FFA4 regulation of pancreatic islet function

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。有一个 对旁分泌反馈控制胰岛素和胰升糖素分泌的认识存在根本差距。长期的 该研究计划的目标是阐明胰岛内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 长链脂肪酸受体4(FFA4)。这种G蛋白偶联受体是一种有希望的T2D药物靶点，它可以增加 胰岛素分泌。然而，关于涉及到的胰岛细胞类型(S)，有相互矛盾的信息 FFA4的行动。中心假设是，在胰岛内，FFA4主要在d细胞中表达， 它的激活抑制了生长抑素(SST)的释放，减轻了对胰岛素和胰升糖素分泌的紧张性抑制。这 假设是根据初步和最近发表的数据提出的，这些数据是 这两种PI的实验室都将FFA4主要表达在d细胞上。此外，刺激FFA4抑制d 并刺激b和a细胞中cAMP、钙离子和激素的分泌依赖于d细胞中ffa4的表达。 然而，ffa4激动剂刺激b细胞的增殖不依赖于d细胞，其他的则声称是直接的ffa4刺激。 B细胞的数量。提出这项研究的基本原理是确定FFA4激动剂(S)的作用机制 促进胰岛素和胰升糖素的分泌以及β细胞的增殖。这一假设将在三个具体目标上得到检验。目标1 验证了d细胞中ffa4的激活会降低cAMP的生成并抑制sst分泌的假设，这间接地 促进b和a细胞中cAMP的生成，分别刺激胰岛素和胰升糖素的分泌。SST将被删除 在基因上，或者d细胞将被白喉毒素消融，以确定刺激ffa4是否刺激b和a细胞。 SST被删除，2)d个细胞被消融。目的2检验a或b细胞直接激活FFA4的可能性最小的假设 影响荷尔蒙分泌，但促进b细胞增殖。将在a或b单元格中选择性地删除FFA4以确定 无论FFA4诱导的激素释放、cAMP生成或增殖是a细胞还是b细胞自主的。AIM 3测试 假设FFA4与D细胞中的GAZ偶联以抑制SST的分泌，并与A和/或B细胞中的GAS偶联以刺激B细胞 扩散。参与FFA4介导的胰岛反应的G蛋白将通过GAZ基因缺失来鉴定 特定的胰岛细胞与药物抑制和siRNA介导的特定G蛋白亚型的沉默相结合。 这项研究在概念和技术上都是创新的，因为它评估了d细胞在体内的重要生理作用。 完整的胰岛在减弱a和b细胞活性方面的作用，并应用复杂的功能成像来量化 在完整的胰岛内，a、b和d细胞内的cAMP和Ca~(2+)在单个细胞分辨率下。这些实验以前提供了 无法达到的单细胞分辨率结合实验中断SST介导的胰岛内反馈的方法 阐明FFA4刺激对胰岛素分泌和b细胞有益作用的机制 扩散。这一点意义重大，因为对调解有益行为的途径有充分的机械性理解 FFA4的研究将提供新的治疗方法，以改善和保护功能性b细胞团和胰岛素的分泌能力。

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 the paracrine feedback in control of insulin and glucagon secretion. The long-term goal of the research program 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 the mechanisms of action for the long chain fatty acid receptor 4 (FFA4). This G protein-coupled receptor is a promising T2D drug target that increases insulin secretion. However, there is conflicting information with regards to the islet cell type(s) involved in the salutary actions of FFA4. The central hypothesis is that within pancreatic islets FFA4 is predominantly expressed in d cells where its activation inhibits somatostatin (SST) release and alleviates tonic inhibition of insulin and glucagon secretion. This hypothesis was formulated on the basis of preliminary and recently published data produced collaboratively in the laboratories of both PIs that place FFA4 expression predominantly on d cells. Furthermore, FFA4 stimulation inhibits d cells and stimulates cAMP, Ca2+ and hormone secretion in b and a cells - effects that depend on d cell expression of FFA4. Nevertheless, FFA4 agonists stimulate b cell proliferation independently of d cells and others claim direct FFA4 stimulation of b cells. The rationale for the proposed research is to definitively resolve the mechanism(s) by which FFA4 agonists promote insulin and glucagon secretion and beta cell proliferation. This hypothesis will be tested in 3 specific aims. Aim 1 tests the hypothesis that FFA4 activation in d cells lowers cAMP generation and inhibits SST secretion, which indirectly enhances cAMP generation in b and a cells to stimulate insulin and glucagon secretion, respectively. SST will be deleted genetically, or d cells will be ablated by diphteria toxin, to determine if stimulation of FFA4 stimulates b and a cells if 1) SST is deleted, 2) d cells are ablated. Aim 2 test the hypothesis that direct FFA4 activation of a or b cells has minimal impact on hormone secretion, but promotes b-cell proliferation. FFA4 will be selectively deleted in a or b cells to determine whether FFA4-induced hormone release, cAMP generation, or proliferation is a or b cell-autonomous. Aim 3 tests the hypothesis that FFA4 couples to Gaz in d cells to inhibit SST secretion and to Gas in a and/or b cells to stimulate b-cell proliferation. The G proteins involved in FFA4-mediated islet responses will be identified by genetic deletion of Gaz in specific islet cells combined with pharmacological inhibition and siRNA-mediated silencing of select G-protein isoforms. The research is conceptually and technically innovative as it evaluates the important physiological role of d cells within intact islets in attenuating a- and b-cell activity and applies sophisticated functional imaging to quantify the kinetics of cAMP and Ca2+ in a, b, and d cells at single cell resolution within intact islets. These experiments provide previously unattainable single-cell resolution combined with approaches to experimentally interrupt SST-mediated intra-islet feedback to elucidate the mechanisms that underlie the beneficial effects of FFA4 stimulation on insulin secretion and b-cell proliferation. This is significant because a full mechanistic understanding of the pathways that mediate the salutary actions of FFA4 will inform new therapies to improve and preserve functional b-cell mass and insulin secretory capacity.