Regulatory Mechanisms of SNAP-25-Associated Insulin Release Revealed by Live-Cell Confocal and Single-Molecule Localization Imaging

Impaired insulin release is the key feature of type 2 diabetes. Insulin secretion, mainly mediated by SNARE proteins, is closely related to the blood glucose level. However, the mechanism underlying how glucose controls SNARE proteins to regulate insulin release is largely unexplained. Herein, we investigated the effects of glucose on the subcellular localization and spatial distribution on the plasma membrane (PM) of t-SNAREs (SNAP-25 and STX-1A) using a live-cell confocal microscope and the single-molecule localization imaging technique. Live-cell confocal and dSTORM imaging first revealed that SNAP-25 was mostly localized to the PM as clusters under the basal glucose concentration condition and demonstrated significant colocalization with STX-1A clusters. Furthermore, our data showed that the elevated glucose concentration increased the expression of SNAP-25 and induced more and larger SNAP-25 clusters on the PM, whereas glucotoxicity severely inhibited SNAP-25 transport to the PM and caused fewer and smaller SNAP-25 clusters on the PM. Additionally, we found that glucotoxicity also had an inhibitory effect on the colocalization between SNAP-25 and STX-1A, indicating a decrease of their interactions. Our study sheds light on the regulatory effects of glucose on the functional organization of t-SNAREs at a subcellular and molecular level, thus providing new insights into the mechanisms by which SNAREs regulate insulin release.

胰岛素释放受损是2型糖尿病的核心特征。胰岛素分泌主要由SNARE蛋白（SNARE proteins）介导，其过程与血糖水平密切相关。然而，葡萄糖如何调控SNARE蛋白以调节胰岛素释放的潜在机制，目前仍未得到充分阐明。本研究借助活细胞共聚焦显微镜与单分子定位成像技术，探究了葡萄糖对t-SNARE蛋白（包括SNAP-25与STX-1A）在质膜（PM）上的亚细胞定位与空间分布的影响。活细胞共聚焦成像与dSTORM成像首先揭示：在基础葡萄糖浓度条件下，SNAP-25主要以簇状形式定位于质膜，并与STX-1A簇存在显著共定位。此外，本研究数据显示，高葡萄糖浓度会提升SNAP-25的表达水平，并诱导质膜上形成更多、更大的SNAP-25簇；而糖毒性则会显著抑制SNAP-25向质膜的转运，导致质膜上的SNAP-25簇数量更少、体积更小。此外，我们还发现糖毒性对SNAP-25与STX-1A之间的共定位也具有抑制作用，表明二者的相互作用有所减弱。本研究从亚细胞与分子层面阐明了葡萄糖对t-SNARE蛋白功能组装的调控作用，从而为SNARE蛋白调控胰岛素释放的机制提供了全新的研究视角。