Modulation of microRNA‐375 expression alters voltage‐gated Na⁺ channel properties and exocytosis in insulin‐secreting cells

Abstract

AimMiR-375 has been implicated in insulin secretion and exocytosis through incompletely understood mechanisms. Here we aimed to investigate the role of miR-375 in the regulation of voltage-gated Na+ channel properties and glucose-stimulated insulin secretion in insulin-secreting cells.MethodsMiR-375 was overexpressed using double-stranded mature miR-375 in INS-1 832/13 cells (OE375) or downregulated using locked nucleic acid (LNA)-based anti-miR against miR-375 (LNA375). Insulin secretion was determined using RIA. Exocytosis and ion channel properties were measured using the patch-clamp technique in INS-1 832/13 cells and beta-cells from miR-375KO mice. Gene expression was analysed by RT-qPCR, and protein levels were determined by Western blot.ResultsVoltage-gated Na+ channels were found to be regulated by miR-375. In INS-1 832/13 cells, steady-state inactivation of the voltage-gated Na+ channels was shifted by approx. 6 mV to a more negative membrane potential upon down-regulation of miR-375. In the miR-375 KO mouse, voltage-gated Na+ channel inactivation was instead shifted by approx. 14 mV to a more positive membrane potential. Potential targets differed among species and expression of suggested targets Scn3a and Scn3b in INS-1 832/13 cells was only slightly moderated by miR-375. Modulation of miR-375 levels in INS-1-832/13 cells did not significantly affect insulin release. However, Ca2+ dependent exocytosis was significantly reduced in OE375 cells.ConclusionWe conclude that voltage-gated Na+ channels are regulated by miR-375 in insulin-secreting cells, and validate that the exocytotic machinery is controlled by miR-375 also in INS-1 832/13 cells. Altogether we suggest miR-375 to be involved in a complex multifaceted network controlling insulin secretion and its different components.

Modulation of Ionic Channels and Insulin Secretion by Drugs and Hormones in Pancreatic Beta Cells

“…The authors observed that the overexpression of microRNA-375 in INS-1 832/13 cells led to a decrease in the protein levels of Scn3a and Scn3b. Nevertheless, the relevance of this regulation is not completely understood in a more physiologic context, because glucose-induced insulin secretion was not affected by knocking down microRNA-375 (Salunkhe et al, 2015).…”

Confluence does not affect the expression of miR-375 and its direct targets in rat and human insulin-secreting cell lines

“…Cells were maintained in RPMI 1640 medium containing 11.1 mM glucose (HyClone, UT, USA) as previously described (Salunkhe et al, 2015). Both cell lines were incubated in a humidified atmosphere with 5% CO 2 at 37 °C for 48 h. All experiments were performed within culture passages in which the cell lines respond robustly to glucose-stimulated insulin secretion assay.…”

Potential relevance of microRNAs in inter-species epigenetic communication, and implications for disease pathogenesis

“…42,45 Only a modest increment in the plasma concentration of these 2 miRNAs was noted, and the authors concluded that milk miRNAs serve just as a dietetic source. However, both miR-375 and miR-200c might also affect exosome formation via exocytotic/endocytotic pathways, 42,48,49 and the required amount of miRNAs for biological activity is also intensively debated. 32,50 It would be necessary to develop models that are more physiological, e.g.…”