Mechanism and dynamics of islet GABA signaling

γ-氨基丁酸（GABA）是一种有效的神经递质，在胰岛中产生的水平与大脑中一样高。虽然GABA在神经系统中的功能已被充分理解，但对胰岛GABA系统的描述被数十个描述不同分泌途径和效应器功能的对立报告所掩盖。现在很清楚，GABA不直接调节β细胞质量，因此GABA在胰岛中的最终作用仍然没有解决。我们最近描述了一种从人类胰岛分泌GABA的新机制，挑战了30年的概念现状，即胰岛GABA分泌是通过突触样囊泡发生的。相反，β细胞通过体积调节阴离子通道（VRAC）直接从胞质溶胶释放GABA。接下来，我们发现β细胞以规律的脉冲释放GABA，提供周期性反馈，以帮助同步激素分泌。GABA也通过称为GABA分流的途径在β细胞中代谢，这可以加速ATP的产生。我们进行了药理学研究，以操纵GABA的合成和catalysts，这深刻地影响葡萄糖反应性胰岛素分泌。这些结果表明，GABA是重要的正常胰岛功能。从这里开始，我们未来五年的目标是（1）分析GABA从人类β细胞流出的详细机制，（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条件性基因敲除小鼠：β细胞特异性缺失VRAC，负责GABA释放的通道;和β细胞特异性缺失GAD 67，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，胰岛芯片微流控装置，以及用于细胞溶质Ca 2+，膜电位和VRAC活性的光学探针，以动态测量胰岛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，包括使用活的人类胰腺器官型切片。这项研究与人类健康有关。我们以前发现，GABA含量和分泌受损的人胰岛供体与1型和2型糖尿病，这表明GABA水平与糖尿病发病机制。我们建议的工作将确定GABA和胰岛功能之间是否存在因果关系。如果成功阐明影响胰岛激素分泌的GABA机制，则可以提出调节GABA系统的现有药物作为新的干预策略，以促进糖尿病预防或管理中的胰岛功能。这项研究的直接影响将是最终澄清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，扩展到其他神经递质（VRAC也可渗透甘氨酸，谷氨酰胺和牛磺酸）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。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，能够在这项奋进中取得成功。

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.