Modulation of insulin and glucagon secretion by GPCR ligands

描述(申请人提供)：葡萄糖稳态是由郎格汉斯胰岛分泌的激素调节的，主要是胰岛素和胰升糖素。虽然葡萄糖刺激胰岛素分泌(GSIS)的机制已经确立，但G蛋白偶联受体(GPCRs)调节胰岛素和胰升糖素分泌的分子机制尚不清楚。考虑到GPCR配体(即GLP-1类似物)已成为治疗2型糖尿病的一种有前途的药物，这一点尤其重要。神经肽-Y(NPY)是一种普遍存在的多肽信使，在胰岛局部产生，通过GI介导的腺酰环化酶抑制和cAMP减少而降低GSIS。相比之下，NPY已被证明能刺激小鼠和大鼠胰岛分泌胰高血糖素。生长抑素(SST)由胰腺细胞分泌，有SST-14和SST-28两种活性形式。这两种形式都与SSTR2和SSTR5结合，SSTR2和SSTR5在胰岛大量表达。与NPY一样，SST通过胃肠道途径抑制胰岛素的分泌，但据报道它也对胰高血糖素的分泌有抑制作用。为了了解这两种GPCR配体的不同作用，我们建议测量NPY和SST信号对胰岛细胞代谢、电活动和激素分泌的影响。在这里，假设NPY抑制胰岛素下游的分泌，刺激钙信号上游的胰升糖素的分泌。SST改变了钙信号的上下游通路，通过类似的机制抑制胰岛素和胰升糖素的分泌。提出了两个具体的目标：1)确定NPY和SST抑制细胞胰岛素分泌的机制；2)确定NPY和SST调节胰升糖素分泌的信号通路。拟议的实验结合了新的和传统的成像和电生理学技术来解决这些目标。转基因小鼠模型的使用允许明确的细胞鉴定，并增强了我们表征胰岛信号通路的能力。NADH和NADPH(NAD(P)H)的联合自发荧光信号将被用来测量细胞的氧化还原状态，该方法将用于研究NPY和SST对细胞代谢的影响。电压门控钙通道的激活和随后的膜去极化是胰岛素和胰升糖素分泌的必要组成部分，这些事件可以用电生理和荧光方法来测量。深入了解离子通道功能、钙信号和胞吐作用在神经肽Y或SST存在或不存在的情况下，将有助于阐明这些抑制配体调节胰岛素和胰升糖素分泌的机制(S)。这些生物物理技术将与传统的激素分泌测量和生化分析相关联。由此得到的定量数据将使我们能够缩小范围，并最终确定受这些GPCR配体调控的精确分子途径。 公共卫生相关性：为了全面了解美国流行的2型糖尿病，我们需要了解胰岛激素分泌的复杂调节。胰岛素和胰升糖素分泌的变化可能是机体试图克服2型糖尿病期间胰岛素抵抗的一种代偿机制。深入了解其他激素和神经系统优化胰岛素和胰升糖素分泌的机制，将使人们能够更全面地了解2型糖尿病的病理，从而有助于未来治疗方法的开发和国家的整体健康。

DESCRIPTION (provided by applicant): Glucose homeostasis is regulated by the orchestrated secretion of hormones, primarily insulin and glucagon, from the pancreatic islet of Langerhans. Although the mechanism of glucose-stimulated insulin secretion (GSIS) is well-established, the molecular mechanisms that underlie the modulation of insulin and glucagon secretion by G-protein coupled receptors (GPCRs) are not clearly understood. This is especially significant considering GPCR ligands (i.e. GLP-1 analogs) have emerged as a promising treatment of Type-2 diabetes. Neuropeptide-Y (NPY) is a ubiquitous peptide messenger that is locally produced in pancreatic islets and decreases GSIS through Gi-mediated inhibition of adenylyl cyclase and reduced cAMP. In contrast, NPY has been shown to stimulate glucagon secretion from mouse and rat islets. Somatostatin (SST) is secreted by the pancreatic / cells and has two active forms-SST-14 and SST-28. Both forms bind with high affinity to SSTR2 and SSTR5, which are expressed abundantly in islets. Like NPY, SST inhibits insulin secretion through the Gi pathway, but it is reported also to have an inhibitory effect on glucagon secretion. To understand the differential effects of these two GPCR ligands, we propose to measure the effects of NPY and SST signaling on islet cellular metabolism, electrical activity, and hormone secretion. Here, it is hypothesized that NPY inhibits insulin secretion downstream and stimulates glucagon secretion upstream of Ca2+ signaling. SST alters pathways both upstream and downstream of Ca2+ signaling, inhibiting insulin and glucagon secretion through a similar mechanism. Two specific aims are proposed: 1) Determine the mechanisms by which NPY and SST inhibit -cell insulin secretion; 2) Determine the signaling pathways of glucagon secretion modulated by NPY and SST. The proposed experiments combine novel and conventional imaging and electrophysiology techniques to address the aims. The use of a transgenic mouse model permits definitive .-cell identification and enhances our ability to characterize islet signaling pathways. The cellular redox state will be measured by the combined autofluorescence signal from NADH and NADPH (NAD(P)H); this assay will be used to investigate the effects of NPY and SST on cellular metabolism. Activation of voltage-gated Ca2+ channels and subsequent membrane depolarization are necessary components of insulin and glucagon secretion, and these events can be measured using electrophysiological and fluorescence approaches. Insight into ion channel function, Ca2+ signaling, and exocytosis in the presence and absence of NPY or SST will help elucidate the mechanism(s) by which these inhibitory ligands modulate insulin and glucagon secretion. These biophysical techniques will be correlated with traditional hormone secretion measurements and biochemical assays. The resulting quantitative data will allow us to narrow down and, eventually, determine the precise molecular pathways modulated by these GPCR ligands. PUBLIC HEALTH RELEVANCE: To wholly understand the American epidemic that is Type-2 diabetes, we need to understand the complex regulation of hormone secretion from pancreatic islets. Changes in insulin and glucagon secretion may be a compensatory mechanism by which the body attempts to overcome insulin-resistance during Type-2 diabetes. Insight into the mechanisms by which insulin and glucagon secretion are optimized by other hormones and the nervous system will allow for a more comprehensive understanding of the pathology of Type-2 diabetes, and will thus contribute to the development of future therapies and the overall health of the nation.