In vivo systems to discover mechanisms regulating human islet alpha cell function

1型和2型糖尿病被认为是双激素疾病，在产生胰岛素的胰岛b细胞和产生胰高血糖素的胰岛a细胞中都反映了病理。虽然全球密集的努力集中在b细胞生物学和替代上，但人们对a细胞活动在健康或糖尿病中的遗传或分子效应知之甚少，特别是在人类胰岛中。迫切需要发现控制人类a细胞基因调控和生理功能的分子机制，如胰高血糖素的输出。我们已经开发出新的工具，可以在体内以单细胞分辨率测量人类a细胞的功能和调节。我们提出了以下科学上及时和医学上相关的目标来定义人类a细胞的调节：1.确定调节人类a细胞功能的机制。为了能够对人类胰高血糖素的分泌和调节进行前所未有的研究，我们产生了NOD.Cg-Prkdc cerdIl2rgtm1WjlSz小鼠(NSG)，其前胰高血糖素前外显子3的框内缺失，编码GGLG1-29；称为GKO-NSG。这些小鼠允许量化从移植的人类a细胞中调节的胰升糖素分泌。体外和体内的遗传学、生理学和基因组学方法将被用来识别控制人类a细胞功能的机制。具体地说，我们将分析转录因子MAFB和RFX6是如何影响a-细胞的，我们的分析表明，这两种转录因子在一个独特的、功能活跃的细胞群中富含。2.探讨脂滴在人a细胞对脂毒性适应性反应中的作用。脂肪毒性和伴随的胰岛素抵抗是人类糖尿病发病的既定风险。我们将使用遗传方法和体外和体内的生理测试来确定控制人类胰岛细胞器脂滴(LDS)积累的机制。这些研究将检验这样一种假设，即低密度脂蛋白调节受到脂毒性和胰岛素抵抗挑战的人类a细胞的功能。这项工作使得以前无法实现的对控制和调节人类a细胞的生理机制的研究成为可能。糖尿病和胰岛生物学研究的一个根本性进展将是识别和表征导致糖尿病患者或有发生糖尿病风险的人的胰岛细胞功能下降的因素。人类胰岛a细胞的功能调控机制应该通过这里提出的研究来揭示。我们的工作应该建立一个范例，将基因转录和细胞器调节与控制健康和患病胰岛的关键a细胞生理功能联系起来。适当调节胰岛a细胞功能是健康的基本特征，我们的工作将通过描述在正常和病理条件下改善胰岛a细胞功能的方法产生广泛的影响。

Type 1 and Type 2 diabetes mellitus are recognized as bi-hormonal diseases reflecting pathology in both insulin-producing islet b-cells and glucagon-producing islet a-cells. While intensive global efforts have focused on b-cell biology and replacement, little is known about the genetic or molecular effectors of a-cell activity in health or in diabetes, especially in human islets. Discovery of molecular mechanisms that govern human a-cell gene regulation and physiological functions like glucagon output are urgently needed. We have generated new tools to measure human a-cell function and regulation in vivo, and at single-cell resolution. We propose the following scientifically-timely and medically-relevant Aims that will define regulators of human a-cells: 1. Identify mechanisms regulating human a-cell function. To enable unprecedented studies of human glucagon secretion and regulation, we generated NOD.Cg-Prkdc scidIl2rgtm1WjlSz mice (NSG) with an in-frame deletion of preproglucagon exon 3 that encodes Glucagon 1-29; termed GKO-NSG. These mice permit quantification of regulated glucagon secretion from transplanted human a-cells. Genetic, physiological, and genomic approaches in vitro and in vivo will be used to identify mechanisms governing human a-cell function. Specifically, we will analyze how a-cells are impacted by transcription factors, MAFB and RFX6, which our analysis indicates are enriched in a distinct, functionally active cell population. 2. Investigate the role of lipid droplets in human a-cell adaptive responses to lipotoxicity. Lipotoxicity and accompanying insulin resistance is an established risk for diabetes pathogenesis in humans. We will use genetic approaches and physiological assays in vitro and in vivo to identify mechanisms governing accumulation of lipid droplets (LDs), an organelle in human islets. These studies will test the hypothesis that LDs regulate function of human a-cells challenged by lipotoxicity and insulin resistance. This work allows previously unattainable investigation of physiological mechanisms governing and regulating human a-cells. A fundamental advance in diabetes and islet biology research would be the identification and characterization of factors that lead to decreased islet cell function in those with diabetes, or at risk for developing diabetes. Mechanisms governing function of human islet a-cells should be revealed by the studies proposed here. Our work should establish paradigms that connect gene transcriptional and organelle regulation to control of key a-cell physiological functions in healthy and diseased pancreatic islets. Appropriately regulated islet a-cell function is an essential feature of health, and our work will have broad impact by describing ways to ameliorate islet a-cell function, in both normal and pathological conditions.