models of the disease, which contributes to disrupted glucagon and insulin secretion. It is generally accepted that Sst secretion from -cells occurs in response to elevated intracellular Ca2+, which primarily results from endoplasmic reticulum (ER) Ca2+ (Ca2+ER) release. However, the mechanisms that control -cell Ca2+ER handling and how they are altered in T2D are largely unknown. Data from our lab finds that the islet-enriched two-pore- domain K+ channel, TALK-1, is an ER localized channel in that provides a countercurrent for -cell Ca2+ER release and Ca2+ER leak. TALK-1-mediated augmentation of the electrochemical driving force for -cell Ca2+ER leak con- strains Ca2+ER storage, which limits glucose-stimulated Ca2+ER release and Sst secretion. Further data show that -cell Ca2+ER release and Sst secretion are amplified by glucose-induced allosteric activation of -cell Ca2+-sens- ing receptors (CaSRs). Finally, our preliminary data provide the first evidence that diabetic conditions diminish -cell Ca2+ER storage, which contributes to perturbations in glucose-stimulated Ca2+ handling and Sst secretion under diabetic conditions. Based on these exciting preliminary data, the overall objective of this proposal is to elucidate how -cell Ca2+ER is controlled and becomes disrupted during the pathogenesis of diabetes. This project will test the central hypothesis that glucose-stimulated -cell Sst secretion is amplified by CaSR-mediated Ca2+ER release, which is controlled by TALK-1 channel constraint of Ca2+ER storage. The rationale that underlies this project is that understanding how CaSR and TALK-1 control -cell Ca2+ER handling and Sst secretion will expose novel therapeutic targets for restoring glucose-stimulated Sst secretion and islet hormone secretion in T2D. This project will be accomplished with the following two specific aims: 1) Determine how -cell CaSR controls Ca2+ER handling, Sst secretion, and islet hormone secretion; and 2) Determine how TALK-1 channel control of Ca2+ER release modulates -cell function and dysfunction. Under the first aim, transgenic mice with -cell ablation of CaSR as well as human pseudoislets with ShRNA knockdown of -cell CaSR will be utilized to assess the roles of the Ca2+-sensing receptor during secretagogue modulation of -cell Ca2+ handling and Sst secretion. Aim1 will also determine how depletion of -cell Ca2+ER stores under diabetic conditions impacts CaSR signaling and Sst secretion. Under the second aim, the function TALK-1 channels on -cell Ca2+ER handling and function will be determined in mice with -cell specific ablation of TALK-1 and in human pseudoislets containing either -cells with knockdown of TALK-1 or expressing dominant negative TALK-1 channel subunits. Furthermore, Aim2 will determine how TALK-1 augmentation of -cell Ca2+ER depletion under the stressful conditions associated with diabetes contributes to -cell dysfunction. This project is significant because it is expected to illuminate mecha- nisms that alter -cell Ca2+ER handling and disrupt islet hormone secretion in T2D. Moreover, this project will identify pharmacological strategies for normalizing Sst secretion and reducing islet dysfunction in T2D.