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.