Targeted disruption the enzymes of O-GlcNAc cycling: Animal models of Disease

靶向破坏 O-GlcNAc 循环酶：疾病动物模型

• 批准号: 8148882
• 负责人: John A. Hanover
• 金额: $ 50.73万
• 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/8148882
• 关键词: Animal Disease Models; Enzymes

The hexosamine signaling pathway terminating in O-GlcNAc cycling has been implicated in cellular signaling cascades and regulation of transcription and translation. We seek to understand the biological functions of O-GlcNAc-dependent signaling and to determine whether altered O-GlcNAc metabolism contributes to human diseases such as diabetes mellitus and neurodegeneration. Transgenic overexpression of an isoform of OGT in muscle and fat induced Insulin resistance and Hyperleptinemia in mice. These data demonstrate a central role for OGT in the insulin and leptin-signaling cascades. The findings suggest a more general role for glycan-dependent signaling in nutrient sensing and the pathogenesis of type-2 diabetes. Using reverse genetics, knockout, and other mouse transgenic models, we are currently exploring the role of the enzymes of O-GlcNAc metabolism in signal transduction and the pathogenesis of diabetes mellitus. Using cre/lox technology, we have made knockout and hypomorphic alleles of OGT in the mouse. OGT knockout animals are embryonic lethal. However, mouse embryo fibroblasts derived from these mice are being used to examine the insulin signaling cascade. Embryonic stem cells with a hypomorphic OGT allele are used for in vitro differentiation into a number of lineages including the pancreatic Beta cells. Interestingly, Beta cells derived from the hypomorphic OGT allele produce much more insulin mRNA than control cells suggesting a role for OGT in regulating insulin secretion. Recently, the human O-GlcNAcase gene was identified as a non-insulin dependent diabetes mellitus (NIDDM) susceptibility locus in Mexican Americans. We have now targeted the O-GlcNAcase gene (MGEA5) in the mouse. Using tissue specific promoters to drive expression of cre-recombinase in various target tissues, we are examining the physiological impact of O-GlcNAcase disruption. Knockout of O-GlcNAcase during early development leads to embryonic cell death. Fibroblasts derived from these knockout animals show dramatically altered O-GlcNAc levels and slower growth. Tissue-specific disruptions of the O-GlcNAcase gene are in progress. Our goal is to understand how interference with O-GlcNAc cycling may impact nutrient sensing pathways deregulated in type-2 diabetes and neurodegeneration. Analysis is being pursued in three model systems: fly, mouse and nematode. To examine the function of hexosamine signaling in a more genetically amenable organism, we have examined null alleles of OGT and the O-GlcNAcase in Caenorhabditis elegans that are viable and fertile. In nematodes, a highly conserved insulin-like signaling cascade regulates macronutrient storage, longevity and dauer formation. We demonstrate that the OGT and OGA null mutants exhibit striking metabolic changes manifested in an elevation in trehalose levels and glycogen stores with a concomitant decrease in triglycerides levels. The OGT knockout suppresses dauer larvae formation induced by a temperature sensitive allele of the insulin-like receptor gene daf-2. The OGA knockout enhances dauer formation suggesting the development of insulin resistance in the absence of O-GlcNAcase activity. Our findings demonstrate that OGT and O-GlcNAcase modulate insulin action in C. elegans and provide a unique genetic model for examining the role of O-GlcNAc in cellular signaling, insulin resistance and obesity. These studies have been extended by examining the transcriptional changes associated with interference of O-GlcNAc cycling in C. elegans. Both expression microarrays and chromatin immunoprecipitation studies argue that defects in O-GlcNAc cycling dramatically impact gene expression. It is likely that these transcriptional changes are normally linked to the nutrient sensing hexosamine-signaling pathway. Our data point to an impact on stem cell fate, which is linked to germline stem cells in C. elegans. Caenorhabditis elegans is also an excellent model system in which to examine neurodegeneration. The hexosamine signaling pathway terminating in O-GlcNAc addition has been proposed to play a key role in neurodegeneration. In these disorders, the proteins accumulating as aggregates such as tau and amyloid precursor protein are heavily modified with O-GlcNAc and are also phosphorylated. To examine the role of O-GlcNAc in tauopathy we have developed a C. elegans model of tauopathy in which the enzymes of hexosamine signaling have been systematically deleted. This strategy is based on previous work demonstrating the utility of C. elegans in modeling the one form of tauopathy, FTDP-17. We find that the loss of OGT-1, the O-GlcNAc transferase protects the nematode from human tau-induced neuropathy. This protection is associated with a decrease in the hyperphosphorylation of tau associated with aggregate formation (See Figure 2). This genetically amenable model of tauopathy is being exploited to examine how removal of OGT-1 exerts its protective effect on tau-induced neuropathy.

终止于O-GlcNAc循环的己糖胺信号通路与细胞信号级联以及转录和翻译调控有关。我们试图了解O-GlcNAc依赖性信号的生物学功能，并确定O-GlcNAc代谢的改变是否与糖尿病和神经变性等人类疾病有关。OGT在肌肉和脂肪中的转基因过表达诱导小鼠胰岛素抵抗和高瘦素血症。这些数据证明了OGT在胰岛素和瘦素信号级联反应中的核心作用。研究结果表明，在2型糖尿病的营养感知和发病机制中，聚糖依赖信号的作用更为普遍。利用反向遗传学、基因敲除等小鼠转基因模型，我们目前正在探索O-GlcNAc代谢酶在糖尿病信号转导和发病机制中的作用。利用cre/lox技术，我们在小鼠体内制备了OGT的基因敲除和半胚等位基因。OGT敲除的动物是胚胎致死的。然而，来自这些小鼠的小鼠胚胎成纤维细胞被用于检查胰岛素信号级联。具有半胚性OGT等位基因的胚胎干细胞被用于体外分化成许多谱系，包括胰腺β细胞。有趣的是，源自半胚性OGT等位基因的β细胞比对照细胞产生更多的胰岛素mRNA，这表明OGT在调节胰岛素分泌方面发挥了作用。