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.