Molecular Basis of Antidiabetogenic Hormone Action

抗糖尿病激素作用的分子基础

• 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
• 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

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）：本文提出的研究涉及一种新型磷脂酶c - ε (plcε)，我们建议该酶介导2型糖尿病（T2DM）患者胰高血糖素样肽-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，胰岛β细胞GLP-1R与cAMP产生存在偶联，随后plc柱被激活，从而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_1控制下的胞吐“后期”来恢复T2DM患者的胰岛素分泌。使用人类胰岛或plcε KO小鼠的胰岛，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柱是否介导Byetta的作用，以增强第一阶段和/或第二阶段的GSIS，或增强GSIS的“触发”和“放大”机制。接下来，将使用单细胞膜片钳法结合双光子共聚焦显微镜观察分泌颗粒动力学，以测试plc柱活化是否解释了二酰基甘油（DAG）和蛋白激酶C （PKC）介导的Byetta促进胞外分泌的作用。为了评估Byetta的体内作用，我们将使用Pdx-1-hGLP1R:Glpr-/-小鼠进行血糖调节研究，其中存在plc柱的β细胞特异性KO。由于Pdx-1-hGLP1R:Glpr-/-小鼠仅在胰腺中表达GLP-1R，因此给予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在体内增强GSIS的作用。目的2:Byetta也可能通过使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，我们提出Byetta通过Epac2、Rap1和plc柱起作用，恢复T2DM患者β细胞中葡萄糖代谢依赖的K-ATP通道关闭。我们的假设包含了一种新的刺激-分泌偶联模型，其中K-ATP通道的磺酰脲受体-1 （SUR1）亚基作为分子支架，允许由Epac2、Rap1和plcλ组成的信号转导复合物的形成。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H-Ras GTPase作用于Epac2的Ras-association （RA）结构域促进的。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plc柱，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。这一关于离子通道调制新机制的假设将在使用人胰岛或Epac2和plcε敲除（KO）小鼠的胰岛进行K-ATP通道活性测定中得到验证。总结：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。

项目成果

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

Epac2A makes a new impact in β-cell biology.

• DOI: 10.2337/db13-0796
• 发表时间: 2013-08
• 期刊: Diabetes
• 影响因子: 7.7
• 作者: Holz GG;Chepurny OG;Leech CA
• 通讯作者: Leech CA

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
• 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。

• DOI: 10.1074/jbc.h119.007622
• 发表时间: 2019
• 期刊: The Journal of biological chemistry
• 作者: Holz,GeorgeG;Chepurny,OlegG;Leech,ColinA
• 通讯作者: Leech,ColinA