Dufurrena Quinn, Bäck Nils, Mains Richard, Hodgson Louis, Tanowitz Herbert, Mandela Prashant, Eipper Betty, Kuliawat Regina
Department of Medicine, Stony Brook University School of Medicine, Stony Brook, New York, USA.
Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
J Mol Endocrinol. 2019 Jan;62(1):47-65. doi: 10.1530/JME-18-0048.
Key features for progression to pancreatic β-cell failure and disease are loss of glucose responsiveness and an increased ratio of secreted proinsulin to insulin. Proinsulin and insulin are stored in secretory granules (SGs) and the fine-tuning of hormone output requires signal-mediated recruitment of select SG populations according to intracellular location and age. The GTPase Rac1 coordinates multiple signaling pathways that specify SG release, and Rac1 activity is controlled in part by GDP/GTP exchange factors (GEFs). To explore the function of two large multidomain GEFs, Kalirin and Trio in β-cells, we manipulated their Rac1-specific GEF1 domain activity by using small-molecule inhibitors and by genetically ablating Kalirin. We examined age-related SG behavior employing radiolabeling protocols. Loss of Kalirin/Trio function attenuated radioactive proinsulin release by reducing constitutive-like secretion and exocytosis of 2-h-old granules. At later chase times or at steady state, Kalirin/Trio manipulations decreased glucose-stimulated insulin output. Finally, use of a Rac1 FRET biosensor with cultured β-cell lines demonstrated that Kalirin/Trio GEF1 activity was required for normal rearrangement of Rac1 to the plasma membrane in response to glucose. Rac1 activation can be evoked by both glucose metabolism and signaling through the incretin glucagon-like peptide 1 (GLP-1) receptor. GLP-1 addition restored Rac1 localization/activity and insulin secretion in the absence of Kalirin, thereby assigning Kalirin's participation to stimulatory glucose signaling.
胰腺β细胞功能衰竭和疾病进展的关键特征是葡萄糖反应性丧失以及分泌的胰岛素原与胰岛素的比例增加。胰岛素原和胰岛素储存在分泌颗粒（SGs）中，激素输出的微调需要根据细胞内位置和年龄通过信号介导的方式募集特定的SG群体。GTP酶Rac1协调多种指定SG释放的信号通路，并且Rac1的活性部分受GDP / GTP交换因子（GEFs）控制。为了探究两种大型多结构域GEFs，即Kalirin和Trio在β细胞中的功能，我们通过使用小分子抑制剂和基因敲除Kalirin来操纵它们的Rac1特异性GEF1结构域活性。我们采用放射性标记方案研究了与年龄相关的SG行为。Kalirin / Trio功能丧失通过减少2小时龄颗粒的组成型样分泌和胞吐作用来减弱放射性胰岛素原的释放。在更长的追踪时间或稳态下，对Kalirin / Trio的操作会降低葡萄糖刺激的胰岛素输出。最后，在培养的β细胞系中使用Rac1荧光共振能量转移（FRET）生物传感器表明，Kalirin / Trio GEF1活性是葡萄糖刺激后Rac1正常重排至质膜所必需的。葡萄糖代谢和通过肠促胰岛素胰高血糖素样肽1（GLP - 1）受体的信号传导均可诱发Rac1激活。在缺乏Kalirin的情况下，添加GLP - 1可恢复Rac1定位/活性和胰岛素分泌，从而确定Kalirin参与刺激葡萄糖信号传导。
