RESUMO
AIM: MicroRNAs (miRNAs) regulate ß-cell function, and ß-cell mitochondria and insulin secretion are perturbed in diabetes. We aimed to identify key miRNAs regulating ß-cell mitochondrial metabolism and novel ß-cell miRNA-mitochondrial pathways. METHODS: TargetScan (http://www.targetscan.org/) was used to predict if 16 miRNAs implicated in ß-cell function target 27 cis-eGenes implicated in mitochondrial activity. The expression of candidate miRNAs and insulin secretion after 24 and 1 h pre-incubation in 2.8, 11.1- and 16.7-mM glucose was measured in clonal INS-1 832/13 ß-cells. MiR-29 silenced INS-1 832/13 cells were assessed for insulin secretion (glucose, pyruvate, and K+), target cis-eGene expression (Ndufv3 and Ndufa10 components of mitochondrial complex I (CI)), OXPHOS (CI-V) protein expression, and mitochondrial OXPHOS respiration/activity. The expression of differentially expressed miR-29 miRNAs was evaluated in Goto-Kakizaki (GK) rat, db/db mouse and type 2 diabetic (T2D) human islets, as well as NMRI mouse islets cultured under glucolipotoxic conditions. RESULTS: MiR-29, miR-15 and miR-124 were predicted to regulate ~20 cis-eGenes, while miR-29 alone was predicted to regulate ≥12 of these in rat and human species. MiR-29 expression and insulin secretion were reduced in INS-1 832/13 cells after 24 h in elevated glucose. MiR-29 knockdown increased all tested insulin secretory responses, Nudfv3, Ndufa10, complex I and II expression, and cellular mitochondrial OXPHOS. MiR-29 expression was reduced in db/db islets but increased in GK rat and T2D human islets. CONCLUSION: We conclude miR-29 is a key miRNA in regulating ß-cell mitochondrial metabolism and insulin secretion via underlying miR-29-OXPHOS complex pathways. Furthermore, we infer reduced miR-29 expression compensatorily enhances insulin secretion under glucotoxicity.
RESUMO
AIM: MiR-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. METHODS: MiR-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. RESULTS: Voltage-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, Ca(2+) dependent exocytosis was significantly reduced in OE375 cells. CONCLUSION: We 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.
