Elucidating the role of G6PC2 in glucose-regulation of glucagon secretion in pancreatic alpha-cells

项目摘要 血糖动态平衡是脊椎动物的中心代谢挑战，由 内分泌胰腺内产生胰升糖素的α细胞和产生胰岛素的β细胞的协调作用。 这种调节网络的紊乱会导致代谢紊乱，如2型糖尿病(T2D)- 其患病率、合并症和医疗费用构成了一个重大的公共卫生问题。尽管 定义T2D潜在的生理异常是不适当的低胰岛素分泌反应 血糖水平升高和胰岛素抵抗，T2D患者也表现出高血糖素血症 通过刺激肝脏葡萄糖的产生而加重高血糖。而葡萄糖的作用机制- 刺激的胰岛素分泌已经确立，葡萄糖对胰高血糖素的分泌有抑制作用 人们对此仍然知之甚少。支持糖酵解通量在调节胰高血糖素分泌中的关键作用 来自最近的一项研究，在该研究中，α细胞特异性地消融葡萄糖激酶损害了葡萄糖诱导的胰高血糖素 压制。此外，凯斯特纳实验室还发现使用来自T2D器官的胰岛单细胞RNAseq 供者认为，胰岛特异的葡萄糖-6-磷酸酶2(G6PC2)，它反对葡萄糖激酶的作用，是 T2D患者-细胞显著上调。这种上调在功能上很重要，因为 Kaestner实验室证实，G6PC2在小鼠假性胰岛中的过度表达增加 胰高血糖素分泌。这项研究提案将检验α细胞糖酵解通量介导胰高血糖素的假设 在2型糖尿病患者中观察到高血糖素血症的基础是其分泌及其失调。 G6PC2在调节胰高血糖素分泌中的作用将在体内进行研究 正常和糖尿病条件下可诱导的细胞特异性遗传小鼠模型。携带细胞的小鼠 特定的G6PC2消融将使用葡萄糖耐量试验来评估G6PC2在 体内血糖水平的变化。胰岛随后将被分离，并受到静态和 动态激素分泌实验验证G6PC2在体外对胰升糖素分泌的影响。此外，- 富含细胞的假性胰岛将被制备，并使用海马XFe96分析仪进行糖酵解分析 在胰高血糖素刺激和抑制条件下评估G6PC2基因操作的影响 -细胞糖酵解通量和三磷酸腺苷产生率的水平。为了确定这些发现如何应用于人类， 这些研究也将通过互补的功能丧失和功能获得研究在人类细胞中进行 使用前面描述的体外激素分泌和糖酵解率分析。 最终，研究结果将有助于我们对-细胞生物学和 T2D病理生理学，并确定改善T2D高血糖素血症和 恢复血糖动态平衡。

Project Summary Blood glucose homeostasis is a central metabolic challenge in vertebrates and is regulated by the coordinated action of glucagon-producing α-cells and insulin-producing β -cells within the endocrine pancreas. Disturbances in this regulatory network cause metabolic disorders such as type 2 diabetes mellitus (T2D) – whose prevalence, comorbidities, and medical costs constitute a significant public health concern. Although the defining physiological abnormalities underlying T2D are an inappropriately low insulin secretory response to elevated glucose levels and insulin resistance, patients with T2D also exhibit hyperglucagonemia which exacerbates hyperglycemia through stimulation of hepatic glucose production. While mechanisms of glucose- stimulated insulin secretion have been established, the suppression of glucagon secretion by glucose has remained poorly understood. Support for a critical role of glycolytic flux in regulating glucagon secretion comes from a recent study in which α-cell-specific ablation of glucokinase impaired glucose-induced glucagon suppression. Additionally, the Kaestner lab has found using single cell RNAseq of islets from T2D organ donors that the islet-specific glucose-6-phosphatase 2 (G6PC2), which opposes the action of glucokinase, is dramatically upregulated in -cells in T2D patients. This upregulation is functionally important, because overexpression of G6PC2 in murine pseudo-islets has been demonstrated by the Kaestner lab to increase glucagon secretion. This research proposal will test the hypothesis that α-cell glycolytic flux mediates glucagon secretion, and its dysregulation underlies hyperglucagonemia observed in Type 2 Diabetes patients. The function of G6PC2 in the regulation of glucagon secretion will be investigated in vivo using an inducible, -cell specific genetic mouse model under normal and diabetogenic conditions. Mice with -cell specific G6PC2 ablation will be evaluated using glucose tolerance tests to assess glucagon secretion under varying blood glucose levels in vivo. Pancreatic islets will then be isolated and subjected to both static and dynamic hormone secretion assays to verify the role of G6PC2 on glucagon secretion ex vivo. Additionally, - cell-enriched ‘pseudo islets’ will be prepared and analyzed for glycolysis using the Seahorse XFe96 Analyzer under glucagon stimulating and suppressing conditions to assess the impact of genetic manipulation of G6PC2 levels on -cell glycolytic flux and ATP production rate. To determine how these findings translate to humans, these studies will also be performed in human  cells via complementary loss- and gain-of-function studies using the previously described ex vivo hormone secretion and glycolytic rate assays. Ultimately, the results from study will contribute to our fundamental understanding of -cell biology and T2D pathophysiology, and identify a potential therapeutic target for improving T2D hyperglucagonemia and restoring blood glucose homeostasis.