NOS1AP及其信号通路对胰岛β细胞功能和容量的调控作用及机制

Type 2 diabetes (T2D) is caused by relative insulin deficiency due to the reduction of pancreatic beta-cell mass and function. The exact mechanisms underlying the loss of functional beta-cell mass are not fully understood. Identification of key signal molecules involved in regulating functional beta-cell mass is, thus, essential to reveal potential therapeutic targets in diabetes. We reported previously that rsl2742393 of NOS1AP was associated with the pathogenesis of T2D, and C allele carriers have higher risk for T2D in Chinese. NOS1AP, encoding the nitric oxide synthase (nNOS)1 adaptor protein. As an adaptor, it interacts with nNOS via the PDZ domain helping regulate nNOS activity. It also interacts with Dexras1 via the PTB domain, which in turn to form a complex with NOS1AP and nNOS. We found previously that NOS1AP was highly expressed in both human and mouse liver and islets. Liver specific overexpression of NOS1AP in high fat diet-induced obese and ob/ob mice improved glucose tolerance and reduced triglyceride accumulation in the liver. However, the involvement of NOS1AP in the regulation of beta-cells is poorly understood. We showed in our pilot study that overexpression of NOS1AP in beta-cells potentiated glucose stimulated insulin secretion and protected the cells from FFA-induced ER stress and apoptosis. These findings, together with our previous reports, led to the hypothesis that NOS1AP may regulate beta-cell function and mass, thereby contributing to the development of T2D. Thus, we propose here to elucidate the role of the NOS1AP in the functional beta-cell mass failure under condition of nutrient overload and characterize the underlying molecular mechanisms. The dependency of NOS1AP effect on nNOS-NO and /or Dexras1 will also be explored. Completion of the project will shed light on our understanding the mechanisms involving in the regulation of functional beta-cell mass failure induced by nutrition overload in the development of T2D, and provide a novel link between NOS1AP, pancreatic beta-cells and diabetes, which could provide opportunities for treating the disease.

胰岛β细胞功能障碍和容量减低是2型糖尿病（T2D）发病核心，调控机制目前不明。我们前期研究显示NOS1AP的SNP位点是T2D发病易感基因。其蛋白在人、小鼠肝脏和胰岛中高度表达，并参与调节肝脂代谢。然而，胰岛β细胞内NOS1AP功能作用还不清楚。申请人预实验结果显示，INS-1细胞过表达NOS1AP可增加葡萄糖刺激胰岛素分泌，并降低糖、脂毒性导致的细胞内质网应激和凋亡。提示NOS1AP可能调控β细胞命运，参与T2D发病。为此，本申请拟利用NOS1AP胰岛β细胞特异性敲除小鼠和NOS1AP过表达β细胞从分子、细胞、组织和整体水平，明确NOS1AP对β细胞分泌功能和容量调控作用及分子机制。并探讨NOS1AP作用对nNOS-NO和/或Dexras1等通路的依赖性。研究结果对于深入探讨T2D 发病中β细胞功能障碍和容量降低的分子机制、开发潜在T2D 防治干预靶点，有着重要的理论意义和临床转化前景。

NOS1AP是一种转接器蛋白，在细胞信号传递中起重要作用。我们以往曾报道NOS1AP是中国人2型糖尿病（T2D）发病密切关联的易感基因。通过组织表达谱检测发现，NOS1AP在胰岛细胞和肝脏中高度表达。为此本研究对其是否对胰岛细胞功能有影响作用以及作用机制；是否对肝脏胰岛素抵抗有调控作用及其机制进行了深入探讨。研究首先发现，在小鼠整体水平上，胰腺内NOS1AP的特异敲除导致小鼠葡萄糖耐量减退、葡萄糖刺激的胰岛素分泌（GSIS）降低。在细胞水平上，过表达NOS1AP则增加GSIS，该作用与葡萄糖有氧氧化有关。胰岛细胞特异性敲除NOS1AP可导致小鼠胰岛细胞增殖率降低。过表达NOS1AP的胰岛β细胞对游离脂肪酸诱导的细胞凋亡有保护作用，该作用与调控β细胞内质网应激有关。在胰岛细胞中，NOS1AP依赖其全长发挥相应作用，单独N-端和C-端片段都不能模拟NOS1AP作用。其次，NOS1AP单核苷酸多态与人脂肪肝关联，AC+CC携带者人脂肪肝发生率显著高于AA携带者。特异敲除小鼠肝脏NOS1AP，导致葡萄糖耐量减退、胰岛素敏感性下降；肝脏脂滴沉积增加，胰岛素信号通路受损。相反，ob/ob小鼠肝脏特异性过表达NOS1AP，其葡萄糖耐量、肝脏丙酮酸耐量明显改善，肝脏脂质沉积减少，胰岛素刺激的IR/Akt 信号通路活性提高。在肝脏NOS1AP活性作用主要与其C端的 PDZ 结合区有关，NOS1AP与nNOS在肝脏共表达，小鼠肝脏过表达nNOS后加重肥胖小鼠胰岛素抵抗，增加p38 MAPK活性。相反增加NOS1AP可降低p38 MAPK活性，改善肝脏胰岛素敏感性。总之，NOS1AP无论在胰岛细胞还是在肝脏中，均参与调控细胞功能。在胰岛细胞中参与胰岛β细胞的抗游离脂肪酸毒性作用，促进葡萄糖刺激的胰岛素分泌；在肝脏通过降低肝细胞葡萄糖异生，参与调控肝脏胰岛素敏感性。我们结果提示NOS1AP可作为预防T2D潜在治疗靶点。