Molecular Basis of Antidiabetogenic Hormone Action

抗糖尿病激素作用的分子基础

基本信息

批准号：
8825035
负责人：
GEORGE G HOLZ
金额：
$ 43.45万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-18 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8825035
关键词：
1,2-diacylglycerol Adverse effects Agonist Beta Cell Binding Biological Assay Blood Glucose Cells Complex Confocal Microscopy Coupled Coupling Cyclic AMP Cyclic AMP-Dependent Protein Kinases Diagnosis Diglycerides Electric Capacitance Exhibits Exocytosis Funding G-Protein-Coupled Receptors Glucose Goals Growth Factor Guanine Nucleotide Exchange Factors Guanosine Triphosphate Phosphohydrolases HRAS gene Hormones Human Hydrolysis Injection of therapeutic agent Insulin Ion Channel Islets of Langerhans Kinetics Knock-out Knockout Mice Link Malignant Neoplasms Mediating Membrane Modeling Molecular Mus Nature Non-Insulin-Dependent Diabetes Mellitus Pancreas Pancreatitis Patients Perfusion Pharmacologic Substance Phase Phosphatidylinositol 4,5-Diphosphate Potassium Channel Production Property Protein Kinase C Receptor Signaling Secretory Vesicles Signal Transduction Signal Transduction Pathway Stimulus Structure of beta Cell of islet Tamoxifen Testing analog base cell type drug discovery exenatide glucagon-like peptide glucagon-like peptide 1 glucose metabolism in vivo insulin secretagogues insulin secretion interest islet mimetics novel patch clamp phospholipase C epsilon public health relevance receptor response scaffold sensor sulfonylurea receptor treatment strategy two-photon

项目摘要

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 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 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 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.

描述（由申请人提供）：这里提出的研究涉及一种新型磷脂酶 C-ε（PLC？），我们建议它介导胰高血糖素样肽 1 受体（GLP-1R）激动剂 Byetta 对患有以下疾病的患者具有有益的降血糖作用：我们提出的中心假设是，胰腺 β 细胞 GLP-1R 与 cAMP 产生存在耦合，从而导致 2 型糖尿病 (T2DM)。激活 PLCε 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 信号传导机制的性质，我们希望进一步开展药物发现工作，寻求鉴定纯 GLP-1R 激动剂。胰岛素促分泌剂，不会引起胰腺炎和癌症等危险副作用。目标 1：Byetta 可能通过促进胞吐作用的“后期”来恢复 T2DM 中的胰岛素分泌。使用人类胰岛或 PLCε KO 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 PLCε 是否介导 Byetta 的作用以增强第一个和/或第一个。或第二阶段 GSIS，或增强 GSIS 的“触发”和“放大”机制接下来，单细胞贴片检测钳与 2 光子共焦相结合。将进行分泌颗粒动力学显微镜检查以测试如果 PLCε 激活解释了二酰基甘油 (DAG) 和蛋白激酶 C (PKC) 介导的 Byetta 促进胞吐作用的作用，为了评估 Byetta 的体内作用，将使用 Pdx-1-hGLP1R:Glpr-/- 小鼠研究血糖调节，其中由于 Pdx-1-hGLP1R:Glpr-/- 小鼠表达 PLCε，因此存在 β 细胞特异性 KO。 GLP-1R 仅在胰腺中，通过施用 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 将破坏 Byetta 的作用，从而增强体内的 GSIS。：Byetta 还可能通过使 β 细胞对葡萄糖代谢的刺激作用敏感来恢复 T2DM 患者的胰岛素分泌。更具体地说，我们建议 Byetta 通过 Epac2、Rap1 和 Epac2 发挥作用。 PLCε 可恢复 T2DM 患者 β 细胞中 K-ATP 通道的葡萄糖代谢依赖性关闭。我们的假设包含一种新的刺激-分泌耦合模型，其中 K-ATP 通道的磺酰脲受体 1 (SUR1) 亚基发挥作用。重要的是，我们证明 cAMP 传感器 Epac2 与 SUR1 结合，并且促进了这种相互作用。通过 H-Ras GTPase 作用于 Epac2 的 Ras 关联 (RA) 结构域，因此，我们研究 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 水解，并调节 ATP 和 Mg。 -K-ATP 通道的 ADP 敏感性，以关闭通道。这一关于离子通道调节新机制的假设将在使用人胰岛或胰岛的 K-ATP 通道活性测定中进行测试。 Epac2 和 PLCε 敲除 (KO) 小鼠摘要：该项目的长期目标是确定 GLP-1R 激动剂对 T2DM 患者有益的降血糖特性的分子基础。