Mechanism and dynamics of islet GABA signaling

γ-氨基丁酸(GABA)是一种在胰岛产生的强大的神经递质，其水平与大脑一样高。虽然GABA在神经系统中的功能已为人所熟知，但对胰岛GABA系统的描述却被数十篇描述不同分泌途径和效应器功能的相反报告所笼罩。现在清楚的是，GABA并不直接调节β细胞质量，因此GABA在胰岛中的最终作用仍未解决。我们最近描述了一种新的人类胰岛分泌GABA的机制，挑战了30年来胰岛通过突触样囊泡分泌GABA的概念现状。相反，β细胞通过体积调节的阴离子通道(VRAC)直接从胞浆中释放GABA。接下来，我们展示了β细胞以规则的脉冲形式释放GABA，这种脉冲提供周期性反馈，帮助同步激素的分泌。GABA还通过一条称为GABA分流的途径在β细胞中代谢，该途径可以加速ATP的产生。我们进行了药理学研究，以控制GABA的合成和分解代谢，这深刻地影响了葡萄糖反应性胰岛素的分泌。这些结果表明，GABA对正常的胰岛功能是重要的。从这里开始，我们在未来五年的目标是(1)分析GABA从人类β细胞外流的详细机制，(2)确定GABA在血糖控制中的总体作用。我们的方法实现了两个品系的Cre-Lox条件性基因敲除小鼠：β细胞特异性的vrac缺失，负责GABA释放的通道；以及β细胞特异性的GAD67，负责GABA生物合成的酶。后一种模型代表了胰岛特异性GABA缺失小鼠的第一个例子。这些模型将与技术创新相结合，包括GABA生物传感器细胞、芯片上胰岛微流控设备，以及用于胞浆钙离子、膜电位和vrac活性的光学探针，以动态测量胰岛GABA的释放及其功能效应。我们将在人类胰岛上验证我们的结论，包括使用活的人胰腺器官型切片。这项研究对人类健康具有相关性。我们之前发现1型和2型糖尿病供者的胰岛GABA含量和分泌受到损害，这表明GABA水平与糖尿病的发病机制有关。我们建议的工作将确定GABA和胰岛功能之间是否存在因果联系。如果成功地阐明了影响胰岛激素分泌的GABA机制，现有的调节GABA系统的药物可以被建议作为新的干预策略，以促进糖尿病预防或治疗中的胰岛功能。这项研究的直接影响将是最终明确GABA系统在胰岛中的作用和机制。然而，这项研究对其他神经递质(vrac对甘氨酸、谷氨酰胺和牛磺酸也具有渗透性)和其他利用GABA的细胞类型(包括神经元、神经胶质细胞、肠内分泌细胞和免疫细胞)具有更广泛的影响。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，能够在这一努力中取得成功。

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.