Paracrine feedback by pancreatic delta cells to control glucagon and insulin release and manage diabetes

一半的美国成年人患有糖尿病或糖尿病前期，这表明迫切需要新的治疗方法。有一个 在对胰岛旁分泌反馈如何控制胰岛素和胰升糖素的理解上存在根本差距。这个 长期的目标是阐明胰岛内的(病理性)生理串扰，以便识别新的 治疗靶点。本应用的总体目标是了解内源性SST如何抑制钙离子 在α细胞和β细胞中同时存在cAMP途径，以平衡对胰岛素和胰升糖素分泌的抑制。 一种单一的抑制性激素如何平衡对β细胞和阿尔法细胞的抑制是一个主要而糟糕的问题。 了解胰岛生理学方面的知识。中心假设是胰腺三角洲细胞与 β细胞通过同时抑制钙离子和cAMP抑制胰岛素和胰升糖素的分泌 重塑F-肌动蛋白细胞骨架以限制胞吐作用。建议进行这项研究的理由是 阐明Delta抑制α细胞和β细胞的机制和环境将建立一个更好的 了解德尔塔细胞的生理作用。这一假设将在三个具体目标上得到检验。AIM 1测试 假设β细胞和德尔塔细胞的反应在很大程度上独立于缝隙连接，通过 旁分泌因子的交换。贝塔和三角洲细胞的钙行为将在数百个贝塔中进行量化 GCaMP6在同一完整的胰岛中检测到三角洲细胞的表达。染料注入实验将确定缝隙连接 可以解释协调的增量和β细胞行为。Aim 2检验了内生增量假设 细胞反馈抑制导致协调α细胞和β细胞的SST在胰岛内强劲上升 通过cAMP和Ca~(2+)的联合抑制行为。β细胞主要通过钙离子对葡萄糖作出反应。 CAMP增强了胰岛素的释放。钙离子和cAMP将在数百个阿尔法和贝塔细胞中进行量化 在有或无SST的胰岛中表达钙和cAMP生物传感器。我们将通过以下方式量化本地SST排放 独特的SSTR2和Sstr3(ANT)激动剂，用于确定纤毛SSTR3和细胞表面SSTR2的相对重要性。 AIM 3验证SSTR2和SSTR3激活对α细胞和β细胞的不同抑制作用的假设 通过激活RhoA GTP酶重塑皮质F-肌动蛋白网络的胞吐作用。F-肌动蛋白重塑将是 使用FRET生物传感器直接检测稳定的MIN6细胞和原代胰岛对 SST，通过LifeAct测量皮质F-肌动蛋白的动态重塑，并通过量化转录变化 SSTR2和SSTR3选择性激动剂对原代α细胞和β细胞的刺激。这项研究是 申请人认为，在概念和技术上的创新，因为它评估了重要的生理 完整胰岛中的三角洲细胞在减弱α和β细胞活性中的作用。它通过应用新的 克服以前阻碍这类研究的障碍的技术。这一点意义重大，因为 三角洲细胞对胰升糖素和胰岛素分泌的控制对新策略具有广泛的翻译重要性 以遏制美国的糖尿病流行。

Half of US adults have diabetes or pre-diabetes, illustrating a critical need for novel treatments. There is a fundamental gap in the understanding of how paracrine feedback in the islet controls insulin and glucagon. The long-term goal is to elucidate the (patho)physiological crosstalk within pancreatic islets in order to identify novel therapeutic targets. The overall objective in this application is to understand how endogenous SST inhibits Ca2+ and cAMP pathways in alpha and beta cells concurrently to balance inhibition of insulin and glucagon exocytosis. How a single inhibitory hormone can balance the inhibition of beta and alpha cells is a major and poorly understood aspect of islet physiology. The central hypothesis is that the pancreatic delta cell coordinates with beta cells to attenuate insulin and glucagon secretion by inhibiting Ca2+ and cAMP while concurrently remodeling the F-actin cytoskeleton to limit exocytosis. The rationale for the proposed research is that elucidating the mechanisms and circumstances of delta inhibition of alpha and beta cells would establish a better understanding of the physiological role of delta cells. This hypothesis will be tested in 3 specific aims. Aim 1 tests the hypothesis that beta and delta cells coordinate responses largely independently of gap junctions via the exchange of paracrine factors. Beta and delta cell calcium behavior will be quantified across hundreds of beta and delta cells in the same intact islets by GCaMP6. Dye injection experiments will determine if gap junctions can account for the coordinated delta and beta cell behavior. Aim 2 tests the hypothesis that endogenous delta cell feedback inhibition leads to robust intra-islet elevations of SST that coordinate alpha and beta cell behaviors via the combined inhibition of cAMP and Ca2+. Beta cells primarily respond to glucose with Ca2+. Insulin release is potentiated by cAMP. Ca2+ and cAMP will be quantified across hundreds of alpha and beta cells expressing biosensors for Ca2+ and cAMP in islets with or without SST. We will quantify local SST release via unique Sstr2 and Sstr3 (ant)agonists to determine the relative importance of cilia SSTR3 and cell-surface Sstr2. Aim 3 tests the hypothesis that activation of SSTR2 and SSTR3 on alpha and beta cells differentially inhibits exocytosis by remodeling the cortical F-actin network by activating RhoA GTPase. F-actin remodeling will be tested directly using FRET biosensors for RhoA expressed by stable MIN6 cells and primary islets in response to SST, by measuring dynamic remodeling of cortical F-actin via LifeAct, and by quantifying transcriptional changes to stimulation of primary alpha and beta cells with SSTR2 and SSTR3-selective agonists. The research is conceptually and technically innovative, in the applicants' opinion, as it evaluates the important physiological role of delta cells within intact islets in attenuating alpha and beta cell activity. It attains this by applying new technologies to overcome the hurdles that have previously precluded such studies. This is significant because delta cell control over glucagon and insulin secretion has broad translational importance towards novel strategies to curb the diabetes epidemic in the US.