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糖尿病治疗方法。在胰岛旁分泌反馈如何控制胰岛素和胰高血糖素的理解上存在根本性的差距。长期目标是阐明胰岛内（病理）生理串扰的关键参与者，以确定新的治疗靶点。在这个应用程序的总体目标是了解delta细胞介导的反馈控制。肽激素尿皮质素3 （Ucn3）促进三角洲细胞分泌生长抑素（Sst），以减弱胰岛素和胰高血糖素。这种反馈决定了血浆葡萄糖的设定点，但在糖尿病中受到干扰，并有助于其病理生理。核心假设是，胰三角洲细胞是一个局部控制中心，决定葡萄糖的稳态设定值，并可靶向重新平衡糖尿病患者的胰高血糖素和胰岛素。这一假设是根据申请人实验室产生的初步数据制定的。该研究的基本原理是了解Ucn3和Sst如何控制胰岛素和胰高血糖素，从而确定delta细胞依赖性反馈作为糖尿病治疗的新靶点。这一假设将在三个具体目标中进行检验。Aim 1验证了delta细胞在Ucn3激活GPCR的控制下分泌大部分Sst的假设，这保证了延迟的β细胞依赖性Sst分泌。胰岛素和Sst释放将通过胰岛灌注并行测量，并在GCaMP6钙指示剂在两种细胞类型中选择性表达后，研究δ和β细胞激活的机制异同。Aim 2验证了β细胞来源的Ucn3在高糖条件下促进Sst释放抑制α细胞的假设，而来自人类α细胞的Ucn3在低糖条件下反映了一个单独的反馈回路，以减弱胰高血糖素。这一假设得到了申请人的初步观察结果的支持，即Ucn3促进Sst分泌，抑制α细胞活性和胰高血糖素释放。完整胰岛内α细胞的胰高血糖素分泌和钙反应将在遗传和药理学抑制Ucn3后进行测量。我们还将通过诱导Ucn3在小鼠α细胞中的表达来测试“人源化”反馈控制在小鼠中的效果。目的3验证了ucn3依赖性负反馈的缺失通过允许不适当的胰高血糖素释放而加重糖尿病的假设。我们将使用GCaMP6在Ucn3存在和不存在的情况下测量糖尿病模型胰岛中的α和β细胞反应，并使用化学遗传学在体内恢复stz诱导的糖尿病继发的delta细胞反馈。在申请人看来，这项研究在概念和技术上都是创新的，因为它评估了完整胰岛内δ细胞在减弱α和β细胞活性方面的重要生理作用。它通过应用新技术来克服以前阻碍此类研究的障碍来实现这一目标。这是很重要的，因为delta细胞控制胰高血糖素和胰岛素分泌具有广泛的翻译重要性，并可能发现抑制美国糖尿病流行的新策略。

Half of US adults have diabetes or pre-diabetes, illustrating a critical need for novel diabetes 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 key players in (patho)physiological crosstalk within pancreatic islets in order to identify novel therapeutic targets. The overall objective in this application is to understand delta cell-mediated feedback control. The peptide hormone urocortin3 (Ucn3) promotes somatostatin (Sst) secretion from delta cells in order to attenuate insulin and glucagon. This feedback determines the set-point for plasma glucose, but is perturbed in diabetes and contributes to its pathophysiology. The central hypothesis is that the pancreatic delta cell is a local control hub that determines the homeostatic set point for glucose and can be targeted to rebalance glucagon and insulin in diabetes. This hypothesis was formulated on the basis of preliminary data produced in the applicants' laboratory. The rationale for the proposed research is to understand how Ucn3 and Sst control insulin and glucagon, thus identifying delta cell-dependent feedback as a novel target for diabetes treatment. This hypothesis will be tested in 3 specific aims. Aim 1 tests the hypothesis that delta cells secrete most of their Sst under control of GPCR activation by Ucn3, which assures delayed, beta cell-dependent Sst secretion. Insulin and Sst release will be measured in parallel by islet perfusion, and the mechanistic similarities and differences of delta and beta cell activation will be investigated following the selective expression of the GCaMP6 calcium indicator in both cell types. Aim 2 tests the hypothesis that beta cell-derived Ucn3 promotes Sst release to inhibit alpha cells under high glucose, while Ucn3 from human alpha cells reflects a separate feedback loop to attenuate glucagon at low glucose. This hypothesis is supported by the applicants' preliminary observations that Ucn3 promotes Sst secretion and inhibits alpha cell activity and glucagon release. Glucagon secretion and calcium responses of alpha cells within intact islets will be measured following genetic and pharmacological inhibition of Ucn3. We will also test the effect of `humanizing' feedback control in mice by the inducible expression of Ucn3 in mouse alpha cells. Aim 3 tests the hypothesis that loss of Ucn3-dependent negative feedback aggravates diabetes by allowing inappropriate glucagon release. We will measure alpha and beta cell responses in islets of diabetes models using GCaMP6 in the presence and absence of Ucn3, and the restoration of delta cell feedback using chemicogenetics in vivo secondary to STZ-induced diabetes. 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 and could uncover novel strategies to curb the diabetes epidemic in the US.