SummaryStimulus-coupled insulin secretion from β-cells involves the fusion of insulin granules to the plasma membrane (PM) via SNARE complex formation—a cellular process key for maintaining whole-body glucose homeostasis. Optimal insulin secretion depends on how the clamping of SNAREs is released, rendering granules fusogenic. We show that an insulin granule protein synaptotagmin-9 (Syt9) deletion in lean mice increased glucose clearance, random-fed plasma insulin levels, and insulin secretion (in vivo and ex vivo islets) without affecting insulin sensitivity. These outcomes demonstrate that Syt9 has an inhibitory function in insulin secretion. Moreover, Syt9 interacts with PM-Stx1A and soluble Tomosyn-1 proteins to form non-fusogenic complexes between PM and insulin granules, preventing Stx1A-SNARE formation and insulin secretion. Furthermore, Syt9 inhibits SNARE-complex formation by posttranscriptional regulation of Tomosyn-1. We conclude that Syt9 and Tomosyn-1 are endogenous inhibitors that modulate Stx1A availability to determine β-cell secretory capacity.HighlightsSynaptotagmin-9 inhibits biphasic insulin secretion from β-cells.Synaptotagmin-9, syntaxin-1A, and Tomosyn-1 forms a molecular complex that decreases the availability of syntaxin-1A to form SNARE complexes in insulin secretion.Synaptotagmin-9–mediated inhibition of insulin secretion occurs through post-transcriptional regulation of Tomosyn-1.
