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着根本性的差距。长期目标是阐明胰岛内(病理性)生理串扰的关键因素，以确定新的治疗靶点。本应用程序的总体目标是了解Delta细胞介导的反馈控制。多肽激素Ucn3(Ucn3)促进三角洲细胞分泌生长抑素(SST)，以抑制胰岛素和胰高血糖素。这种反馈决定了血糖的设定点，但在糖尿病中受到干扰，并有助于其病理生理学。中心假设是，胰腺三角洲细胞是一个局部控制中枢，它决定了血糖的稳态设定点，并可作为糖尿病患者重新平衡胰高血糖素和胰岛素的靶点。这一假设是根据申请人实验室提供的初步数据提出的。这项拟议研究的基本原理是了解Ucn3和SST是如何控制胰岛素和胰升糖素的，从而确定三角洲细胞依赖的反馈是糖尿病治疗的新靶点。这一假设将在三个具体目标上得到检验。目的1验证一种假说，即三角洲细胞在Ucn3的控制下分泌大部分SST，这确保了延迟的、依赖于β细胞的SST的分泌。胰岛素和SST的释放将通过胰岛灌流平行测量，并将在GCaMP6钙指示剂在这两种类型的细胞中选择性表达后，研究Delta和β细胞激活的机制的异同。目的2验证β细胞来源的Ucn3促进SST释放以抑制高糖下的α细胞的假说，而来自人α细胞的Ucn3反映了一个独立的反馈环以抑制低糖时的胰高血糖素。申请人的初步观察支持这一假设，即Ucn3促进SST分泌，抑制阿尔法细胞活性和胰高血糖素释放。在Ucn3的遗传和药物抑制后，将测量完整胰岛内α细胞的胰高血糖素分泌和钙反应。我们还将通过在小鼠阿尔法细胞中诱导Ucn3的表达，来测试在小鼠中“人性化”反馈控制的效果。目的3验证Ucn3依赖的负反馈丢失通过允许不适当的胰高血糖素释放而加重糖尿病的假说。我们将使用GCaMP6在存在和不存在Ucn3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检测继发于STZ诱导的糖尿病的三角洲细胞反馈。申请者认为，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中的三角洲细胞在减弱α细胞和β细胞活动方面的重要生理作用。它通过应用新技术来克服以前排除此类研究的障碍来实现这一点。这一点意义重大，因为Delta细胞控制胰升糖素和胰岛素分泌具有广泛的翻译重要性，并可能发现遏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.