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：
10540311
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/10540311
关键词：
Acceleration Affect Anabolism Anions Beta Cell Biology Biosensor Blood Glucose Brain Catabolism Cell Volumes Cells Complement Coupled Cre-LoxP Cytosol Dependence Development Devices Diabetes prevention Disease Enteroendocrine Cell Enzymes Event Feedback Fluorescence Resonance Energy Transfer Genetic Genetic Models Glucose Glutamine Glycine Goals Health Heterozygote High Pressure Liquid Chromatography Hormone secretion Human Immune Immunomodulators Impairment Insulin-Dependent Diabetes Mellitus Intervention Islet Cell 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 Neuroglia Neurons Neurotransmitters Non-Insulin-Dependent Diabetes Mellitus Optics Pancreas Paracrine Communication Pathway interactions Periodicals Periodicity Permeability Pharmacologic Substance Pharmacology Study Phenotype Physiologic pulse Physiological Physiology Production Protein Isoforms Reporting Research Role 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 neural neurotransmitter release novel paracrine pharmacologic restoration small hairpin RNA technological innovation tool type I and type II diabetes

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）是在胰岛中产生的潜在神经递质，其水平与大脑一样高。尽管GABA在神经系统中的功能是充分理解的，但胰岛GABA系统的描述被描述不同分泌途径和效应子功能的数十个相反的报告所蒙上阴影。现在很明显，GABA并不直接调节β细胞量，因此GABA在胰岛中的最终作用仍未解决。我们最近描述了人类胰岛的GABA分泌的一种新机制，该机制挑战了30年前的概念现状，该概念性现状是通过类似突触的蔬菜发生的，该胰岛分泌是GABA分泌的。取而代之的是，β细胞通过体积调节的阴离子通道（VRACS）直接从细胞质释放GABA。接下来，我们表明β细胞在常规脉冲中释放GABA，这些脉冲提供了定期反馈以帮助同步GABA的Beta细胞中的GABA通过称为GABA - 方案的途径在Beta细胞中代谢，该途径可以加速ATP的产生。我们进行了药物研究以操纵GABA合成和分解代谢，这深刻影响了葡萄糖反应性胰岛素分泌。这些结果表明，GABA对于正常的胰岛功能很重要。从这里开始，我们未来五年的目标是（1）分析人类β细胞中GABA外排的详细机制，（2）确定GABA在血糖控制中的总体作用。我们的方法实现了两种Cre-Lox条件敲除小鼠的菌株：VRAC的Beta细胞特异性缺失，VRAC是负责GABA释放的通道； GAD67（负责GABA生物合成的酶）的Beta细胞特异性缺失。后一个模型代表了胰岛特异性GABA-NULL小鼠的第一个示例。这些模型将与包括GABA生物传感器细胞，芯片微流体胰岛胰岛和胞质CA2+的光学问题，膜电位和VRAC活性的光学问题结合使用，以动态测量Islet GABA释放及其功能效应。我们将在人类胰岛中验证我们的结论，包括使用人类胰有有机切片。这项研究与人类健康有关。我们先前发现，GABA含量和分泌在1型和2型糖尿病的供体的人类胰岛受损，这表明GABA水平与糖尿病发病机理相关。我们提出的工作将确定GABA和胰岛功能之间是否存在因果关系。如果成功阐明了影响胰岛分泌的GABA机制，则可以将调节GABA系统的现有药物作为促进糖尿病预防或管理中胰岛功能的新型干预策略提出。这项研究的直接影响将是最终使GABA系统在胰岛中的作用和机制清晰。然而，这项研究具有更广泛的影响，扩展到其他神经递质（VRAC也可以渗透到甘氨酸，谷氨酰胺和牛磺酸）和其他利用包括神经元，神经胶质细胞，肠内分泌细胞和免疫细胞的GABA的细胞类型。我们的团队对人类胰岛生物学和独特的技术专长有广泛的了解，可以在这项工作中取得成功。