Nutrient Flux and Development

养分流动与发育

• 批准号: 8939552
• 负责人: Michael Krause
• 金额: $ 79.83万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939552
• 关键词: Acetylglucosamine; Affect; Aging; Alleles; Animal Model; Animals; Antibodies; Bioinformatics; Biological Assay; C. elegans genome; Caenorhabditis elegans; Cells; Chromatin; Collaborations; Complex; Development; Diabetes Mellitus; Disease; Enzymes; Excision; Fertility; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Models; Genome; Genomics; Glucose; Hereditary Disease; Hexosamines; Homeostasis; Human; Human Genetics; Insulin; Knock-out; Link; Longevity; Maintenance; Mammals; Mediating; Metabolic; Metabolic Diseases; Metabolism; Modeling; Mutation; National Institute of Diabetes and Digestive and Kidney Diseases; Nematoda; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Pore; Nutrient; Nutritional; Organism; Pathway interactions; Phosphotransferases; Physiology; Pore Proteins; Process; Proteins; RNA; Regulation; Role; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism; Stress; System; Testing; Time; Toxic effect; blood glucose regulation; chromatin immunoprecipitation; detection of nutrient; fly; forward genetics; human INSR protein; human disease; in vivo; insight; insulin signaling; man; mutant; novel; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; prevent; promoter; receptor; reproductive; response; sensor; transcription factor

A dynamic cycle of O-linked N-acetylglucosamine (O-GlcNAc) addition and removal acts on nuclear pore proteins, transcription factors, and kinases to modulate cellular signaling cascades. This nutrient sensing hexosamine signaling pathway is conserved from nematodes to man. A single nucleotide polymorphism in the human O-GlcNAcase gene is linked to type 2 diabetes, suggesting that perturbation of this pathway results in disease. We continue to explore the many functions of this signaling pathway in modulating aspects of nutrient sensing, development, and metabolic functions. In collaboration the Hanover lab (NIDDK), we showed that the C. elegans genome encodes the two evolutionarily conserved enzymes that mediate O-GlcNAc cycling, with the genes called ogt-1 and oga-1. We previously characterized knockout alleles of ogt-1 and oga-1 genes. Using a combination of genomic expression arrays and chromatin immunoprecipitation (ChIP) we are looking for genes that respond to nutrient flux differently in the mutants with the hope of identifying pathways of importance. The expression analysis has revealed widespread de-regulation of gene expression in the mutants, identify affected pathways including longevity and aging. We have tested these pathways in the mutants and find alteration in function that are consistent with the gene expression patterns we observe. From the ChIP studies, we have identified a discrete number of genes associated with O-GlcNAcylated proteins. These associations are pronounced at the promoters of the genes and show some overlap with ChIP signals using RNA PolII antibodies. We are currently investigating the functional roles, if any, of these restricted O-GlcNAc chromatin marks. These marks have the potential to link nutritional flux in the cell directly to gene regulation, offering a novel insight into the role of O-GlcNAc cycling in animal physiology and development. In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. During the past year, we have demonstrated that O-GlcNAc cycling and insulin signaling are both essential components of the C. elegans response to glucose stress. A number of insulin-dependent processes were found to be sensitive to glucose stress, including fertility, reproductive timing, and dauer formation, yet each of these differed in their threshold of sensitivity to glucose excess. Our findings suggest that O-GlcNAc cycling and insulin signaling are both required for a robust and adaptable response to glucose stress, but these two pathways show complex and interdependent roles in the maintenance of glucose and insulin homeostasis. We have also begun to explore the utility of C. elegans in modeling rare human genetic disorders of metabolism. In collaboration with Dr. Semple (Univ of Cambridge, UK), we have initiated a proof-of-principle study using the DAF-2 insulin-like receptor in the worm to model mutations in the human insulin receptor (INSR). Combining bioinformatic analysis and in vivo assays, we are testing mutations located throughout DAF-2 for phenotypic consequences and correlating those changes with human disease alleles. Our aim is to exploit the relatively quick and easy forward genetics, genome editing, and phenotypic assays of the C. elegans system to gain insight into human insulin receptor functional domains.

O-连接的N-乙酰葡糖胺（O-GlcNAc）添加和去除的动态循环作用于核孔蛋白、转录因子和激酶，以调节细胞信号级联。这种营养传感己糖胺信号通路是保守的从线虫到man.A单核苷酸多态性在人类O-GlcNAcase基因与2型糖尿病，这表明该通路的扰动导致疾病。 我们将继续探索这种信号通路在调节营养感测、发育和代谢功能方面的许多功能。 在与汉诺威实验室（NIDDK）的合作中，我们证明了C。线虫基因组编码两种进化上保守的酶，其介导O-GlcNAc循环，基因称为ogt-1和oga-1。 我们以前的特点敲除等位基因ogt-1和oga-1基因。使用基因组表达阵列和染色质免疫沉淀（ChIP）的组合，我们正在寻找基因，响应不同的营养通量的突变体，希望确定的重要途径。表达分析揭示了突变体中广泛的基因表达失调，确定了受影响的途径，包括长寿和衰老。 我们已经在突变体中测试了这些途径，并发现与我们观察到的基因表达模式一致的功能改变。 从ChIP研究中，我们已经鉴定了与O-GlcNAc酰化蛋白相关的离散数量的基因。 这些关联在基因的启动子处是明显的，并且使用RNA PolII抗体显示出与ChIP信号的一些重叠。 我们目前正在研究这些限制性O-GlcNAc染色质标记的功能作用（如果有的话）。 这些标记有可能将细胞中的营养通量直接与基因调控联系起来，为O-GlcNAc循环在动物生理学和发育中的作用提供了新的见解。 在各种生物体中，包括蠕虫、苍蝇和哺乳动物，葡萄糖稳态是通过在一个强大的反向和互补信号传导途径网络中的胰岛素样信号传导来维持的。己糖胺信号通路终止于O-连接-N-乙酰葡萄糖胺（O-GlcNAc）循环，是营养状态的关键传感器，并且在秀丽隐杆线虫中与胰岛素信号调节遗传相关。在过去的一年中，我们已经证明了O-GlcNAc循环和胰岛素信号传导都是C. elegans响应葡萄糖应激。许多胰岛素依赖性过程被发现对葡萄糖应激敏感，包括生育力，生殖时间和dauer形成，但每个过程对葡萄糖过量的敏感性阈值不同。我们的研究结果表明，O-GlcNAc循环和胰岛素信号都需要一个强大的和适应性的葡萄糖应激反应，但这两个途径显示复杂和相互依赖的作用，在维持葡萄糖和胰岛素的稳态。 我们也开始探索C的实用性。elegans在建模罕见的人类遗传代谢紊乱。 与Semple博士（英国剑桥大学）合作，我们启动了一项原理验证研究，使用蠕虫中的β-2胰岛素样受体模拟人胰岛素受体（INSR）的突变。 结合生物信息学分析和体内测定，我们正在测试位于整个突变的表型后果，并将这些变化与人类疾病等位基因。 我们的目标是利用相对快速和容易的正向遗传学，基因组编辑和C的表型测定。elegans系统，以深入了解人胰岛素受体功能域。