1. Figure 1. GLP-1 induces miR-132 and miR-212 expressions in pancreatic β-cells in vitro. A, miRNA expression profiling of GLP-1-treated INS-1 832/3 cells. miRNA profiling was performed by real-time qRT-PCR. Data represent the replicate-combined average of 3 independent measurements with cells at different passages. The x-axis of the scatter plot represents the copy number of miRNAs per 10-pg total RNA, and the y-axis is the ratio of miRNA expression changes in response to 50nM GLP-1 for 24 hours. Data from replicates of the same treatment group were combined by using the Rosetta Resolver. B, Time course of miRNA induction by GLP-1 in INS-1 832/3 cells. The 832/3 cells were treated with GLP-1 (50nM) for the time periods as labeled. Levels of miRNAs were measured by TaqMan with specific primer/probes (ABI system) and normalized to 4.5S RNA. The relative expression levels in GLP-1-treated cells were expressed relative to that observed for vehicle alone. Data represent mean ± SE of 3 independent treatment groups. C, miRNAs 132, 212, and 375 levels in GLP-1-treated rat islets. Pancreatic islets were isolated from male Sprague-Dawley rats and cultured in RPMI 1640 medium with or without 50nM GLP-1 for 24 hours. Levels of the miRNAs were determined by TaqMan PCR and normalized to 4.5S RNA. Data are the mean ± SE of 3 independent islet preparations. D, miRNAs 132, 212, and 375 levels in GLP-1-treated mouse islets. Islets were isolated from male C57Bl/6N mice and cultured in RPMI 1640 medium with or without 50nM GLP-1 for 48 hours. Data are the mean ± SE of 6 independent islet preparations. E, miRNAs 132, 212, and 375 levels in GLP-1-treated human islets. Islets were isolated from human donors and cultured in complete CMRL-1066 medium in the present or absence of GLP-1 for 24 hours. Data are the mean ± SE of 3–6 replicates of each islet preparation. For B–E, 2-tailed Student's t test was performed for the GLP-1-treated group vs the vehicle group comparison; *, P < .05; **, P < .01; ***, P < .001.
From: Induction of miR-132 and miR-212 Expression by Glucagon-Like Peptide 1 (GLP-1) in Rodent and Human Pancreatic β-Cells
Mol Endocrinol. 2015;29(9): doi: /me
Mol Endocrinol | Copyright © 2015 by the Endocrine Society

2. Figure 2. Infusion of Ex-4 in mice improves glucose homeostasis and increases the expression levels of miRNAs 132 and 212 in pancreatic islets. C57Bl/6N lean mice were infused with Ex-4 (10 or 30 μg/kg·d) or saline through sc implanted osmotic minipumps. Glucose (A) and insulin (B) levels were measured during an OGTT after Ex-4 infusion. C, Pancreatic islets were isolated after Ex-4 infusion for RNA extraction. miRNAs 132, 212, and 375 were determined by TaqMan PCR. Data are the mean ± SE of 5 mice per group. One-way ANOVA followed by Dunnett's post hoc test was performed for the 2 Ex-4-treated and the saline groups; *, P < .05; **, P < .01 as compared with the saline group.
From: Induction of miR-132 and miR-212 Expression by Glucagon-Like Peptide 1 (GLP-1) in Rodent and Human Pancreatic β-Cells
Mol Endocrinol. 2015;29(9): doi: /me
Mol Endocrinol | Copyright © 2015 by the Endocrine Society

3. Figure 3. GLP-1 induces miRNAs 132 and 212 in a PKA-dependent manner
Figure 3. GLP-1 induces miRNAs 132 and 212 in a PKA-dependent manner. A, Effects of cAMP enhancing agents on miR-212 and miR-132 expression in INS-1 832/3 cells. INS-1 832/3 cells were treated in RPMI medium (containing 16mM glucose) with 50nM GLP-1, 50nM Ex-4, 5μM IBMX, or 1μM forskolin (Fsk) for 24 hours before being harvested for RNA extraction and TaqMan analysis of miR-132 and miR-212 expression. B, Effects of cAMP-enhancing agents on intracellular cAMP levels. The INS-1 832/3 cells, grown to near confluence in 96-well plates, were treated with 50nM GLP-1, 50nM Ex-4, or 1μM Fsk for 1 hour in Hank's Balanced Salt Solution buffer containing 0.5mM IBMX. Intracellular cAMP accumulation during the 1-hour stimulation was measured with immunoassay using the Lance cAMP detection kit from PerkinElmer. C, Effects of PKA/Epac modulators on miRNAs 132 and 212 expression. INS-1 832/3 cells were treated during tissue culture in RPMI 1640 medium (with 11mM glucose and 10% fetal bovine serum) with GLP-1 (50nM) with or without H-89 (PKA inhibitor, 10μM), ESCA (Epac-specific activator, 10μM) for 24 hours before being harvested for RNA extraction and miRNA measurement. Data represent the mean ± SE of 3 independent experiments. D, Effects of 16mM glucose on miR132 and miR-212 expression in INS-1 832/3 cells. The 832/3 cells were cultured in RPMI 1640 medium with either 2mM or 16mM glucose for 24 hours before RNA extraction and real-time qRT-PCR analysis. Data represent the mean ± SE of 3 independent experiments. For A–C, one-way ANOVA followed by Dunnett's post hoc test compared with the vehicle group was performed; *, P < .05; **, P < .01; ***, P < .001 as compared with the vehicle control; #, P < .05, as compared with the GLP-1-treated group. For D, 2-tailed Student's t test was performed for the comparison of the high-glucose group vs the low-glucose group; *, P < .05.
From: Induction of miR-132 and miR-212 Expression by Glucagon-Like Peptide 1 (GLP-1) in Rodent and Human Pancreatic β-Cells
Mol Endocrinol. 2015;29(9): doi: /me
Mol Endocrinol | Copyright © 2015 by the Endocrine Society

4. Figure 4. miRNA-132 augments GLP-1-stimulated insulin secretion in INS-1 832/3 cells. A, Effects of miRNA overexpression on GLP-1-stimulated insulin secretion. INS-1 832/3 cells were transfected by electroporation with precursors for miR-132, miR-375, or a Pre-miR negative control, and insulin secretion was determined 48 hours after transfection in response to 16mM glucose in the absence or presence of GLP-1 (10nM). Data are the mean ± SE of 3 independent experiments. B, Effects of miRNA antagonist on glucose and GLP-1-stimulated insulin secretion. The INS-1 832/3 cells were transfected with anti-miRNA oligonucleotides or negative control oligonucleotides by electroporation. Forty-eight hours after transfection, insulin secretion was measured in response to 2mM or 16mM glucose, or 16mM glucose plus 10nM GLP-1. Data are the mean ± SE of 3 replicate. One-way ANOVA followed by Dunnett's post hoc test compared with the control group was performed for both A and B; **, P < .01 as compared with the negative control.
From: Induction of miR-132 and miR-212 Expression by Glucagon-Like Peptide 1 (GLP-1) in Rodent and Human Pancreatic β-Cells
Mol Endocrinol. 2015;29(9): doi: /me
Mol Endocrinol | Copyright © 2015 by the Endocrine Society

5. Figure 5. GLP-1 induces the expression of miRNAs 132 and 212 and enhances insulin secretion in parallel with an increase in cellular cAMP. A, Potentiation of GSIS by GLP-1 in the INS-1 832/3 and 832/13 cells. Insulin secretion was measured from both INS-1 clonal cell lines in response to 16mM glucose in the absence and presence of varying concentrations of GLP-1. Data are the mean ± SE of 3 replicates. B, cAMP responses to Ex-4 in the 832/3 and 832/13 cells. The INS-1 clonal cells were incubated with varying concentrations of Ex-4 for 1 hour at 37°C. Cells were then lysed and cAMP concentration measured as described in Materials and Methods. Data are the mean ± SE of 4 independent experiments. C, miR-132 and miR-212 expression changes in response to GLP-1 in the 832/3 and 832/13 cells. The INS-1 clonal cells were treated with GLP-1 (50nM) for 24 hours, followed by RNA extraction and miRNA expression measurements by TaqMan PCR. Data are the mean ± SE of 3 independent experiments. Student's t test was performed for the GLP-1-treated group vs the vehicle group comparison in each cell line; *, P < .05. D, Effects of miR-132 and miR-212 overexpression on glucose and GLP-1-stimulated insulin secretion in the INS-1 832/13 cells. Cells were transfected with precursors of miR-132, miR-212, or Pre-miR control oligonucleotides. Insulin secretion was measured as described in Figure 4. Data are mean ± SE of 5 replicates. One-way ANOVA followed by Dunnett's post hoc test was performed for the comparisons of the miRNA precursor-transfected groups vs the control oligonucleotides-transfected group under each treatment condition; *, P < .05; **, P < .01 as compared with the negative control under the same treatment condition.
From: Induction of miR-132 and miR-212 Expression by Glucagon-Like Peptide 1 (GLP-1) in Rodent and Human Pancreatic β-Cells
Mol Endocrinol. 2015;29(9): doi: /me
Mol Endocrinol | Copyright © 2015 by the Endocrine Society