The highly expressed calcium-insensitive synaptotagmin-11 and synaptotagmin-13 modulate insulin secretion.

AIM: SYT11 and SYT13, two calcium-insensitive synaptotagmins, are downregulated in islets from type 2 diabetic donors, but their function in insulin secretion is unknown. To address this, we investigated the physiological role of these two synaptotagmins in insulin-secreting cells. METHODS: Correlations between gene expression levels were performed using previously described RNA-seq data on islets from 188 human donors. SiRNA knockdown was performed in EndoC-ßH1 and INS-1 832/13 cells. Insulin secretion was measured with ELISA. Patch-clamp was used for single-cell electrophysiology. Confocal microscopy was used to determine intracellular localization. RESULTS: Human islet expression of the transcription factor PDX1 was positively correlated with SYT11 (p = 2.4e-10 ) and SYT13 (p < 2.2e-16 ). Syt11 and Syt13 both co-localized with insulin, indicating their localization in insulin granules. Downregulation of Syt11 in INS-1 832/13 cells (siSYT11) resulted in increased basal and glucose-induced insulin secretion. Downregulation of Syt13 (siSYT13) decreased insulin secretion induced by glucose and K+ . Interestingly, the cAMP-raising agent forskolin was unable to enhance insulin secretion in siSYT13 cells. There was no difference in insulin content, exocytosis, or voltage-gated Ca2+ currents in the two models. Double knockdown of Syt11 and Syt13 (DKD) resembled the results in siSYT13 cells. CONCLUSION: SYT11 and SYT13 have similar localization and transcriptional regulation, but they regulate insulin secretion differentially. While downregulation of SYT11 might be a compensatory mechanism in type-2 diabetes, downregulation of SYT13 reduces the insulin secretory response and overrules the compensatory regulation of SYT11 in a way that could aggravate the disease.

MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles.

MicroRNAs contribute to the maintenance of optimal cellular functions by fine-tuning protein expression levels. In the pancreatic ß-cells, imbalances in the exocytotic machinery components lead to impaired insulin secretion and type 2 diabetes (T2D). We hypothesize that dysregulated miRNA expression exacerbates ß-cell dysfunction, and have earlier shown that islets from the diabetic GK-rat model have increased expression of miRNAs, including miR-335-5p (miR-335). Here, we aim to determine the specific role of miR-335 during development of T2D, and the influence of this miRNA on glucose-stimulated insulin secretion and Ca2+-dependent exocytosis. We found that the expression of miR-335 negatively correlated with secretion index in human islets of individuals with prediabetes. Overexpression of miR-335 in human EndoC-ßH1 and in rat INS-1 832/13 cells (OE335) resulted in decreased glucose-stimulated insulin secretion, and OE335 cells showed concomitant reduction in three exocytotic proteins: SNAP25, Syntaxin-binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11). Single-cell capacitance measurements, complemented with TIRF microscopy of the granule marker NPY-mEGFP demonstrated a significant reduction in exocytosis in OE335 cells. The reduction was not associated with defective docking or decreased Ca2+ current. More likely, it is a direct consequence of impaired priming of already docked granules. Earlier reports have proposed reduced granular priming as the cause of reduced first-phase insulin secretion during prediabetes. Here, we show a specific role of miR-335 in regulating insulin secretion during this transition period. Moreover, we can conclude that miR-335 has the capacity to modulate insulin secretion and Ca2+-dependent exocytosis through effects on granular priming.