运用新型荧光cAMP探针研究腺苷酸环化酶6在胰岛素分泌中的作用

Diabetes is a serious disease with long-term complications causing suffering and premature death, and affects global healthcare. Type-2 diabetes is characterized by impaired insulin secretion and insulin resistance. Two major pathways are involved in insulin secretion, which are triggering pathway and amplifying pathway. The amplifying pathway promotes additional insulin secretion and is verified to be more important and critical in consistent insulin secretion. However, the mechanism underlying the amplifying pathway has not been clarified. Many factors have been suggested to be involved, and our previous studies have shown that the second messenger cAMP is one of the most important factors. The inhibition of cAMP formation will entirely terminate insulin secretion from pancreatic islet cells. cAMP is generated from ATP by adenylyl cyclases (AC), and AC6 is the most abundant AC in both mouse and human islets. Therefore, in this study we are trying to investigate the role of AC6, by monitoring cAMP and insulin secretion kinetics using a novel fluorescent indicator and total internal reflection fluorescent microscope. The differential signaling between control and AC6 gene knock-out cells will be analyzed to clarify their function in insulin secretion. Furthermore, we will utilize the diabetic mouse model and investigate whether the impaired insulin secretion will be rescued by increasing AC6 activity. Based on results, the potential of AC6 as a new anti-diabetes therapy target will be evaluated. This project will be the first attempt to real-time monitor cAMP and insulin signaling changes at the same time in intact AC6 knock-down islet cells. It is therefore important for understanding the mechanisms of insulin secretion, and identifying new anti-diabetes drug targets.

糖尿病已成为世界范围内威胁人类健康的重要疾病，它是胰岛素分泌缺陷或其生物作用受损引起的代谢性疾病，胰岛素分泌通过激发和扩增通路两个途径。近期研究表明后者对胰岛素分泌数量更关键，但此通路机制尚不明确。申请人前期研究发现环磷酸腺苷（cAMP）是扩增通路中最关键因子之一，cAMP合成的抑制会终止胰岛素分泌。本课题拟在胰岛中敲除cAMP合成酶腺苷酸环化酶6（AC6），结合申请人构建的荧光探针和荧光显微镜同步实时监测cAMP和胰岛素分泌变化，通过对照和敲除组的差异来明确AC6在胰岛素分泌中作用，探索AC6作为糖尿病药物靶点的可能性。并在胰岛素分泌缺陷的糖尿病小鼠中刺激AC6活性，来验证AC6 对胰岛素分泌的调控作用。该项目将首次在完整胰岛细胞中同步实时监测cAMP和胰岛素在AC6缺乏时的变化，从单细胞生物学角度加强对胰岛素分泌机制的研究，并结合对糖尿病小鼠模型研究为发现治疗糖尿病新药物奠定理论基础。

糖尿病是世界范围内威胁人类健康的重要疾病。环磷酸腺苷（cAMP）在胰岛素的分泌中具有重要调控作用。本项目发现使用棕榈酸处理小鼠胰岛细胞后，cAMP合成酶腺苷酸环化酶家族（ACs）中的AC9 mRNA水平明显下调，棕榈酸处理同时造成了葡萄糖诱导的胰岛素分泌的算上。当在小鼠胰岛细胞中敲除AC9后，结合荧光探针和荧光显微镜同步实时监测cAMP和胰岛素分泌变化，发现不管葡萄糖还是GLP-1诱导的cAMP信号振荡都受到了明显损伤，同时，胰岛素的分泌诱导的PIP3的活化也受到了明显损伤，而外源cAMP的添加又可以恢复胰岛素分泌诱导的PIP3的活化。实验结果证明AC9能通过cAMP信号通路调控胰岛素的分泌。本研究结果从单细胞生物学角度加强了胰岛素分泌扩增通路的研究，对于糖尿病靶点药物的研发提供了新思路。本研究还资助了全新的高通量蛋白质表达水平分析技术-定量斑点印记分析技术（QDB）的研发，为实现大规模样品中蛋白质水平的快速定量检测提供了可行性，对于临床疾病的快速诊断提供指导作用。本研究所提出的以高分辨率高敏感度质谱仪为基础，并与生物信息学相结合的蛋白质组学分析方法可应用于胃癌等多种重大慢性疾病的生物特异标记物的筛选工作，可为今后各类重大慢性疾病临床诊断和治疗提供实验和理论基础。