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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 葡萄糖代谢的控制是一个复杂的过程，任何水平的失调都可能导致糖耐量受损和胰岛素抵抗。 这两种缺陷是与肥胖和 2 型糖尿病发病相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，这是一种由 Huber 实验室鉴定并克隆的丝氨酸蛋白酶。 我们培育了一只条件性 DPP2 敲低 (kd) 小鼠，并将其与神经生成素 3 (NGN3)-Cre 转基因 (tg) 小鼠杂交，从而在大脑下丘脑和胃肠道 (GI) 中产生特定的 DPP2 kd (NGN3-DPP2 kd)。 这些小鼠在 4 个月大时自发出现高胰岛素血症、葡萄糖不耐受和胰岛素抵抗。 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。 随着年龄的增长和高脂肪饮食的挑战，这种表型会加剧。 因此，我们得出结论，DPP2 酶活性对于维持葡萄糖稳态至关重要。 这项工作是塔夫茨大学波士顿校区两位科学家之间持续进行的合作：Brigitte T. Huber，病理学系的 PI，一位免疫学家，DPP2 方面的专家，并培育出了条件性 DPP2 kd 小鼠；以及 Ronald M. Lechan，医学系内分泌、糖尿病和代谢科联合首席研究员，神经内分泌学家和神经解剖学家，研究了大脑中 DPP2 表达模式的特征。 这种新的 2 型糖尿病动物模型的表征已经发表了一份联合出版物。 为了确定 DPP2 酶活性是否通过中枢（大脑）或外周（胃肠道）机制控制葡萄糖代谢，我们最近将条件 DPP2-loxP 小鼠与 Sf1-Cre tg 小鼠杂交，这导致 DPP2 在下丘脑的腹内侧核 (VMN)，但不是胃肠道 (Sf1-DPP2 kd)。 这些小鼠出现与 NGN3-DPP2 kd 小鼠类似的葡萄糖不耐症。 因此，我们现在处于有利地位，可以确定 VMN 中 DPP2 的特异性底物，从而预防高胰岛素血症和肥胖。 我们正在申请 Russo 资助，为该项目奠定基础，为计划于今年秋季向 NIDDK 申请双重研究者 RO1 提供坚实的基础（具体目标在摘要部分概述）。 我们的主要目标是评估下丘脑 VMN 解剖的后勤情况，为最终 DPP2 底物识别提供足够的材料。 该底物很可能是一种分泌的神经肽。 因此，对于我们的初步方法，我们建议使用鸟枪蛋白质组学来分析在 37°C 短暂培养后由解剖的 VMN 分泌的蛋白质。这项工作将与位于洛杉矶斯克里普斯研究所的 John R. Yates（第 3 名）合作进行。 Jolla 是通过这种方法识别神经肽领域的顶尖专家之一。 由于 N 末端控制蛋白质周转，我们预计两个菌株中 DPP2 底物蛋白质水平存在定量差异。 此外，我们将在野生型而非突变型小鼠中寻找被 DPP2 修饰的 N 端肽。然后通过 VMN 切片的原位杂交来分析候选底物。该应用程序的特殊优势在于两位 PI 的独特专业知识，这是高度互补的。