RESISTIN INHIBITS GLUCOSE-STIMULATED INSULIN SECRETION THROUGH MIR-494 BY TARGET ON STXBP5

Abstract

Aims: Resistin has been reported to impair the pancreatic beta cells and associated with insulin resistance. MicroRNAs (miRNAs) are short, endogenously produced non-coding ribonucleotides that bind mRNAs and function mainly as negative regulators in mammals. MiRNAs have been implicated in many diseases, including insulin resistance and diabetes. A considerable body of evidence has indicated an important function for miRNAs in insulin secretion. The current study was designed to investigate the effects of miR-494 in the reductions in insulin secretion attributable to resistin.

Methods: Insulin secretion was determined by ELISA, and expressions of genes were identified using quantitative RT-PCR (qRT-PCR) or Western blot analysis.

Results: Insulin secretion was significantly reduced by resistin. Overexpression of miR-494 inhibited insulin secretion both in diet culture and high glucose medium in MIN6 cell lines. MiR-494 down-regulated the protein level of STXBP5 by pairing with sites in the 3'UTR.

Conclusion: miR-494 is involved in the insulin secretion regulated by resistin via its effects on STXBP5 in MIN6 cells.

Keywords: STXBP5; insulin secretion; miR-494; resistin.

Figure 1.

Resistin Inhibits Glucose-stimulated Insulin Secretion in MIN6 Cells. The insulin concentration in the culture medium which treated with or without resistin was measured, and insulin secretion was assessed in the presence of resistin under low-glucose (5.5 mM) and high-glucose (25 mM) condition respectively. (A) MIN6 cells were treated with resistin at doses of 25, 50, and 100 ng/mL for 24 h. Cellular insulin secretion was measured by ELISA assay. (B) MIN6 cells were treated with 50 ng/mL resistin and incubated with either 5.5 mM or 25 mM glucose medium for 24 h. Insulin secretion was measured by ELISA assay. Data are here presented as means ± SD of percentage of controls. (*, P<0.05, significant; **, P<0.01, very significant; ns, not significant).

Figure 2.

MiR-494 Overexpression Inhibits Insulin Secretion. The expression of miR-494 was detected in MIN6 cells after resistin treatment and the role of miR-494 in glucose-induced insulin secretion was studied.(A) The expression of miR-494 was examined by real-time PCR. (B) MIN6 cells were transfected with NC, 100 nM, 150 nM, or 200 nM miR-494 mimics, respectively, then incubated with either 5.5 mM or 25 mM glucose medium for 24 h. Insulin secretion was measured by ELISA assay. (C) The expression of miR-494 was examined using real-time PCR. (D) MIN6 cells were treated with recombinant resistin (50 ng/mL) for 24 h, then transfected with miR-494 inhibitor, cells were incubated with either 5.5 mM or 25 mM glucose medium for 24 h. Insulin secretion was measured by ELISA assay. (E) MIN6 cells were transfected with miR-494 mimics or miR-494 inhibitor for 24 h. The expression of genes involved in insulin secretion was examined by real-time PCR. (F) MIN6 cells were treated with recombinant resistin for 24 h, then transfected with miR-494 inhibitor. The expression of genes involved in insulin secretion was examined using real-time PCR. Data are here presented as means ± SD of percentage of controls. (*, P<0.05, significant; **, P<0.01, very significant; ns, not significant).

Figure 3.

MiR-494 Inhibit Insulin Secretion Through Target on STXBP5. The target genes of miR-494 were predicted and analyzed, and also the target gene was verified.(A) The result of bioinformatics prediction shows that miR-494 can bind to the 5253–5259 bp sites of the Stxbp5 mRNA 3′UTR region. (B) miR-494 and Stxbp5 3′UTR report vector or binding site mutant vector (Stxbp5 -3′UTR-M) were co-transfected into NIH-3T3 cells and luciferase activity was assessed after 24 h according to the instructions included with the Dual Luciferase Reporter Assay System. (C) Effect on Stxbp5 protein level of miR-494 by Western blot. (D) miR-494 and Stxbp5 were co-transfected into MIN6 cells, which were incubated with either 5.5 mM or 25 mM glucose medium for 24 h. Insulin secretion was measured by ELISA assay. Data are here presented as means ± SD of percentage of controls. (*, P<0.05, significant; **, P<0.01, very significant; ns, not significant).