Stress-Induced MicroRNA-708 Impairs β-Cell Function and Growth
Article Figures & Tables
Figures
- Figure 1
miR-708, Odz4, and Chop are upregulated in islets cultured at low glucose concentrations. A–C: Glucose regulation pattern of the three most upregulated miRNAs at G3 with respect to G11. Quantitative RT-PCR analysis of miR-708 (A), miR-451a (B), and miR-339-5p (C) in islets cultured for 2.5 days at indicated glucose concentrations (G3, G5, G11, and G25). Glucose regulation pattern of Odz4 (D) and Chop (E) in islets cultured at the indicated glucose concentrations for 2.5 days. Time course of Chop (F), Odz4 (G), and miR-708 (H) expression in pancreatic islets exposed to a low glucose concentration (G3). Gene expression data were normalized against Hprt1, and miRNAs were normalized against Let7i, RNU1A1, and RNU5G and are shown relative to levels at G11, which were set arbitrarily to 1. Results are expressed as the mean ± SEM from at least three independent experiments (n = 7–15). *P < 0.05; **P < 0.01; ***P < 0.001.
- Figure 2
ER stress induces miR-708 activation. Expression of Chop (A), Odz4 (B), and miR-708 (C) was quantified by quantitative RT-PCR in pancreatic islets cultured at G11 and treated with 1 µmol/L Thp for 24 h. D and E: Chop was knocked down using siRNA (i.e., siChop; 20 µmol/L) in INS1E cells cultured at G11. Twenty-four hours after transfection, cells were treated with 1 µmol/L Thp for 24 h and the expression of Chop (D) and miR-708 (E) was analyzed. Scrambled (Scrbl) siRNA was used as a control (Thp Scrbl). F–H: Chop, Odz4, and miR-708 expression in islets cultured at G11 or G25 for 2.5 days or 1 week. I–K: PBA ameliorates the stress response in islets cultured at low glucose levels. Chop, Odz4, and miR-708 expression in islets cultured at the indicated glucose concentrations and were treated (PBA) or not (Con) with 2.5 mmol/L PBA for 2.5 days. Results are expressed as the mean ± SEM from three independent experiments (n = 7–12). *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001 comparing PBA treatment (white bar) with its respective control at the same glucose concentration (black bar).
- Figure 3
miR-708 overexpression reduces Nnat levels. Nnat expression in islets (A) and DICs (B) transduced with AdmiR-708 (20 MOI) or AdGFP (20 MOI), or nontransduced (Con). C: Representative immunoblot of Nnat in MIN6 transduced with AdmiR-708 (20 MOI) or AdGFP (20 MOI), or nontransduced (control). Scatter plots show mean and SD of the quantification of the Western blot bands for Nnat normalized to β-actin (n = 3). D: Glucose regulation pattern of Nnat in islets cultured at G3, G5, and G11 not treated (Con) or treated with PBA (2.5 mmol/L) for 2.5 days. E: Nnat expression in islets cultured at G11 in the presence or absence of 1 µmol/L Thp. F: Transfection of siChop (20 μmol/L) in INS1E cells treated with 1 µmol/L Thp leads to increases in mRNA levels of Nnat. Scrambled (Scrbl) siRNA was used as a control. Gene expression data were normalized against Hprt1 and are shown relative to levels at G11, which were set arbitrarily to 1. A, B, and D–F: Results are expressed as the mean ± SEM from three or more independent experiments (n = 7–12). *P < 0.05, **P < 0.01, ***P < 0.001; ###P < 0.001 comparing PBA treatment (white bars) with its respective control at the same glucose concentration (black bars).
- Figure 4
miR-708 expression is increased in ob/ob mice islets. Islets from 14-week-old male ob/ob mice (open circles) and heterozygous male littermates (ob/+, black circles) were isolated. Expression levels of miR-708 (A), miR-375 (B), Odz4 (C), Nnat (D), Pdx1 (E), Chop (F), Xbp1s (G), Atf6 (H), and Atf3 (I) were analyzed. Results are expressed as the mean and SD of four mice per group. *P < 0.05, **P < 0.01.
- Figure 5
miR-708 overexpression reduces insulin secretion. A: GSIS assays were performed in islets cultured for 2.5 days at G11 that were nontransduced (Control) or transduced with AdGFP or AdmiR-708 (20 MOI). For the GSIS assays, islets were incubated for 1 h at 2.8 mmol/L glucose or at 16.7 mmol/L glucose. B: Total insulin content (normalized by protein content) present in islets after the GSIS assay shown in A. C: GSIS assays were performed in nontreated islets (Con, control) or islets treated with a negative control miRNA (Neg. Ctrl.; 500 nmol/L) or an inhibitor of miR-708 (αmiR-708; 500 nmol/L) during a 2.5-day culture at G5 or G11. D: Total insulin content (normalized by protein content) present in islets after the GSIS assay shown in C. E: GSIS assays of islets cultured for 2.5 days at G11 and transduced with AdmiR-708 (20 MOI), AdNnat (5 MOI), and both together. Results are expressed as mean ± SEM from three independent experiments, each one including at least three different replicates per condition. *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ###P < 0.001 compared with its respective control (Con) at G11.
- Figure 6
miR-708 overexpression represses β-cell proliferation and induces β-cell apoptosis. A: Representative images of insulin immunostaining (red) of DICs cultured at G11 for 72 h that were nontransduced (G11), were transduced with AdGFP (G11+AdGFP), or were transduced with AdmiR-708 (G11+AdmiR-708). Scale bar = 400 µm. B: Quantification of the insulin-positive area of DICs treated as in A. Data are shown relative to control samples (G11), which were arbitrarily set to 1. C: Insulin-positive area of DICs cultured at G3, G5, G11, G16 (16.7 mmol/L glucose), and at G11 in the presence of 1 µmol/L Thp. Data are shown relative to levels at G11. Note that the reduction of β-cell growth at G3 and in Thp-treated cells is similar to that observed in miR-708–overexpressing cells. D: Percentage of Ki67-positive β-cells with respect to the total number of β-cells in DICs cultured for 72 h at the indicated glucose concentrations and at G11 and transduced with AdGFP or AdmiR-708 (20 MOI). E: Insulin-positive area of DICs cultured at G5 or G11 for 72 h that were nontreated (Con, control) or treated with a negative control miRNA (Neg. Ctrl.; 500 nmol/L) or an inhibitor of miR-708 (αmiR-708; 500 nmol/L). ***P < 0.001 compared with its corresponding G11 condition. F: DICs cultured for 72 h at G5 and G11 were nontransduced or transduced with AdmiR-708 (20 MOI), AdNnat (5 MOI), and both together. G: Representative images of TUNEL (green), Hoechst (blue), and insulin (red) staining of pancreatic islets. Islets were not transduced (G11) or were transduced with AdGFP or AdmiR-708 (20 MOI). Arrows point to apoptotic cells identified by TUNEL staining. H: Quantification of β-cell apoptosis calculated as the percentage of TUNEL-positive β-cells with respect to the total number of insulin-positive cells. Results are expressed as the mean ± SEM from at least three independent experiments (n = 7–14). **P < 0.01, ***P < 0.001; ##P < 0.01, ###P < 0.001 compared with the control G11 condition.
Tables
- Table 1
Glucose-regulated miRNAs in mouse pancreatic islets
Fold change P value Ct G3 Ct G11 Upregulated miRNAs at low glucose (G3 vs. G11) mmu-miR-708-5p 6.7 0.0014 29.0 ± 0.13 31.9 ± 0.39 mmu-miR-339-5p 2.5 0.0032 31.8 ± 0.50 33.1 ± 0.30 mmu-miR-451a 2.5 0.0377 34.0 ± 0.06 34.5 ± 0.39 mmu-miR-224-5p 2.1 0.0433 34.2 ± 0.20 35.4 ± 0.29 mmu-miR-129-2-3p 2.0 0.0062 28.7 ± 0.48 30.0 ± 0.05 mmu-miR-467a-5p 1.9 0.0323 31.8 ± 0.25 33.0 ± 0.27 mmu-miR-151-3p 1.9 0.0250 30.1 ± 0.05 31.3 ± 0.06 Downregulated miRNAs at low glucose (G3 vs. G11) rno-miR-346 −3.2 0.0017 33.9 ± 0.28 32.5 ± 0.15 mmu-miR-138-5p −2.5 0.0300 35.0 ± 0.20 34.0 ± 0.32 mmu-miR-33-5p −2.1 0.0246 29.7 ± 0.77 28.7 ± 0.12 mmu-miR-541-5p −1.8 0.0297 28.9 ± 0.39 28.3 ± 0.10 mmu-miR-370-3p −1.6 0.0118 30.8 ± 0.39 30.3 ± 0.08 The expression of miRNAs was analyzed by a high-throughput screening of miRNA expression using quantitative PCR 384-well panels. RNA was extracted from mouse pancreatic islets cultured at 2.8 mmol/L glucose (G3) and at 11.1 mmol/L glucose (G11) for 2.5 days. Ct G3 and Ct G11 values indicate the cycle threshold ± SEM from qPCR analysis. cDNA synthesis for each sample was performed using 20 ng of total RNA. The listed miRNAs are those whose expression is significantly different between the G3 and G11 conditions. Statistical significance was determined by multiple t tests corrected for multiple comparisons using the Holm-Sidak method (P < 0.05).
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