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Type-2 diabetes (T2D) is characterized by progressive β-cell dysfunction and impaired insulin secretion, yet the molecular mechanisms remain incompletely understood. In pancreatic β-cells, insulin secretion occurs through exocytosis, a process whereby insulin-containing secretory granules dock at the plasma membrane, recruit proteins that make up the SNARE-dependent exocytosis machinery (priming), and ultimately fuse with the membrane to release their content to the extracellular space. This thesis investigates the molecular machinery governing insulin granule dynamics and exocytosis in both healthy and diabetic conditions. Through analysis of secretory machinery components, we demonstrate distinct roles for SNARE binding protein Munc18 isoforms. Both Munc18 isoforms support granule docking, but Munc18-1 is strikingly required for exocytosis. On a molecular level, both isoforms bind to syntaxin, but are not recruited to the granule release site to the same extent. Our investigation of the v-SNARE protein VAMP8 reveals its predominant localization to endosomal compartments rather than insulin granules. Furthermore, we identify VAMP8 as a negative regulator of insulin secretion, likely by competing with VAMP2 at the release site, suggesting a new regulatory mechanism in β-cell function. We further examine phosphatidylinositol transfer protein alpha (PITPNA) as a critical regulator of insulin granule maturation. Modulation of PITPNA levels in human islets directly impacts insulin granule exocytosis, its silencing impairs secretion while overexpression enhances it. Importantly, restoring PITPNA expression in T2D islets reverses diabetes-related secretory defects, suggesting its loss may contribute to β-cell failure. Finally, using a novel ATP biosensor, we demonstrate that insulin granules can undergo either complete fusion, releasing both peptides and small molecules, or partial fusion that selectively releases only small transmitter molecules. This differential cargo release is regulated through cellular polarity and becomes dysregulated in T2D. Collectively, these findings provide insight into the molecular mechanisms controlling insulin granule trafficking and release, revealing multiple points of dysregulation in T2D.