Redox compartmentalization drives functional heterogeneity of mature insulin secretory vesicles in pancreatic β-cells.

Pancreatic β-cell function requires precise regulation of insulin secretory vesicles (ISVs), yet the redox heterogeneity within mature ISVs remains poorly defined. Here, we implement a novel oxidation-sensing system using NPY-fused DsRed1-E5 (Timer) targeted to mature ISVs in INS-1E and human Endoc-βH5 β-cell models. Leveraging Timer's oxidative color transition from green (Low-oxidative) to yellow-red (High-oxidative), supported by independent measurements using the established redox sensor Grx1-roGFP2, we resolve distinct ISV subpopulations. Strikingly, Krebs-Ringer Bicarbonate HEPES (KRBH) Buffer treatment amplified ISV redox heterogeneity through increasing cytosolic oxidation. Factor screening identified glutamine deprivation as the principal driver of this diversification. Spatial analysis revealed Low-oxidative ISVs predominantly docked peripherally (0-1 μm from plasma membrane), while High-oxidative ISVs localized deeper (>1 μm) and exhibited 1.7-fold higher mobility. TIRF microscopy and volumetric imaging both demonstrated superior glucose-responsive secretion from Low-oxidative ISVs during both first and second phases of glucose-stimulated insulin release. Lysotracker co-localization showed High-oxidative ISVs were preferentially targeted for lysosomal degradation (2.3-fold higher association). These findings establish an oxidation-based taxonomy for mature ISVs, linking redox states to distinct functional fates: secretion-competent Low-oxidative vesicles versus degradation-prone High-oxidative vesicles, redefining ISV heterogeneity as a fundamental organizational principle in β-cell physiology and its dysregulation in metabolic stress.