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

项目摘要/摘要 O-谷氨酸氨基转移酶是一种在胰腺β-细胞中高表达的营养感受性蛋白。这 该酶专一催化目标胞浆蛋白和核蛋白(O- GlcN酰化)。β-细胞OGT基因敲除小鼠出现严重的糖尿病表型，表明OGT在 塑造血糖动态平衡。这项研究的长期目标是了解 蛋白质O-GlcN酰化形成β-细胞健康和功能。我们的数据表明OGT和OGT之间存在很强的联系 以及雷帕霉素复合体1的机制靶点mTORC1mTORC1.mTORC1.在β细胞中是一种关键的营养敏感蛋白。而当 在人类糖尿病患者的胰岛中观察到异常的自噬和慢性mTORC1激活， 这些信号通路如何变得失调的机制尚不清楚。我们建议，作为回应， 对于营养胁迫，蛋白质O-GlcN酰化直接调节mTORC1活性及其下游 线粒体通过自噬过程发挥作用。我们的中心假设是，在营养胁迫中 条件下，O-GlcN酰化减少通过mTORC1的低激活导致胰岛素分泌不足 以及自噬的过度激活。该提案的主要目标是描述OGT之间的信号传递 和mTORC 1-自噬及其对β-细胞健康和功能的贡献。特定目标1将调查如何 O-GlcN酰化调节mTORC 1活性以及β-细胞的质量和功能。特定目标2将测试O- GlcN酰化调控β细胞自噬并确定自噬失调的后果 在细胞器动态平衡中。这项F31建议是创新的，因为目前还没有研究将OGT 和mTORC_1在胰岛中的串扰及其对依赖自噬的β细胞团和胰岛素的调节 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 如高血糖和高脂血症导致细胞器功能异常改变，并鉴定特异性β细胞 OGT和mTORC1信号之间的调节节点，可以作为治疗的靶点，以防止 二型糖尿病的发展。该提案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，以及技术技能，包括基于灌流的胰岛素 分泌物分析、特殊成像技术(透射电子显微镜、共聚焦、活细胞)和海马XF线粒体 呼吸测定。在口头和书面陈述中进行科学交流，以及指导领导力 成为一名成功的院士的培训中不可或缺的一部分。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敬业导师是 可帮助学员成功完成其F31计划，并帮助其实现其职业生涯 和学术目标。

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.