Molecular Mechanisms Regulating Pancreatic Delta Cell Function and Dysfunction

项目概要 2 型糖尿病 (T2D) 患者和动物中葡萄糖刺激的胰岛生长抑素 (Sst) 分泌丧失 该疾病的模型会导致胰高血糖素和胰岛素分泌紊乱。这是普遍接受的  细胞分泌 Sst 是为了响应细胞内 Ca2 升高，这主要是由于 内质网（ER）Ca2+（Ca2+ER）释放。然而，控制  细胞 Ca2+ ER 处理的机制 它们在 T2D 中如何改变目前还不得而知。我们实验室的数据发现，富含胰岛的双孔- 域 K 通道 TALK-1 是一种 ER 局部通道，为  细胞 Ca2+ ER 释放提供逆流 和 Ca2 ER 泄漏。 TALK-1 介导增强  细胞 Ca2 ER 泄漏的电化学驱动力 菌株 Ca2 ER 储存，限制葡萄糖刺激的 Ca2 ER 释放和 Sst 分泌。进一步数据表明 葡萄糖诱导的  细胞 Ca2 -sens- 变构激活可放大  细胞 Ca2 ER 释放和 Sst 分泌 ing 受体（CaSR）。最后，我们的初步数据提供了糖尿病病情减轻的第一个证据  细胞 Ca2 ER 储存，有助于干扰葡萄糖刺激的 Ca2 处理和 Sst 分泌 在糖尿病条件下。基于这些令人兴奋的初步数据，该提案的总体目标是 阐明  细胞 Ca2 ER 在糖尿病发病过程中如何受到控制并被破坏。这个项目 将检验以下中心假设：CaSR 介导的 Ca2 ER 会放大葡萄糖刺激的  细胞 Sst 分泌 释放，这是由 Ca2ER 存储的 TALK-1 通道约束控制的。这背后的基本原理 项目的目的是了解 CaSR 和 TALK-1 如何控制  细胞 Ca2 ER 处理和 Sst 分泌将揭示 恢复 T2D 中葡萄糖刺激的 Sst 分泌和胰岛激素分泌的新治疗靶点。这个 项目将实现以下两个具体目标： 1) 确定 -cell CaSR 如何控制 Ca2 ER 处理、Sst 分泌和胰岛激素分泌； 2) 确定 TALK-1 通道如何控制 Ca2 ER 释放调节细胞功能和功能障碍。在第一个目标下，具有  细胞消融的转基因小鼠 CaSR 以及 ShRNA 敲低  细胞 CaSR 的人类伪胰岛将用于评估其作用 促分泌剂调节  细胞 Ca2 处理和 Sst 分泌过程中 Ca2 感应受体的变化。目标1将 还确定糖尿病条件下  细胞 Ca2 ER 储存的消耗如何影响 CaSR 信号传导和 Sst 分泌。在第二个目标下，TALK-1 功能通道对  细胞 Ca2 ER 处理和功能的影响将是 在具有 TALK-1  细胞特异性消融的小鼠和含有任一  细胞的人伪胰岛中测定 敲低 TALK-1 或表达显性负性 TALK-1 通道亚基。此外，Aim2 将 确定 TALK-1 在应激条件下如何增强  细胞 Ca2 ER 消耗 糖尿病会导致细胞功能障碍。这个项目意义重大，因为它有望阐明机械 改变 T2D 中  细胞 Ca2 ER 处理并破坏胰岛激素分泌的现象。此外，该项目将 确定使 Sst 分泌正常化和减少 T2D 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.