Figure 1
MRPS6 enhances glucose-stimulated insulin secretion (GSIS) in β-cells. (A) Schematic diagram of two IGT-associated SNPs on two overlapping genes: MRPS6 and SLC5A3. Lines indicate introns and boxes show exon, with empty boxes showing untranslated region (UTR) and black boxes showing protein coding region. Red bars indicate the region targeted by siRNAs. (B–C) siRNAs indiscriminately down-regulated the protein levels of both MRPS6 and SLC5A3. The insulinoma β-cells were transfected with siRNAs for 72 h and protein levels were examined by Western blotting and quantified by ImageJ. (D–E) MRPS6 and SLC5A were ectopically overexpressed in the insulinoma β-cells. Cells were transfected with plasmid bearing a cDNA of MRPS6 or SLC5A3 driven by CMV promoter for 72 h. Protein levels were examined by Western blotting. (F–H) MRPS6 or SLC5A3 knockdown reduced GSIS. The insulinoma β-cells were treated with siRNA as in (B) and GSIS was performed. Secreted insulin levels were quantified by ELISA (F) and intracellular proinsulin levels were examined by Western blot (G–H). (I–K) MRPS6 but not SLC5A3 overexpression enhanced GSIS. Overexpression was achieved as in (D). Secreted insulin levels were quantified by ELISA (I) and intracellular proinsulin levels were examined by Western blot (J–K). Shown immunoblots are representative data. (L–O) Glucose-induced insulin genes gene expression was downregulated and upregulated, respectively, by MRPS6 knockdown (L–M) and overexpression (N–O). Glucose stimulation was performed as in (F) and INS1 and INS2 mRNA levels were quantified by RT-qPCR. All experiments were performed > 3 biological repeats and error bars show standard deviation (SD) of the mean. P values are based on One-way ANOVA Turkey’s multiple comparison test or Student’s t-test (L–O): ns denotes not significant, *denotes P < 0.05, **denotes P < 0.01, ***denotes P < 0.001, ****denotes P < 0.0001.
