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 在理解胰岛旁分泌反馈如何控制胰岛素和胰高血糖素方面存在根本性的差距。的 长期目标是阐明胰岛内的（病理性）生理串扰，以鉴定新的 治疗目标本申请的总体目标是了解内源性SST如何抑制Ca 2 + 以及α和β细胞中的cAMP途径，以平衡对胰岛素和胰高血糖素胞吐的抑制。 单一的抑制激素如何平衡β细胞和α细胞的抑制是一个主要的， 了解胰岛生理学方面。核心假设是胰腺δ细胞与 通过抑制Ca 2+和cAMP同时抑制β细胞的胰岛素和胰高血糖素分泌， 重塑F-肌动蛋白细胞骨架以限制胞吐作用。拟议研究的理由是， 阐明α和β细胞的δ抑制的机制和情况将建立一个更好的 了解δ细胞的生理作用。这一假设将在3个具体目标中进行检验。Aim 1测试 假设β和δ细胞协调反应很大程度上独立于间隙连接，通过 旁分泌因子的交换。β和δ细胞钙行为将在数百个β细胞中进行量化。 和δ细胞在相同的完整胰岛中通过GCaMP 6。染料注射实验将确定缝隙连接是否 可以解释δ细胞和β细胞的协同行为目的2检验了内源性δ 细胞反馈抑制导致协调α和β细胞的SST的强烈胰岛内升高 通过cAMP和Ca ~（2+）的联合抑制来实现。β细胞主要以Ca 2+响应葡萄糖。 胰岛素释放由cAMP增强。将在数百个α和β细胞中定量Ca 2+和cAMP 在有或没有SST的胰岛中表达Ca 2+和cAMP的生物传感器。我们将通过以下方式量化当地SST释放： 独特的Sstr 2和Sstr 3（ant）激动剂，以确定纤毛Sstr 3和细胞表面Sstr 2的相对重要性。 目的3检验α细胞和β细胞上SSTR 2和SSTR 3的激活差异性抑制 通过激活RhoA GTPase重塑皮质F-肌动蛋白网络来进行胞吐作用。F-肌动蛋白重塑将是 使用FRET生物传感器直接测试由稳定的MIN 6细胞和原代胰岛表达的RhoA， 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 涉及用SSTR 2和SSTR 3选择性激动剂刺激原代α和β细胞。这项研究是 在申请人看来，这在概念上和技术上都是创新的，因为它评估了重要的生理功能， 完整胰岛内的δ细胞在减弱α和β细胞活性中的作用。它通过应用新的 技术，以克服以前排除此类研究的障碍。这一点意义重大，因为 δ细胞对胰高血糖素和胰岛素分泌的控制对于新策略具有广泛的转化重要性 来遏制美国糖尿病的流行。

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.