Mechanism and dynamics of islet GABA signaling

胰岛 GABA 信号传导机制和动力学

基本信息

批准号：
10318211
负责人：
Edward Phelps
金额：
$ 36.72万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10318211
关键词：
Affect Anabolism Anions Beta Cell Biology Biosensor Blood Glucose Brain Catabolism Cell Volumes Cells Complement Coupled Cre-LoxP Cytosol Dependence Development Diabetes prevention Disease Enteroendocrine Cell Event Feedback Fluorescence Resonance Energy Transfer GAD67 enzyme Genetic Genetic Models Glucose Glutamine Glycine Goals Health High Pressure Liquid Chromatography Hormone secretion Human Immune Impairment Insulin-Dependent Diabetes Mellitus Intervention Islets of Langerhans Knock-out Knockout Mice Knowledge Link LoxP-flanked allele Measurement Measures Mediating Membrane Potentials Metabolic Metabolism Microfluidic Microchips Modeling Mus Nervous system structure Neuroglia Neurons Neurotransmitters Non-Insulin-Dependent Diabetes Mellitus Optics Pancreas Paracrine Communication Pathway interactions Periodicity Permeability Pharmacologic Substance Pharmacology Pharmacology Study Phenotype Physiologic pulse Physiological Physiology Production Protein Isoforms Reporting Research Role Salvelinus Secretory Vesicles Shunt Device Signal Transduction Slice Synapses System Taurine Technical Expertise Time Vesicle Whole Organism Work blood glucose regulation cell type conditional knockout diabetes management diabetes pathogenesis enzyme biosynthesis extracellular gamma-Aminobutyric Acid glucose tolerance glycemic control insulin secretion islet neurotransmitter release novel paracrine relating to nervous system restoration small hairpin RNA technological innovation tool

项目摘要

Gamma-aminobutyric acid (GABA) is a potent neurotransmitter produced in the islet at levels as high as in the brain. While the function of GABA in the nervous system is well-understood, the description of the islet GABA system is clouded by dozens of antithetical reports describing differing secretion pathways and effector functions. It is now clear that GABA does not directly regulate beta cell mass, so GABA’s ultimate role in the islet remains unresolved. We recently described a new mechanism for GABA secretion from human islets that challenges the 30-year-old conceptual status quo that islet GABA secretion occurs via synaptic-like vesicles. Instead, beta cells release GABA directly from the cytosol via volume regulated anion channels (VRACs). Next, we showed that beta cells release GABA in regular pulses that provide periodic feedback to help synchronize hormone secretion. GABA is also metabolized in the beta cell through a pathway called the GABA-shunt, which can accelerate ATP production. We conducted pharmacological studies to manipulate GABA synthesis and catabolism, which profoundly impacted glucose-responsive insulin secretion. These results establish that GABA is important for normal islet function. From here, our Aims over the next five years are to (1) analyze the detailed mechanism of GABA efflux from human beta cells and (2) determine the overall role of GABA in glycemic control. Our approach implements two strains of Cre-Lox conditional knockout mice: beta cell-specific deletion of VRAC, the channel responsible for GABA release; and beta cell-specific deletion of GAD67, the enzyme responsible for GABA biosynthesis. The latter model represents the first example of an islet-specific GABA-null mouse. These models will be combined with technological innovations including GABA biosensor cells, islet-on-a-chip microfluidic devices, and optical probes for cytosolic Ca2+, membrane potential, and VRAC activity to dynamically measure islet GABA release and its functional effects. We will validate our conclusions in human islets including the use of live human pancreas organotypic slices. This research has relevance for human health. We previously found that GABA content and secretion are impaired in human islets from donors with type 1 and type 2 diabetes, suggesting GABA levels correlate with diabetes pathogenesis. Our proposed work will establish if there is a causal linkage between GABA and islet function. If successful in elucidating GABA mechanisms that affect islet hormone secretion, existing pharmaceuticals that modulate GABA systems can be proposed as novel intervention strategies to promote islet function in diabetes prevention or management. The immediate impact of this study will be to finally bring clarity to the role and mechanisms of the GABA system in islets. However, this research has broader impacts that extend to other neurotransmitters (VRAC is also permeable to glycine, glutamine, and taurine) and other cell type that utilize GABA including neurons, glial cells, enteroendocrine cells, and immune cells. Our team has extensive knowledge of human islet biology and unique technical expertise to succeed in this endeavor.

γ-氨基丁酸 (GABA) 是一种强效神经递质，在胰岛中产生的水平与大脑中的水平一样高。描述不同分泌途径和效应器功能的对立报告现在很清楚，GABA 不直接调节 β 细胞质量，因此我们最近描述的 GABA 在胰岛中的最终作用仍未解决。人类胰岛分泌 GABA 的新机制挑战了 30 年来胰岛 GABA 分泌通过突触样囊泡发生的概念现状，相反，β 细胞通过容量调节阴离子通道 (VRAC) 直接从细胞质中释放 GABA。接下来，我们发现β细胞以规律的脉冲释放GABA，从而提供周期性反馈以帮助同步激素分泌。GABA也在β细胞中通过称为“GABA”的途径进行代谢。 GABA 分流，可以加速 ATP 的产生。我们进行了药理学研究来操纵 GABA 的合成和分解代谢，这对葡萄糖反应性胰岛素的分泌产生了深远的影响。这些结果表明，GABA 对于正常的胰岛功能非常重要。五年的时间是（1）分析 GABA 从人类 β 细胞流出的详细机制，以及（2）确定 GABA 在血糖控制中的总体作用。我们的方法实施了两种 Cre-Lox 菌株。条件性基因敲除小鼠：VRAC（负责 GABA 释放的通道）的 β 细胞特异性缺失；以及 GAD67（负责 GABA 生物合成的酶）的 β 细胞特异性缺失。后一种模型代表了胰岛特异性 GABA 缺失的第一个例子。这些模型将与技术创新相结合，包括 GABA 生物传感器细胞、芯片上的胰岛微流体装置以及用于胞质 Ca2+、膜电位和 VRAC 的光学探针。我们将在人类胰岛中验证我们的结论，包括使用活的人类胰腺器官切片，我们之前发现人类的 GABA 含量和分泌受到损害。来自 1 型和 2 型糖尿病捐献者的胰岛，表明 GABA 水平与糖尿病发病机制相关。如果成功阐明影响 GABA 的机制，我们将确定 GABA 和胰岛功能之间是否存在因果关系。胰岛激素分泌，调节 GABA 系统的现有药物可以作为促进糖尿病预防或管理中胰岛功能的新干预策略，这项研究的直接影响将是最终阐明 GABA 系统在胰岛中的作用和机制。然而，这项研究具有更广泛的影响，延伸到其他神经递质（VRAC 也可渗透甘氨酸、谷氨酰胺和牛磺酸）和利用 GABA 的其他细胞类型，包括神经元、神经胶质细胞、肠内分泌细胞我们的团队拥有丰富的人类胰岛生物学知识和独特的技术专长，可以在这一努力中取得成功。