IDENTIFICATION OF DPP2 SUBSTRATE IN THE VMN OF THE HYPOTHALAMUS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 糖代谢的控制是一个复杂的过程，任何水平的调节失调都会导致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因子，N-末端二肽酶DPP2，这是一种由Huber实验室鉴定和克隆的丝氨酸蛋白酶。我们已经产生了一只条件性DPP2基因敲除(Kd)小鼠，并将其与神经原蛋白-3(NGN3)-Cre转基因(TG)小鼠杂交，导致了大脑下丘脑和胃肠道(NGN3-DPP2kd)中DPP2的特异性kd。这些小鼠在4个月大的时候会自发地出现高胰岛素血症、葡萄糖耐受和胰岛素抵抗。此外，我们观察到NGN3-DPP2kd小鼠的食物摄入量增加，这与脂肪组织质量的显著增加有关。这种表型随着年龄的增长和高脂肪饮食的挑战而加剧。因此，我们得出结论，DPP2酶的活性对于维持葡萄糖稳态是必不可少的。这项工作是塔夫茨大学波士顿校区两位科学家的持续合作：Brigitte T.Huber，Pi，病理学系，DPP2方面的免疫学家，并培育出了条件DPP2kd小鼠；Ronald M.Lechan，内分泌学，糖尿病和新陈代谢部门，医学系，联席，神经内分泌学家和神经解剖学家，研究了DPP2在大脑中的表达模式。这一新的2型糖尿病动物模型的特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(脑)或外周(GI束)机制控制葡萄糖代谢，我们最近将条件性DPP2-loxP小鼠与SF1-CRE TG小鼠杂交，这导致DPP2kd选择性地位于下丘脑腹内侧核(VMN)，而不是GI束(SF1-DPP2kd)。这些小鼠的葡萄糖耐量与NGN3-DPP2kd小鼠相似。因此，我们现在处于一个很好的位置来确定VMN中DPP2的特定底物(S)，它可以预防高胰岛素血症和肥胖。我们正在申请RUSSO的拨款，为这个项目奠定基础，为计划于今年秋季向NIDDK提出的双调查员RO1申请奠定坚实的基础(具体目标在总结部分概述)。我们的主要目标是评估下丘脑VMN解剖的后勤条件，为最终的DPP2底物鉴定提供足够的材料。这种底物很可能是一种分泌的神经肽。因此，对于我们的初始方法，我们建议使用鸟枪蛋白质组学来分析在37℃的短时间培养后被解剖的VMN分泌的蛋白质。这项工作将与位于拉霍亚的斯克里普斯研究所的约翰·R·耶茨合作进行，他是通过这种方法识别神经肽领域的顶级专家之一。由于N末端控制蛋白质的周转，我们预计在两个菌株中DPP2亚基蛋白质的水平会有数量上的差异。此外，我们将在野生型小鼠中寻找被DPP2修饰的N端肽，而不是突变小鼠。候选底物随后将通过VMN切片的原位杂交进行分析。这一应用程序的特殊优势是两个PI的独特专业知识，具有很强的互补性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.