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，这说明了对新型糖尿病治疗的迫切需求。在了解胰岛中的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。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。本申请的总体目标是理解δ细胞介导的反馈控制。肽激素尿皮质素3（Ucn3）促进生长抑素（Sst）从δ细胞分泌以减弱胰岛素和胰高血糖素。这种反馈决定了血糖的设定点，但在糖尿病中会受到干扰，并导致其病理生理学。核心假设是胰腺δ细胞是决定葡萄糖稳态设定点的局部控制中心，并且可以靶向重新平衡糖尿病中的胰高血糖素和胰岛素。这一假设是根据申请人实验室的初步数据提出的。这项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δ细胞依赖性反馈作为糖尿病治疗的新靶点。这一假设将在3个具体目标中进行检验。目的1测试的假设，S细胞分泌的大部分Sst的GPCR激活Ucn3的控制下，这确保延迟，β细胞依赖的Sst分泌。将通过胰岛灌注平行测量胰岛素和Sst释放，并在两种细胞类型中选择性表达GCaMP6钙指示剂后研究δ和β细胞活化的机制相似性和差异。目的2测试以下假设：β细胞衍生的Ucn3促进Sst释放以在高葡萄糖下抑制α细胞，而来自人α细胞的Ucn3反映单独的反馈回路以在低葡萄糖下减弱胰高血糖素。该假设得到了申请人的初步观察结果的支持，即Ucn3促进Sst分泌并抑制α细胞活性和胰高血糖素释放。在Ucn 3的遗传和药理学抑制后，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α细胞中诱导表达Ucn 3来测试小鼠中“人源化”反馈控制的效果。目的3检验Ucn 3依赖性负反馈的丧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。我们将在存在和不存在Ucn3的情况下使用GCaMP6测量糖尿病模型胰岛中的α和β细胞反应，并使用体内继发于STZ诱导的糖尿病的化学遗传学恢复δ细胞反馈。在申请人看来，该研究在概念上和技术上是创新的，因为它评估了完整胰岛内的δ细胞在减弱α和β细胞活性中的重要生理作用。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。这是重要的，因为δ细胞对胰高血糖素和胰岛素分泌的控制具有广泛的转化重要性，并且可以揭示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.