IDENTIFICATION OF DPP2 SUBSTRATE IN THE VMN OF THE HYPOTHALAMUS

下丘脑 VMN 中 DPP2 底物的鉴定

• 批准号: 8365792
• 负责人: Brigitte T. Huber
• 金额: $ 1.28万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365792
• 关键词: Adipose tissue; Age; Age-Months; Animal Model; Biology; Boston; Brain; Cell Nucleus; Characteristics; Collaborations; Complex; Defect; Diabetes Mellitus; Diet; Dipeptidases; Dissection; Eating; Endocrinology; Fatty acid glycerol esters; Funding; Fungal Genome; Gastrointestinal tract structure; Glucose Intolerance; Goals; Grant; Hyperinsulinism; Hypothalamic structure; Immunologist; In Situ Hybridization; Insulin Resistance; Joints; Logistics; Medicine; Metabolism; Mus; Mutant Strains Mice; N-terminal; National Center for Research Resources; National Institute of Diabetes and Digestive and Kidney Diseases; Neuropeptides; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Pathology; Pattern; Peptides; Peripheral; Phenotype; Positioning Attribute; Principal Investigator; Process; Proteins; Proteomics; Publications; Research; Research Infrastructure; Research Institute; Research Personnel; Resources; Scientist; Serine Protease; Shotguns; Solid; Source; Transgenic Mice; United States National Institutes of Health; Work; base; blood glucose regulation; cost; enzyme activity; falls; glucose metabolism; impaired glucose tolerance; knock-down; mouse Neurog3 protein; novel; prevent; protein degradation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The control of glucose metabolism is a complex process, and dysregulation at any level can cause impaired glucose tolerance and insulin resistance. These two defects are well-known characteristics associated with obesity and onset of type 2 diabetes. We have recently discovered a novel regulator of glucose metabolism, the N-terminal dipeptidase, DPP2, a serine protease that had been identified and cloned by the Huber lab. We have generated a conditional DPP2 knock down (kd) mouse and crossed it with a neurogenin-3 (NGN3)-Cre transgenic (tg) mouse that led to specific kd of DPP2 in the hypothalamus of the brain and the gastrointestinal (GI) tract (NGN3-DPP2 kd). These mice spontaneously develop hyperinsulinemia, glucose intolerance and insulin resistance by 4 months of age. In addition, we observed an increase in food intake in NGN3-DPP2 kd mice, which was associated with a significant increase in adipose tissue mass. This phenotype was exacerbated with age and when challenged with a high fat diet. We conclude, therefore, that DPP2 enzyme activity is essential for maintaining glucose homeostasis. This work constitutes an ongoing collaboration between two scientists on the Boston campus of Tufts: Brigitte T. Huber, PI, Department of Pathology, an immunologist who is an expert on DPP2 and has produced the conditional DPP2 kd mouse, and Ronald M. Lechan, co-PI, Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, a neuroendocrinologist and neuroanatomist who has characterized the DPP2 expression pattern in the brain. A joint publication has already resulted from the characterization of this new animal model for type 2 diabetes. In order to define whether the DPP2 enzyme activity controls glucose metabolism over a central (brain) or peripheral (GI tract) mechanism, we have recently crossed the conditional DPP2-loxP mice with Sf1-Cre tg mice, which leads to DPP2 kd selectively in the ventromedial nucleus (VMN) of the hypothalamus, but not the GI tract (Sf1-DPP2 kd). These mice develop similar glucose intolerance as the NGN3-DPP2 kd mice. Thus, we are now in an excellent position to identify the specific substrate(s) of DPP2 in the VMN that prevents hyperinsulinemia and obesity. We are applying for a Russo grant to lay the groundwork for this project, providing a solid basis for a dual investigator RO1 application to NIDDK that is planned for fall of this year (the specific aims are outlined in the summary section). Our main goal is to assess the logistics of VMN dissection in the hypothalamus in terms of yielding sufficient material for eventual DPP2 substrate identification. It is highly likely that this substrate is a secreted neuropeptide. Thus, for our initial approach, we propose to use shotgun proteomics to analyze the proteins secreted by the dissected VMN after short culture at 37 C. This work will be performed in collaboration with John R. Yates, 3rd, at Scripps Research Institute in La Jolla, one of the top experts in the field of identifying neuropeptides by this approach. Since the N-terminus controls protein turnover, we expect to see a quantitative difference in the level of a DPP2-subsrate protein in the two strains. In addition, we will look for N-terminal peptides that are modified by DPP2 in wild type, but not mutant mice. Candidate substrates will then be analyzed by in situ hybridization of sections of the VMN. The special strength of this application is the unique expertise of the two PIs, which is highly complementary.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 葡萄糖代谢的控制是一个复杂的过程，任何水平的失调可能会导致葡萄糖耐受性和胰岛素抵抗受损。 这两个缺陷是与肥胖和2型糖尿病发作有关的众所周知的特征。 我们最近发现了一种新型的葡萄糖代谢调节剂，即N末端二肽酶DPP2，DPP2是Huber Lab鉴定和克隆的丝氨酸蛋白酶。 我们已经产生了有条件的DPP2敲低（KD）小鼠，并用神经蛋白蛋白3（NGN3）-CRE转基因（TG）小鼠交叉，该小鼠在大脑下丘脑和胃肠道（GI）（GI）（NGN3-DPP2 KD）（NGN3-DPP2 KD）中导致了DPP2的特异性KD。 这些小鼠自发发展高胰岛素血症，葡萄糖不耐症和胰岛素抵抗，到4个月大。 此外，我们观察到NGN3-DPP2 KD小鼠的食物摄入量增加，这与脂肪组织质量的显着增加有关。 这种表型随着年龄的增长而加剧，并在高脂肪饮食中受到挑战。 因此，我们得出的结论是，DPP2酶活性对于维持葡萄糖稳态至关重要。 This work constitutes an ongoing collaboration between two scientists on the Boston campus of Tufts: Brigitte T. Huber, PI, Department of Pathology, an immunologist who is an expert on DPP2 and has produced the conditional DPP2 kd mouse, and Ronald M. Lechan, co-PI, Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, a neuroendocrinologist and神经解剖学家表征了大脑中DPP2表达模式。 这种新动物模型的2型糖尿病的表征已经引起了共同的出版物。 为了定义DPP2酶活性是否控制着中央（脑）或外围（GI段）机制上的葡萄糖代谢，我们最近越过条件DPP2-LOXP小鼠SF1-CRE TG鼠标，该小鼠用SF1-CRE TG鼠标跨越了dpp2 kd，从而导致腹膜内核（VME）的DPP2 kd（vme neftract），但注意到了p. hypoth and tract of and dpp tg tg。 （SF1-DPP2 KD）。 这些小鼠产生的葡萄糖不耐症与NGN3-DPP2 KD小鼠相似。 因此，我们现在处于良好的位置，可以在VMN中识别DPP2的特定底物，以防止高胰岛素血症和肥胖。 我们正在申请RUSSO赠款，以为该项目奠定基础，为今年秋季计划的双重研究者RO1申请提供了可靠的基础（在摘要部分中概述了特定目的）。 我们的主要目标是评估下丘脑中VMN解剖的物流，以产生足够的材料来最终DPP2底物鉴定。 该底物很有可能是分泌的神经肽。 因此，对于我们的初始方法，我们建议使用shot弹枪蛋白质组学来分析37 C短期文化后解剖VMN分泌的蛋白质。这项工作将与第三名的约翰·耶茨（John R. 由于N末端控制蛋白质周转率，因此我们期望这两种菌株中DPP2-甲酸盐蛋白的水平存在定量差异。 此外，我们将寻找通过野生型（而不是突变小鼠）修饰的N末端肽。然后，将通过对VMN部分的原位杂交来分析候选底物。该应用的特殊优势是两个PI的独特专业知识，这是高度互补的。