衔接蛋白APPL2对胰岛β细胞生理功能的分子调节机制研究

Type 2 diabetes mellitus (T2DM) and its related complications are the top healthcare burdens worldwide. Pancreatic ?-cell dysfunction, characterized by defective glucose-stimulated insulin secretion (GSIS) and decline of ?-cell mass, has been considered as one of the earliest defect during the pathogenesis of T2DM. Recent evidence indicate that impairment of autophagy, a conserved degradative and defensive process responsible for cellular homeostasis under stress condition, leads to hyperglycemia and ?-cell dysfunction in mice, yet its molecular underpinnings are unclear. The adaptor proteins APPL1 and APPL2, a pair of signaling molecules with same domain organization and high sequence identity, have been shown to regulate glucose metabolism via adiponectin and insulin signaling. We and other recently demonstrate that APPL1 acts as a positive regulator of action and secretion of insulin. Overexpression of APPL1 protects mice from insulin resistance and glucose intolerance, whereas knockout of APPL1 produces an opposite phenotype. Such beneficial actions of APPL1 are largely attributed to its potentiating effect on GSIS by up-regulating the expression of exocytotic proteins in ?-cells. Like APPL1, APPL2 is abundantly expressed in pancreatic ?-cells, and its expression is dramatically reduced in dietary-induced obese and db/db diabetic mouse models. Knockdown of APPL2 expression potentiates the inhibitory effects of the saturated fatty acid palmitate on GSIS, accompanied with reduced autophagy flux in pancreatic ?-cells. On the contrary, overexpression of APPL2 enhances autophagic flux. Further analysis revealed that APPL2 is translocated to autophagosome upon autophagy induction. Interestingly, APPL2 interacts with microtubule-associated protein light chain 3 (LC3), an ubiquitin-like protein involves in transport and maturation of the autophagosome. Based on these results, we propose that APPL2 protects ?-cell functions by promoting autophagy in response to metabolic stress. In this study, we will test this hypothesis by characterizing the metabolic changes in ?-cell-specific APPL2 knockout mice generated in our laboratory. We will use both in vitro, ex vivo and in vivo approaches to elucidate how APPL2 modulates ?-cell mass and insulin secretion by controlling the autophagy process. Furthermore, we will investigate whether overexpression of APPL2 alleviates hyperglycemia and ?-cell failure in db/db diabetic mice by using adeno-associated virial gene transfer system. The results are expected to provide new insight into the fundamental mechanisms regulating ?-cell function and insulin secretion, and to shed new light on the pathogenesis of glucose dysregulation in T2DM. In addition, such information may be helpful for the development of novel therapeutics to combat T2DM in human.

细胞功能异常是2型糖尿病的早期病理特征。自噬功能障碍可能导致β细胞功能异常，但机制不明。衔接蛋白APPL1和APPL2是一对具有近似结构的信号分子，我们已经报道了APPL1调节β细胞功能的机制。我们近期发现，APPL2在胰腺β细胞表达丰富，在肥胖及糖尿病小鼠显著降低。APPL2下调通过抑制自噬加重棕榈酸对葡萄糖刺激胰岛素分泌的抑制作用；APPL2与微管相关蛋白轻链3（LC3）存在相互作用。我们推测代谢应激状态下APPL2通过诱导自噬保护β细胞功能。我们将通过已经建立的β细胞特异性APPL2敲除小鼠模型，并分别在动物，离体胰岛和体外培养β细胞水平阐明APPL2如何通过对自噬过程的调控影响β细胞功能；并通过腺病毒相关病毒基因转运系统，确认过表达APPL2是否可以缓解db/db 糖尿病小鼠β细胞损伤。本研究将阐明APPL2调控自噬及其对β细胞功能的作用机制，为2型糖尿病的防治提供新的靶点。

纤维状肌动蛋白（F-肌动蛋白）动力学对于胰岛β细胞中葡萄糖刺激的胰岛素分泌（Glucose-stimulated insulin secretion [GSIS]）是必不可少的，但是其潜在的调节机制仍然知之甚少。衔接蛋白APPL1通过胞吐作用促进第一相GSIS，但其密切同源物APPL2在β细胞中的作用尚不清楚。我们的研究结果发现，APPL2通过调节胰岛β细胞中F-肌动蛋白的重塑来调节第一相和第二相GSIS。β细胞特异性敲除APPL2小鼠（β-APPL2-KO）在衰老过程中表现出受损的GSIS和葡萄糖耐受不良，这些缺陷在喂食高脂肪饮食的小鼠中进一步恶化。体外实验的结果表明，缺乏APPL2的胰岛或敲低APPL2的INS-1E β细胞株显示出双相GSIS的显著减少。另一方面，精氨酸或氯化钾诱导的胰岛素分泌不受APPL2缺乏的影响。APPL2敲除导致的GSIS缺陷不是由于葡萄糖诱导的ATP产生、钙内流及胰岛素颗粒停靠的改变引起的。进一步分析显示APPL2缺乏导致葡萄糖刺激时Rac1活化和F-肌动蛋白重塑的缺陷。重要的是，红海海绵素A 依赖的F-肌动蛋白的解聚可以恢复β-APPL2-KO小鼠胰岛中的GSIS，这表明F-肌动蛋白重塑在胰岛β细胞中处于APPL2的控制之下。蛋白质组学分析发现，APPL2与Rac GTPase活化蛋白1（RacGAP1）相互作用，负责感受β细胞中的葡萄糖刺激。随着RacGAP1的敲低或组成型活性形式Rac1的过表达，在APPL2下调的β细胞中GSIS、F-肌动蛋白重塑和Rac1活化的损害在很大程度上得到了补救。此外，在db/db糖尿病小鼠的胰岛中，APPL2的表达显著降低，并伴随着GSIS和F-肌动蛋白重塑的损伤。腺病毒介导的APPL2过表达能够逆转db/db糖尿病小鼠的β细胞功能障碍。我们的数据表明，APPL2-RacGAP1-Rac1轴在通过F-肌动蛋白重塑调节双相GSIS中起着重要作用。