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，这说明了对新型糖尿病治疗的迫切需求。有一个 在控制胰岛素和胰高血糖素分泌的旁分泌反馈的理解上存在根本性的差距。长期 研究计划的目标是阐明胰岛内（病理性）生理串扰的关键参与者， 来确定新的治疗靶点。本申请的总体目标是了解以下作用机制： 长链脂肪酸受体4（FFA 4）。这种G蛋白偶联受体是一种有前途的T2D药物靶点， 胰岛素分泌然而，关于有益的胰岛细胞类型存在相互矛盾的信息。 FFA4的作用。中心假设是在胰岛内FFA 4主要在d细胞中表达， 它的激活抑制生长抑素（SST）的释放并增强胰岛素和胰高血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， 实验室的两个PI，放置FFA 4表达主要在d细胞。此外，FFA 4刺激抑制了细胞凋亡。 细胞并刺激B和a细胞中的cAMP、Ca2+和激素分泌-依赖于FFA 4的d细胞表达的效应。 然而，FFA 4激动剂刺激B细胞增殖独立于d细胞，而其他人声称直接FFA 4刺激 B细胞。拟议研究的基本原理是明确解决FFA 4激动剂 促进胰岛素和胰高血糖素分泌和β细胞增殖。这一假设将在3个具体目标中进行检验。要求1 检验了d细胞中FFA 4活化降低cAMP生成并抑制SST分泌的假设， 增强B和a细胞中cAMP的产生，以分别刺激胰岛素和胰高血糖素分泌。SST将被删除 或d细胞将被白喉毒素消融，以确定FFA 4的刺激是否刺激B和a细胞，如果1） SST被删除，2）d细胞被消融。目的2检验a或B细胞的直接FFA 4活化具有最小的 影响激素分泌，但促进b细胞增殖。FFA 4将在a或B细胞中选择性缺失，以确定 FFA 4诱导的激素释放、cAMP产生或增殖是否是a或B细胞自主的。目标3测试 假设FFA 4与d细胞中Gaz偶联以抑制SST分泌，并与a和/或B细胞中的Gas偶联以刺激b细胞 增殖参与FFA 4介导的胰岛应答的G蛋白将通过在细胞中Gaz的遗传缺失来鉴定。 特异性胰岛细胞结合药理学抑制和siRNA介导的选择G蛋白亚型沉默。 这项研究在概念上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，并应用复杂的功能成像来量化的动力学， 完整胰岛内单细胞分辨率下a、B和d细胞中的cAMP和Ca2+。这些实验提供了以前 无法达到的单细胞分辨率结合实验性中断SST介导的胰岛内反馈的方法 为了阐明FFA 4刺激对胰岛素分泌和b细胞增殖的有益作用的机制， 增殖这是重要的，因为一个完整的机械理解的途径，调解有益的行动， FFA 4的发现将为改善和保护功能性B细胞群和胰岛素分泌能力的新疗法提供信息。

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.