Single Fusion Pore Analysis via Single Cell Amperometry Uncovers Impaired Pore Expansion That Restricts Insulin Exocytosis in Human Type 2 Diabetes

Abstract Insulin secretion from vesicles within pancreatic beta cells occurs through the rapid (≤10 ms) process of exocytosis. A crucial final step in this process is the formation of a fusion pore, which connects the insulin vesicle interior to the extracellular space and insulin secretion rate. We employed real‐time single‐cell amperometry (SCA) with microsensors to quantitatively and dynamically monitor serotonin secretion (used as a proxy for insulin) and alterations in the dynamics of fusion pore formation, including opening, duration, and closing times, during single exocytosis events. Additionally, total internal reflection fluorescence (TIRF) microscopy was used to track docked vesicles and optically measure insulin release. In beta cells from human organ donors with type 2 diabetes (T2D), the fusion pore opening was prematurely aborted, leading to diminished cargo release. Furthermore, the number of docked vesicles per beta cell differed between healthy donors and individuals with T2D. In conclusion, our combined super‐sensitive optical and electrochemical analyses from a new perspective highlight a link between defective fusion pore dynamics and reduced insulin secretion (a hallmark of T2D) that has not been previously reported. These findings reveal the role of impaired fusion pore dynamics in T2D, independent of its underlying etiology, with potential therapeutic implications.