Molecular Basis of Antidiabetogenic Hormone Action

描述(申请人提供)：这里提出的研究涉及一种新的磷脂酶C-epsilon(PLCϵ)，我们建议该研究介导胰升糖素样肽-1受体(GLP-1R)激动剂BYETTA在2型糖尿病(T2 DM)患者中的有益降血糖作用。我们提出的中心假设是，存在胰腺β细胞GLP-1R与cAMP产生的偶联，从而激活PLCϵ，以增强郎格汉斯胰岛的葡萄糖刺激的胰岛素分泌。通过了解这种非常规cAMP信号机制的性质，我们希望进一步的药物发现努力寻找GLP-1R激动剂，这些激动剂是纯粹的胰岛素促分泌剂，并且不会引起危险的副作用，如胰腺炎和癌症。目的1：BYETA可能通过促进在PLCϵ控制下的“晚期”胞吐来恢复T2 DM患者的胰岛素分泌。使用人的胰岛或PLCϵKO小鼠的胰岛，这一假说将在GSI的灌流或静态孵育分析中得到验证。第一个目标是确定PLCϵ是否介导了BYETA增强第一和/或第二阶段GSI的作用，或者增强了GSI的“触发”和“放大”机制。接下来，单细胞膜片钳分析结合分泌颗粒动态的双光子共聚焦显微镜将被用来测试PLCϵ的激活是否解释了BYETA介导的二酰甘油和蛋白激酶C促进胞吐的作用。为了评价BYETTA的体内作用，我们将用pDX-1-hGLP1R：Glpr-/-小鼠研究糖调节，在小鼠中存在PLCϵ的β细胞特异性KO。由于PDX-1-hGLP1R：Glpr-/-小鼠仅在胰腺表达GLP-1R，因此BYETTA可以特异性激活β细胞GLP-1R。我们预测，PLCϵ的β细胞特异性KO将在体内阻断BYETA增强GSIS的作用。目的：BYETA还可能通过使β细胞对糖代谢的刺激作用敏感来恢复T2 DM患者的胰岛素分泌。更具体地说，我们建议BYETTA通过Epa2、RAP1和PLCϵ来恢复T2 DM患者β细胞中糖代谢依赖的K-ATP通道的关闭。我们的假设采用了刺激-分泌耦合的新模型，在该模型中，K-三磷酸腺苷通道的磺酰脲受体1亚单位作为分子支架，允许形成由Epac2、RAP1和PLCϵ组成的信号转导复合体。重要的是，我们证明了cAMP传感器Epac2与SUR1结合，并且这种相互作用是由作用于Epac2的RAS关联(RA)区域的H-RAS GTP酶促进的。因此，我们假设BYETTA与生长因子或可能分泌的胰岛素协同作用，激活PLCϵ，刺激PIP2的水解，并调节K-ATP通道的三磷酸腺苷和镁-腺苷二磷酸的敏感性，从而关闭通道。这一关于离子通道调节新机制的假设将在使用人胰岛或Epa2和PLCϵ基因敲除(KO)小鼠的胰岛的K-ATP通道活性分析中得到验证。摘要：该项目的长期目标涉及我们对确定GLP-1R激动剂在T2 DM患者中有益的降血糖特性的分子基础的兴趣。

DESCRIPTION (provided by applicant): Studies proposed here concern a novel phospholipase C-epsilon (PLCϵ) that we propose mediates beneficial blood glucose-lowering actions of the glucagon-like peptide-1 receptor (GLP-1R) agonist Byetta in patients with type 2 diabetes mellitus (T2DM). The central hypothesis we present is that there exists coupling of the pancreatic beta-cell GLP-1R to cAMP production with consequent activation of PLCϵ in order to potentiate glucose-stimulated insulin secretion (GSIS) from the islets of Langerhans. By understanding the nature of this unconventional cAMP signaling mechanism, we hope to further drug discovery efforts that seek to identify GLP-1R agonists that are pure insulin secretagogues and that do not induce dangerous side effects such as pancreatitis and cancer. Aim 1: Byetta might restore insulin secretion in T2DM by facilitating a "late step" of exocytosis that is under te control of PLCϵ. Using human islets or islets of PLCϵ KO mice, this hypothesis will be tested in perfusion or static incubation assays of GSIS. A first goal is to determine if PLCϵ mediates the action of Byetta to potentiate 1st and/or 2nd phase GSIS, or to potentiate "triggering" and "amplification" mechanisms of GSIS. Next, single cell patch clamp assays in combination with 2-photon confocal microscopy of secretory granule dynamics will be performed to test if PLCϵ activation explains diacylglycerol (DAG) and protein kinase C (PKC) mediated actions of Byetta to facilitate exocytosis. To evaluate the in vivo action of Byetta, glucoregulation will be studied using Pdx-1-hGLP1R:Glpr-/- mice in which there is a beta-cell specific KO of PLCϵ. Since Pdx-1-hGLP1R:Glpr-/- mice express the GLP-1R only in the pancreas, specific activation of the beta-cell GLP-1R by administered Byetta will be possible. We predict that a beta-cell specific KO of PLCϵ will disrupt the action of Byetta to potentiate GSIS in vivo. Aim 2: Byetta might also restore insulin secretion in patients with T2DM by sensitizing beta cells to the stimulatory effect of glucose metabolism. More specifically, we propose that Byetta acts via Epac2, Rap1, and PLCϵ to restore glucose metabolism-dependent closure of K-ATP channels in beta cells of T2DM patients. Our hypothesis embraces a new model of stimulus-secretion coupling in which the sulfonylurea receptor-1 (SUR1) subunit of K-ATP channels acts as a molecular scaffold to allow the formation of a signal transduction complex comprised of Epac2, Rap1, and PLCϵ. Importantly, we demonstrate that cAMP sensor Epac2 binds to SUR1, and that this interaction is facilitated by H-Ras GTPase acting at a Ras-association (RA) domain of Epac2. Thus, we hypothesize that Byetta acts in concert with growth factors or possibly secreted insulin to activate PLCϵ, to stimulate PIP2 hydrolysis, and to modulate the ATP and Mg-ADP sensitivity of K-ATP channels in order to close the channels. This hypothesis concerning a novel mechanism of ion channel modulation will be tested in assays of K-ATP channel activity using human islets or islets of Epac2 and PLCϵ knockout (KO) mice. Summary: The long-term goal of this project concerns our interest in determining the molecular basis for beneficial blood glucose-lowering properties of GLP-1R agonists in patients with T2DM.