Molecular Basis of Antidiabetogenic Hormone Action

描述（由申请人提供）：本文提出的研究涉及一种新型磷脂酶C-epsilon (PLC?)，我们建议在2型糖尿病（T2DM）患者中介导胰高血糖素样肽-1受体（GLP-1R）激动剂Byetta有益的降血糖作用。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，并随之激活PLC?？以增强朗格汉斯胰岛的葡萄糖刺激胰岛素分泌（GSIS）。通过了解这种非常规cAMP信号机制的本质，我们希望进一步寻找GLP-1R激动剂的药物发现工作，这些激动剂是纯胰岛素分泌剂，不会引起胰腺炎和癌症等危险副作用。目的1:Byetta可能通过促进PLC控制下的“晚期”胞吐来恢复T2DM患者的胰岛素分泌。用人的胰岛还是PLC的胰岛？在KO小鼠中，这一假设将在GSIS的灌注或静态孵育试验中得到验证。第一个目标是确定PLC是否？？介导Byetta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，单细胞膜片钳法结合2光子共聚焦显微镜的分泌颗粒动力学将进行测试

DESCRIPTION (provided by applicant): Studies proposed here concern a novel phospholipase C-epsilon (PLC?) 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 PLC??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 PLC?. Using human islets or islets of PLC??KO mice, this hypothesis will be tested in perfusion or static incubation assays of GSIS. A first goal is to determine if PLC??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 PLC??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 PLC?. 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 PLC??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 PLC??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 PLC?. 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 PLC?, 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 PLC??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.