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：
9334841
负责人：
GEORGE G HOLZ
金额：
$ 43.72万
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 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9334841
关键词：
Adverse effects Agonist Beta Cell Binding Biological Assay Blood Glucose Cells Complex Confocal Microscopy Coupled Coupling Cyclic AMP Cyclic AMP-Dependent Protein Kinases Dangerousness Diagnosis Diglycerides Electric Capacitance Exhibits Exocytosis Funding G-Protein-Coupled Receptors GLP-I receptor 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 Metabolic 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 drug discovery exenatide glucagon-like peptide 1 glucose metabolism in vivo insulin secretagogues insulin secretion interest islet mimetics novel patch clamp phospholipase C epsilon public health relevance 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 (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.

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 (PLCϵ)，我们建议它介导胰高血糖素样肽 1 受体 (GLP-1R) 激动剂 Byetta 对 2 型糖尿病患者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 的产生存在耦合。随后激活 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，寻求识别 GLP-1R 激动剂。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。目标 1：Byetta 可能通过促进“后期步骤”恢复 T2DM 的胰岛素分泌。使用人胰岛或 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 增强第一个作用。和/或第二阶段 GSIS，或增强 GSIS 的“触发”和“放大”机制接下来，结合单细胞膜片钳测定。将进行分泌颗粒动力学的双光子共聚焦显微镜检查，以测试 PLCϵ 激活是否可以解释二酰基甘油 (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，将研究葡萄糖调节。使用 Pdx-1-hGLP1R:Glpr-/- 小鼠，其中存在 PLCϵ 的 β 细胞特异性 KO。 Pdx-1-hGLP1R：Glpr-/- 小鼠仅在胰腺中表达 GLP-1R，通过施用 Byetta 特异性激活 β 细胞 GLP-1R 将是可能的，我们预测 PLCϵ 的 β 细胞特异性 KO 将是可能的。破坏 Byetta 在体内增强 GSIS 的作用目标 2：Byetta 还可能通过使 β 细胞对葡萄糖的刺激作用敏感来恢复 T2DM 患者的胰岛素分泌。更具体地说，我们提出 Byetta 通过 Epac2、Rap1 和 PLCϵ 恢复 T2DM 患者 β 细胞中葡萄糖代谢依赖性 K-ATP 通道的关闭。 K-ATP 通道的磺酰脲受体 1 (SUR1) 亚基充当分子支架，允许形成由 Epac2、Rap1 和 PLCϵ 组成的信号转导复合物。证明 cAMP 传感器 Epac2 与 SUR1 结合，并且这种相互作用是通过作用于 Epac2 Ras 关联 (RA) 结构域的 H-Ras GTPase 促进的。因此，我们利用 Byetta 与生长因子或可能分泌的胰岛素协同作用来发挥作用。激活 PLCϵ，刺激 PIP2 水解，并调节 K-ATP 通道的 ATP 和 Mg-ADP 敏感性以关闭通道。这一假设涉及离子通道的新机制。将使用人胰岛或 Epac2 和 PLCϵ 敲除 (KO) 小鼠的胰岛来测试 K-ATP 通道活性的调节。摘要：该项目的长期目标涉及我们对确定有益血糖的分子基础的兴趣。降低 T2DM 患者 GLP-1R 激动剂的特性。

项目成果

期刊论文数量（21）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

PI3 kinases p110α and PI3K-C2β negatively regulate cAMP via PDE3/8 to control insulin secretion in mouse and human islets.

DOI：
10.1016/j.molmet.2016.05.003
发表时间：
2016-07
期刊：
Molecular metabolism
影响因子：
8.1
作者：
Kolic J;Manning Fox JE;Chepurny OG;Spigelman AF;Ferdaoussi M;Schwede F;Holz GG;MacDonald PE
通讯作者：
MacDonald PE

Leptin-stimulated KATP channel trafficking: a new paradigm for β-cell stimulus-secretion coupling?

瘦素刺激的 KATP 通道运输：β 细胞刺激分泌耦合的新范例？

DOI：
10.4161/isl.26958
发表时间：
2013
期刊：
Islets
影响因子：
2.2
作者：
Holz,GeorgeG;Chepurny,OlegG;Leech,ColinA
通讯作者：
Leech,ColinA

Intra-islet glucagon confers β-cell glucose competence for first-phase insulin secretion and favors GLP-1R stimulation by exogenous glucagon.

DOI：
10.1016/j.jbc.2021.101484
发表时间：
2022-03
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Cabrera O;Ficorilli J;Shaw J;Echeverri F;Schwede F;Chepurny OG;Leech CA;Holz GG
通讯作者：
Holz GG

"A-kinase" regulator runs amok to provide a paradigm shift in cAMP signaling.

“A-激酶”调节器疯狂运行，以提供 cAMP 信号传导的范式转变。