IDENTIFICATION OF DPP2 SUBSTRATE IN THE VMN OF THE HYPOTHALAMUS

该子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其他NIH来源。 列出的子项目总成本可能 表示子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 葡萄糖代谢的控制是一个复杂的过程，任何水平的失调都可能导致葡萄糖耐量受损和胰岛素抵抗。 这两个缺陷是与肥胖和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的葡萄糖代谢调节剂，N-末端二肽酶，DPP 2，一种丝氨酸蛋白酶，已被Huber实验室鉴定和克隆。 我们已经产生了条件性DPP 2敲低（kd）小鼠，并将其与神经生成素-3（NGN 3）-Cre转基因（tg）小鼠杂交，所述转基因小鼠在脑的下丘脑和胃肠道（GI）道中产生特定的DPP 2 kd（NGN 3-DPP 2 kd）。 这些小鼠在4月龄时自发地发展高胰岛素血症、葡萄糖耐受不良和胰岛素抗性。 此外，我们观察到NGN 3-DPP 2 kd小鼠的食物摄入量增加，这与脂肪组织质量的显著增加相关。 这种表型随着年龄的增长和高脂饮食的挑战而加剧。 因此，我们得出结论，DPP 2酶活性是维持葡萄糖稳态所必需的。 这项工作是塔夫茨大学波士顿校区的两名科学家之间正在进行的合作。Huber，PI，病理学系，一位免疫学家，他是DPP 2方面的专家，并且已经产生了条件性DPP 2 kd小鼠，和罗纳德M. Lechan，共同PI，医学系，内分泌学，糖尿病和代谢部，神经内分泌学家和神经解剖学家，他描述了DPP 2在大脑中的表达模式。 一份联合出版物已经从这种新的2型糖尿病动物模型的特征中产生。 为了确定DPP 2酶活性是否通过中枢（脑）或外周（胃肠道）机制控制葡萄糖代谢，我们最近将条件性DPP 2-loxP小鼠与Sf 1-Cre tg小鼠杂交，这导致DPP 2 kd选择性地在下丘脑的腹内侧核（VMN）中，而不是胃肠道（Sf 1-DPP 2 kd）。 这些小鼠发展出与NGN 3-DPP 2 kd小鼠相似的葡萄糖耐受不良。 因此，我们现在处于一个很好的位置，以确定特定的底物（S）的DPP 2在VMN，防止高胰岛素血症和肥胖。 我们正在申请Russo赠款，为该项目奠定基础，为计划于今年秋季向NIDDK提出的双重研究者RO 1申请提供坚实的基础（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解剖的物流，以产生足够的材料，最终DPP 2底物鉴定。 这种底物很可能是一种分泌的神经肽。 因此，对于我们最初的方法，我们建议使用鸟枪蛋白质组学来分析在37 ℃短时间培养后由解剖的VMN分泌的蛋白质。 这项工作将与John R.耶茨，3日，在斯克里普斯研究所在拉霍亚，在该领域的顶级专家之一，确定神经肽的这种方法。 由于N-末端控制蛋白质周转，我们期望在两种菌株中看到DPP 2底物蛋白水平的定量差异。 此外，我们将在野生型小鼠中寻找被DPP 2修饰的N-末端肽，但在突变小鼠中没有。然后通过VMN切片的原位杂交分析候选底物。该应用程序的特殊优势在于两个PI的独特专业知识，这是高度互补的。

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.