Molecular Basis of Antidiabetogenic Hormone Action

描述（由申请方提供）：本文提出的研究涉及一种新型磷脂酶C-β（PLC β），我们提出其介导胰高血糖素样肽-1受体（GLP-1 R）激动剂Byetta在2型糖尿病（T2 DM）患者中的有益降糖作用。我们提出的中心假设是，胰腺β细胞GLP-1 R与cAMP产生偶联，随后激活PLC β，以增强胰岛葡萄糖刺激的胰岛素分泌（GSIS）。通过了解这种非常规cAMP信号传导机制的性质，我们希望进一步的药物发现工作，寻求确定GLP-1 R激动剂，这些激动剂是纯胰岛素促分泌剂，不会引起危险的副作用，如胰腺炎和癌症。目的1：百泌达可能通过促进PLC控制下的胞吐“晚期”来恢复T2 DM患者的胰岛素分泌。使用人胰岛或PLC ESTK 0小鼠的胰岛，将在GSIS的灌注或静态孵育测定中测试该假设。第一个目标是确定PLC是否介导Byetta的作用以增强第一和/或第二阶段GSIS，或增强GSIS的“触发”和“放大”机制。接下来，将进行单细胞膜片钳测定结合分泌颗粒动力学的双光子共聚焦显微镜，以测试PLC β激活是否解释了二酰基甘油（DAG）和蛋白激酶C（PKC）介导的Byetta促进胞吐作用的作用。为了评价Byetta的体内作用，将使用Pdx-1-hGLP 1 R：Glpr-/-小鼠研究葡萄糖调节，其中存在PLC β细胞特异性KO。由于Pdx-1-hGLP 1 R：Glpr-/-小鼠仅在胰腺中表达GLP-1 R，因此百泌达给药可能特异性激活β细胞GLP-1 R。我们预测，PLC的β细胞特异性KO将破坏Byetta在体内增强GSIS的作用。目的2：百泌达还可能通过使β细胞对糖代谢的刺激作用敏感来恢复2型糖尿病患者的胰岛素分泌。更具体地说，我们提出百泌达通过Epac 2，Rap 1和PLC β 2来恢复T2 DM患者β细胞中K-ATP通道的葡萄糖代谢依赖性关闭。我们的假设包括一个新的刺激-分泌耦合模型，其中K-ATP通道的磺酰脲受体-1（SUR 1）亚基作为分子支架，允许形成由Epac 2，Rap 1和PLC p2p组成的信号转导复合物。重要的是，我们证明了cAMP传感器Epac 2结合到SUR 1，并且这种相互作用是由H-Ras GTdR作用于Epac 2的Ras-缔合（RA）结构域来促进的。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以激活PLC β，刺激PIP 2水解，并调节K-ATP通道的ATP和Mg-ADP敏感性以关闭通道。将在使用人胰岛或Epac 2和PLC β 2敲除（KO）小鼠的胰岛的K-ATP通道活性测定中测试关于离子通道调节的新机制的这一假设。总结：本项目的长期目标是确定GLP-1 R激动剂在T2 DM患者中有益降血糖特性的分子基础。

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.