Regulation of Autophagy-driven Pancreatic Beta Cell Function by Nutrient Sensor Proteins

项目总结/摘要 O-GlcNAc转移酶（OGT）是在胰腺β细胞中高度表达的营养传感蛋白。这 酶专门催化靶细胞溶质和核蛋白（O-）的翻译后糖基化。 GlcNAc酰化）。β-细胞OGT敲除小鼠发展为严重的糖尿病表型，表明OGT在糖尿病中至关重要。 形成葡萄糖稳态。这项研究的长期目标是了解 蛋白质O-GlcNAc化塑造β细胞的健康和功能。我们的数据表明，OGT之间有很强的关系， 和雷帕霉素复合物1的机制靶点，mTORC 1，β细胞中的关键营养传感器激酶。而 在来自人类糖尿病患者的胰岛中观察到异常的自噬和慢性mTORC 1活化， 这些信号通路如何变得失调的机制是未知的。我们建议，作为回应， 在营养胁迫下，蛋白质O-GlcNAc化直接调节mTORC 1活性， 线粒体功能通过自噬的过程。我们的中心假设是在营养胁迫下 在某些条件下，O-GlcNAc化降低通过mTORC 1的低活化导致胰岛素分泌缺陷 和自噬的过度激活。本提案的主要目标是描述OGT与 和mTORC 1-自噬及其对β细胞健康和功能的贡献。具体目标1将研究如何 O-GlcNAc化调节mTORC 1活性和β细胞质量和功能。具体目标2将测试是否O- GlcNAc酰化调节β细胞中的自噬并确定失调的自噬的后果 在细胞器内稳态中的作用这个F31的建议是创新的，因为目前还没有研究联系OGT 胰岛中mTORC 1和mTORC 1的相互作用及其对自噬依赖的β细胞群和胰岛素的调节 分泌物该提案的成功完成将揭示营养胁迫条件之间的联系机制 如细胞器功能障碍异常变化和鉴定特异性β-细胞 OGT和mTORC 1信号传导之间的调节节点，可以作为治疗剂的靶点，以防止 2型糖尿病的治疗该提议纳入了新的和及时需要的概念培训， 胰岛生物学、自噬和线粒体方面的知识，以及包括灌注胰岛素在内的技术技能 分泌测定、专门成像技术（TEM、共聚焦、活细胞）和海马XF线粒体 呼吸测定口头和书面报告中的科学交流，以及指导领导能力 成为培训的一个组成部分，成为一个成功的院士。所有的技术资源以及 明尼苏达大学和综合生物学和生理学系的专职导师是 帮助学员成功完成F31计划并帮助他实现职业生涯 学术目标。

PROJECT SUMMARY/ABSTRACT O-GlcNAc transferase (OGT) is a nutrient sensor protein which is highly expressed in pancreatic β-cells. This enzyme exclusively catalyzes the post-translational glycosylation of target cytosolic and nucleic proteins (O- GlcNAcylation). β-cell OGT knockout mice develop severe diabetic phenotype, suggesting that OGT is crucial in shaping glucose homeostasis. The long-term goal of this research is to understand the mechanisms of how protein O-GlcNAcylation shape β-cell health and function. Our data suggest a strong relationship between OGT and mechanistic target of Rapamycin complex 1, mTORC1, a key nutrient-sensor kinase in β-cells. While aberrant autophagy and chronic mTORC1 activation is observed in islets from human diabetic patients, the mechanisms of how these signaling pathways become dysregulated is unknown. We propose that in response to nutrient stress, protein O-GlcNAcylation directly regulates mTORC1 activity and subsequent downstream mitochondrial function through the process of autophagy. Our central hypothesis is that in nutrient stress conditions, decreased O-GlcNAcylation leads to insulin secretion deficits through hypo-activation of mTORC1 and hyper-activation of autophagy. The main objective of this proposal is to delineate the signaling between OGT and mTORC1-autophagy and their contribution to β-cell health and function. Specific Aim 1 will investigate how O-GlcNAcylation regulates mTORC1 activity and β-cell mass and function. Specific Aim 2 will test whether O- GlcNAcylation modulates autophagy in β-cells and to determine the consequences of dysregulated autophagy in organelle homeostasis. This F31 proposal is innovative because there are currently no studies linking OGT and mTORC1 crosstalk in pancreatic islets and their regulation of autophagy-dependent β-cell mass and insulin secretion. Successful completion of the proposal will reveal the mechanisms linking nutrient stress conditions such as hyper-glycemia and -lipidemia to aberrant changes in organelle dysfunction and identify specific β-cell nodes of regulation between OGT and mTORC1 signaling that can be targeted for therapeutics to prevent the development of type 2 diabetes. The proposal incorporates new and timely-needed training in conceptual knowledge in islet biology, autophagy, and mitochondria, and technical skills including perifusion based insulin secretion assay, specialize imaging techniques (TEM, confocal, live cell), and Seahorse XF mitochondrial respiration assay. Scientific communication in oral and written presentation, as well as mentoring leadership will be an integral part of training to become a successful academician. All the technical resources as well as dedicated mentors at University of Minnesota and the department of Integrative Biology and Physiology are available to assist the trainee to successfully complete his F31 proposal and to assist him achieved his career and academic goals.