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

阐明 G6PC2 在胰腺 α 细胞胰高血糖素分泌的葡萄糖调节中的作用

• 批准号: 10064793
• 负责人: Varun Bahl
• 金额: $ 4.55万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064793
• 关键词: Ablation; Address; Affect; Alpha Cell; Beta Cell; Biological Assay; Blood Glucose; Catalytic Domain; Cause of Death; Cell secretion; Cells; Cellular Metabolic Process; Cellular biology; Diabetes Mellitus; Disease; Endocrine; Equilibrium; Event; Exhibits; Failure; Functional disorder; Futile Cycling; G6PC2 gene; Gene-Modified; Genes; Genetic; Genetic study; Genus Hippocampus; Glucagon; Glucokinase; Glucose; Glucose tolerance test; Glycolysis; Health; Hepatic; Hormonal; Hormone secretion; Human; Hyperglycemia; Impairment; In Vitro; Insulin; Insulin Resistance; Islet Cell; Islets of Langerhans; Knowledge; Mammals; Measures; Mediating; Medical Care Costs; Metabolic; Metabolic Diseases; Molecular; Mus; Non-Insulin-Dependent Diabetes Mellitus; Organ Donor; Patients; Phenotype; Physiological; Prevalence; Production; Public Health; Regulation; Research Proposals; Role; Structure of alpha Cell of islet; Techniques; Testing; Time; Translating; Up-Regulation; Vertebrates; Viral; blood glucose regulation; cell type; comorbidity; diabetogenic; fasting glucose; gain of function; genetic manipulation; glucose metabolism; glucose production; glucose-6-phosphatase; glycemic control; hyperglucagonemia; improved; in vivo; innovation; insulin secretion; islet; mouse genetics; mouse model; novel; overexpression; particle; real time monitoring; response; single-cell RNA sequencing; therapeutic target

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.

项目摘要 血糖稳态是脊椎动物中的中心代谢挑战，并且由 内分泌胰腺内胰高血糖素生成α细胞和胰岛素生成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（T2 D）- 其患病率、合并症和医疗费用构成了重大的公共卫生问题。虽然 定义T2 D潜在的生理异常是不适当的低胰岛素分泌反应， 葡萄糖水平升高和胰岛素抵抗，T2 D患者还表现出高胰高血糖素血症， 通过刺激肝葡萄糖产生而加重高血糖症。而葡萄糖的机制- 刺激胰岛素分泌已经建立，葡萄糖对胰高血糖素分泌的抑制已经建立， 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 根据最近的一项研究，α细胞特异性消融葡萄糖激酶损害葡萄糖诱导的胰高血糖素， 镇压此外，Kaestner实验室发现使用来自T2 D器官的胰岛的单细胞RNAseq 供体，胰岛特异性葡萄糖-6-磷酸酶2（G6 PC 2），它反对葡萄糖激酶的作用， 在T2 D患者的β-细胞中显著上调。这种上调在功能上很重要，因为 Kaestner实验室已经证明，G6 PC 2在鼠假胰岛中的过表达增加了 胰高血糖素分泌。本研究计划将验证α细胞糖酵解通量介导胰高血糖素的假说 高血糖素分泌及其失调是在2型糖尿病患者中观察到的高胰高血糖素血症的基础。 G6 PC 2在胰高血糖素分泌调节中的功能将使用一种新的方法在体内研究。 在正常和致糖尿病条件下的可诱导的、β-细胞特异性遗传小鼠模型。荷瘤小鼠 特异性G6 PC 2消融将使用葡萄糖耐量试验进行评价，以评估胰高血糖素分泌， 改变体内的血糖水平。然后分离胰岛，并进行静态和动态培养。 动态激素分泌测定以验证G6 PC 2对离体胰高血糖素分泌的作用。此外， 将制备富含细胞的“假胰岛”，并使用Seahorse XFe 96分析仪分析糖酵解 在胰高血糖素刺激和抑制条件下，评估G6 PC 2基因操作的影响， 水平对细胞糖酵解通量和ATP产生速率的影响。为了确定这些发现如何转化为人类， 这些研究也将通过功能丧失和获得的补充研究在人胰岛细胞中进行 使用先前描述的离体激素分泌和糖酵解速率测定。 最终，研究结果将有助于我们对细胞生物学的基本理解， T2 D病理生理学，并确定用于改善T2 D高胰高血糖素血症和 恢复血糖稳态