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

1型和2型糖尿病是公认的双激素疾病，反映了产生胰岛素的胰岛b细胞和产生胰高血糖素的胰岛a细胞的病理。虽然密集的全球努力集中在b细胞生物学和替代上，但对健康或糖尿病中a细胞活性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细胞基因调控和胰高血糖素输出等生理功能的分子机制。我们已经产生了新的工具来测量人类a细胞的功能和调节在体内，并在单细胞分辨率。我们提出了以下科学及时和医学相关的目标，这些目标将定义人类a细胞的调节因子：确定调节人类a细胞功能的机制。为了使前所未有的研究人类胰高血糖素分泌和调节，我们产生了NOD。Cg-Prkdc scidIl2rgtm1WjlSz小鼠（NSG），帧内缺失编码胰高血糖素1-29的胰高血糖素前原外显子3；称为GKO-NSG。这些小鼠允许从移植的人a细胞中定量调节胰高血糖素分泌。体外和体内的遗传、生理和基因组方法将用于确定控制人类a细胞功能的机制。具体来说，我们将分析a细胞如何受到转录因子，MAFB和RFX6的影响，我们的分析表明，这些转录因子在一个独特的，功能活跃的细胞群中富集。2. 探讨脂滴在人a细胞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一个确定的危险因素。我们将使用遗传方法和体外和体内的生理试验来确定控制脂滴（ld）积聚的机制，脂滴是人类胰岛中的一种细胞器。这些研究将验证ld调节人类a细胞受到脂肪毒性和胰岛素抵抗挑战的功能的假设。这项工作允许以前无法实现的生理机制的调查控制和调节人类a细胞。糖尿病和胰岛生物学研究的一个基本进展将是识别和描述导致糖尿病患者或有患糖尿病风险的人胰岛细胞功能下降的因素。本文提出的研究应揭示人类胰岛a细胞功能的调控机制。我们的工作应该建立范式，将基因转录和细胞器调控联系起来，以控制健康和患病胰岛的关键a细胞生理功能。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.