Secretagogue and Gi/o-GPCR signaling through the islet Na+/K+-ATPase in health and diabetes

项目摘要 2型糖尿病患者胰岛葡萄糖刺激的胰岛素和生长抑素（SST）分泌紊乱 (T2D)以及在该疾病的动物模型中，其有助于破坏葡萄糖稳态。一般 接受促分泌素刺激β细胞和β细胞的激素分泌，以响应升高的内分泌， 细胞内钙离子然而，通过Gi/o偶联受体控制胰岛Ca 2+处理抑制的机制， (Gi/o-GPCR）以及它们在T2 D中如何改变在很大程度上是未知的。我们实验室的数据发现，Gi/o-GPCR 通过Src酪氨酸激酶介导的Na+/K+-ATP酶（NKA）激活减少胰岛Ca 2+内流， 使膜电位超极化（Δ β p）并限制胰岛素分泌。进一步的数据显示，蛋白激酶A (PKA)通过Gs偶联受体的激活抑制胰岛NKA活性并刺激Ca 2+进入。此外，我们发现， 胰岛SST提供旁分泌信号，通过振荡减缓葡萄糖刺激的胰岛β细胞Ca 2+振荡， NKA活性依赖于Src酪氨酸激酶和PKA的作用。最后，我们的初步数据提供了 第一个证据表明，糖尿病条件下减少胰岛NKA活性，这有助于扰动葡萄糖 和Ca 2+处理的GPCR控制。根据这些令人兴奋的初步数据，本次亲的总体目标是， 本文旨在阐明胰岛NKA在糖尿病发病过程中是如何被控制和破坏的。 该项目将测试中心假设，即酪氨酸激酶激活胰岛NKA限制Ca 2+进入， 激素分泌通过Δ β超极化;而PKA抑制胰岛NKA可增强Ca 2+内流 和激素分泌。这个项目的基本原理是，理解信号- 整合NKA对胰岛细胞Ca 2+处理和激素分泌的调节将揭示新的治疗方法， 恢复T2 D中葡萄糖刺激的激素分泌的治疗目标。该项目将完成与 以下两个具体目标：1）确定调节NKA控制健康β细胞功能的机制 和糖尿病;和2）确定NKA如何调节β细胞功能和功能障碍。在第一个目标下，跨- 用NKA复合物亚单位的α-β-和β-亚单位的β-细胞消融以及人β-细胞 将利用敲低NKA β-和β-亚基来评估NKA在促分泌素和促分泌素分泌过程中的作用， β细胞Ca 2+处理和胰岛素分泌的Gi/o-GPCR调节。AIM 1还将确定糖尿病患者如何... 环境影响NKA信号传导和胰岛素分泌。在第二个目标下，NKA控制细胞Ca 2+处理 将在具有NKA α-β-和β-亚基的β-细胞特异性消融的小鼠中或在人类中测定其功能 具有NKA α-β-和β-亚基的β-细胞特异性敲低的假胰岛。此外，Aim 2将确定如何 在与糖尿病相关的应激条件下，胰岛β细胞中NKA功能的降低有助于胰岛β细胞 功能障碍这个项目是重要的，因为它有望阐明机制，改变β细胞和β细胞 Ca 2+处理和破坏T2 D中的胰岛激素分泌。此外，该项目计划确定潜在的phar- 正常化胰岛激素分泌和减少T2 D胰岛功能障碍的宏观策略。

Project Summary Islet glucose-stimulated insulin and somatostatin (SST) secretion are perturbed in patients with type-2 diabetes (T2D) and in animal models of the disease, which contributes to disrupted glucose homeostasis. It is generally accepted that secretagogues stimulate hormone secretion from -cells and -cells in response to elevated intra- cellular Ca2. However, the mechanisms that control inhibition of islet Ca2+ handling via Gi/o-coupled receptors (Gi/o-GPCRs) and how they are altered in T2D are largely unknown. Data from our lab finds that Gi/o-GPCRs reduce islet Ca2+ entry via Src tyrosine kinase-mediated activation of Na+/K+-ATPase (NKA), which hyperpolarizes membrane potential (Δψp) and limits insulin secretion. Further data show that Protein kinase A (PKA) activation by Gs-coupled receptors inhibits islet NKA activity and stimulates Ca2+ entry. Moreover, we find that islet SST provides paracrine signaling that slows glucose-stimulated -cell Ca2+ oscillations via oscillations in NKA activity, which depends on the action of Src tyrosine kinase and PKA. Finally, our preliminary data provide the first evidence that diabetic conditions diminish islet NKA activity, which contributes to perturbations in glucose and GPCR control of Ca2+ handling. Based on these exciting preliminary data, the overall objective of this pro- posal is to elucidate how islet NKA is controlled and becomes disrupted during the pathogenesis of diabetes. This project will test the central hypothesis that that islet NKA activation by tyrosine kinases limits Ca2+ entry and hormone secretion through Δψp hyperpolarization; whereas, PKA inhibition of islet NKAs enhances Ca2+ entry and hormone secretion by depolarizing Δψp. The rationale that underlies this project is that understanding sig- naling that integrates NKA modulation of islet cell Ca2+ handling and hormone secretion will expose novel thera- peutic targets for restoring glucose-stimulated hormone secretion in T2D. This project will be accomplished with the following two specific aims: 1) Determine the mechanisms regulating NKA control of β-cell function in health and diabetes; and 2) Determine how NKA modulates -cell function and dysfunction. Under the first aim, trans- genic mice with -cell ablation of the - - and -subunits of the NKA complex subunits as well as human -cells with knockdown of NKA - - and -subunits will be utilized to assess the roles of NKA during secretagogue and Gi/o-GPCR modulation of -cell Ca2+ handling and insulin secretion. Aim1 will also determine how diabetic con- ditions impact NKA signaling and insulin secretion. Under the second aim, NKA control of -cell Ca2+ handling and function will be determined in mice with -cell specific ablation of NKA - - and -subunits or in human pseudoislets with -cell specific knockdown of NKA - - and -subunits. Furthermore, Aim2 will determine how reduced NKA function in -cells under the stressful conditions associated with diabetes contributes to -cell dysfunction. This project is significant because it is expected to illuminate mechanisms that alter -cell and -cell Ca2+ handling and disrupt islet hormone secretion in T2D. Moreover, this project plans to identify potential phar- macological strategies for normalizing islet hormone secretion and reducing islet dysfunction in T2D.