二肽基肽酶IV活性增高对胰岛素抵抗的影响及其机制研究

Insulin resistance is one of the main features of type 2 diabetes. Insulin resistance and various types of proinflammatory cytokines secretion from adipose tissue are closely related. Dipeptidyl peptidase IV (DPP4) is a serine rich protease enzyme which exerts its immune cell mediated proinflammatory effect by depending on IL-12 or binding to mannose 6-phosphate receptor. Recent studies have shown that DPP4 is also one of the factors secreted by adipose tissue, and overexpressed DPP4 is closely related to increased insulin resistance. Our population-based study further confirmed that increased DPP4 activity independently predicts an increased risk of insulin resistance and type 2 diabetes, but the exact mechanism is unknown. We speculate that DPP4 may have effect on the corresponding receptor either by 1) directly affecting insulin signal pathway or 2) as role of pro-inflammatory chemokines which increase insulin resistance. Therefore, this study follows transgenic overexpression of DPP4 to explore its impact on insulin resistance at cellular and animals level.Various combination techniques, such as His-tag pull down，yeast two-hybrid，flow cytometry，were used to clarify the possible presence of DPP4 receptors which can promote inflammatory reactions. The study will help to clarify the new idea regarding molecular mechanism of DPP4 on aggravation of insulin resistance and focus on clinical remission of T2DM.

胰岛素抵抗是2型糖尿病的主要特征，脂肪组织分泌的各类促炎细胞因子与胰岛素抵抗关系密切。二肽基肽酶IV(DPP4)是一种功能丰富的丝氨酸蛋白酶，该酶通过IL-12或与6磷酸甘露糖受体结合增强免疫细胞促炎作用。近期研究证明DPP4也是脂肪组织分泌的细胞因子，DPP4过度表达与胰岛素抵抗加重密切相关，我们的人群研究进一步证实DPP4活性增高独立预测胰岛素抵抗发生并增加2型糖尿病发生风险，但具体机制不明。我们推测DPP4可能通过与其受体结合1）直接影响胰岛素信号传导通路2）发挥趋化促炎作用加重胰岛素抵抗。因此，本课题通过转基因技术高表达DPP4，在动物、细胞水平探讨DPP4在胰岛素抵抗中发挥的作用，并结合His-tag pull down、酵母双杂交、流式细胞技术等方法筛查DPP4可能存在的受体和促炎反应的证据。上述研究将有助于阐明DPP4加重胰岛素抵抗的分子机制，为临床缓解胰岛素抵抗提供新思路。

二肽基肽酶IV(DPP4)作为糖尿病的治疗靶点，其非酶学功能越来越得到学界的关注，参考评审专家的意见，我们对所涉及的科学问题进行进一步的凝练。聚焦两个关键科学问题：1、DPP4表达升高是否能抑制胰岛素通路；2、DPP4表达升高是否会促进炎症因子表达。通过构建尾静脉高压注射质粒小鼠肝脏DPP4模型，我们发现：肝脏DPP4高表达能促进炎症因子：IL-6、IL-1β的表达、但对TNF-α表达无明显影响；构建稳定过表达DPP4的肝细胞株；过表达DPP4能够抑制胰岛素下游信号通路Akt的磷酸化，同时促进炎症细胞因子IL-1β的表达。研究DPP4非酶学功能最佳的技术手段是构建酶活性中心点突变，去除DPP4的酶功能，但保留其蛋白结构，课题组成功构建了2个酶活性中心点的突变体，一个保留蛋白结构，一个对蛋白结构有影响。通过上述研究，丰富了我们对DPP4非酶学功能的理解。同时我们发现DPP4诱导的细胞因子表达增高和Th17细胞功能密切相关，后续我们将进一步聚焦DPP4和Th17关系的研究。