Glucagon-like peptide-1 protects beta-cells against apoptosis by increasing the activity of an IGF-2/IGF-1 receptor autocrine loop

Abstract

Objective: The gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect beta-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect.

Research design and methods: We performed comparative transcriptomic analysis of islets from control and GipR(-/-);Glp-1-R(-/-) mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis.

Results: We found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1-induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary beta-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1-induced protection against apoptosis.

Conclusions: An IGF-2/IGF-1 receptor autocrine loop operates in beta-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1-induced protection against apoptosis. These findings may lead to novel ways of preventing beta-cell loss in the pathogenesis of diabetes.

FIG. 1.

Gluco-incretin signaling controls susceptibility to apoptosis and IGF-1R expression. A: Islets from control or GipR^−/−;Glp-1R^−/− mice were exposed or not to cytokines (IL-1β, TNF-α, and IFN-γ) at low or high concentrations (5, 12.5, and 5 ng/ml or 10, 25, and 10 ng/ml, respectively) and in the presence or absence of GLP-1 (100 nmol/l). B: Quantitative RT-PCR analysis of IGF-1R mRNA expression in islets from control (wt) and GipR^−/−;Glp-1R^−/− mice (dKO). C: Reduced expression of IGF-1R expression in GipR^−/−;Glp-1R^−/− mouse islets as detected by Western blot analysis. Lower panel: Quantitation of the expression data. D: GLP-1 (100 nmol/l) treatment for 18 h increased IGF-1R expression as detected by Western blot analysis. Lower panel: Quantitation of the expression data. For all panels, data are means ± SD, n = 3 independent experiments. **P < 0.01; ***P < 0.001.

FIG. 2.

GLP-1 increased IGF-1R expression and signaling in MIN6 cells. A: MIN6 cells were treated with GLP-1 for the indicated periods of time, and IGF-1R expression and Akt phosphorylation were analyzed by Western blot analysis. B: MIN6 cells were transfected with a control (Un) or an IGF-1R siRNA and treated for 18 h with GLP-1. GLP-1 induced IGF-1 receptor expression, Akt phosphorylation on both T308 and S473, and Bad phosphorylation. Preventing IGF-1R expression suppressed GLP-1–induced Akt and Bad phosphorylation. For all panels, data are means ± SD, n = 3 independent experiments. ***P < 0.001.

FIG. 3.

GLP-1–induced Akt phosphorylation depends on IGF-1R but not IR expression. A: MIN6 cells were transfected with control (Un, lanes 3 and 6), IGF-1R (lanes 4 and 7), or IR-specific (lanes 5 and 8) siRNAs and exposed (+) or not (−) to GLP-1 for 18 h before Western analysis. Lanes 1 and 2 show the induction by GLP-1 of IGF-1R expression in nontransfected cells. Lane 3 shows the basal level of IGF-1R and IR expression in transfected cells; reducing IGF-1R expression (lane 4) but not the IR (lane 5) reduced Akt phosphorylation. Lane 6: GLP-1–treated cells showed higher expression of the IGF-1R but not of the IR; reducing IGF-1R expression (lane 7) but not IR (lane 8) reduced Akt phosphorylation. Bottom panel: Quantitation of the data. Data are means ± SD, n = 3 independent experiments. **P < 0.01; ***P < 0.001. B: MIN6 cells were treated or not for 18 h with GLP-1 to increase IGF-1R expression, then incubated with 2 mmol/l glucose for 2 h, and exposed for 15 min to the indicated concentrations of insulin or IGF-2. IGF-1R and total and phosphorylated Akt were determined by Western blot analysis. C: Quantification of the data in B. *P < 0.05, ***P < 0.001 for IGF-2 vs. insulin; §§P < 0.01, §§§P < 0.001 for IGF-2 in control vs. GLP-1–treated cells; ###P < 0.001 for IGF-2 vs. insulin in GLP-1–treated cells. a.u., arbitrary units.

FIG. 4.

Activation of Akt phosphorylation by GLP-1 depends on glucose-induced secretion. A: MIN6 cells were incubated for 18 h with GLP-1 to induce IGF-1R expression, then with 2 mmol/l glucose for 2 h, and finally for 1 h with 2 or 20 mmol/l glucose with or without GLP-1 and nimodipine (1 μmol/l) or diazoxide (200 μmol/l). Bottom panel: Quantitation of the data. B: Pancreatic islets were treated as the MIN6 cells in A. C: qRT-PCR analysis of IGF-1 and IGF-2 mRNA expression in mouse islets and cell sorter–purified β-cells. D: Secretion of IGF-2 by MIN6 cells incubated to 2 or 20 mmol/l glucose and in the presence of nimodipine, diazoxide, or GLP-1, as indicated. Total IGF-2 content was 10 ng/4 × 10⁶ cells. A–D: Data are means ± SD, n = 3 independent experiments. **P < 0.01; ***P < 0.001. a.u., arbitrary units.

FIG. 5.

GLP-1–increased Akt phosphorylation depends on secreted IGF-2. A: MIN6 cells were transfected with an unrelated (Un) or an IGF-2–specific shRNA. Forty-eight hours later, they were stimulated with GLP-1 for 18 h to increase IGF-1R expression and then incubated with 2 mmol/l glucose for 2 h and for 1 h in either 2 or 20 mmol/l glucose. B: MIN6 cells were stimulated with GLP-1 for 18 h and then incubated with 2 mmol/l glucose for 2 h and for 1 h in either 2 or 20 mmol/l glucose and with nonspecific IgGs or an IGF-2 blocking antibody. C: Pancreatic islets were stimulated with GLP-1 for 18 h and then incubated with 2 mmol/l glucose for 2 h and for 1 h in either 2 or 20 mmol/l glucose and with nonspecific IgGs or an IGF-2 blocking antibody. A–C: Data are means ± SD, n = 3 independent experiments. **P < 0.01; ***P < 0.001. a.u., arbitrary units.

FIG. 6.

GLP-1–induced protection against cytokine-induced apoptosis depends on activation of an IGF-2/IGF-1R autocrine loop. A: MIN6 cells were transfected with an unrelated or an IGF-1R siRNA, and 48 h later they were incubated in the presence or absence of GLP-1 for 4 h before addition of cytokines (IL-1β, 10 ng/ml; TNF-α, 25 ng/ml; IFN-γ, 10 ng/ml); the incubations were continued for 24 h in the continuous presence of GLP-1. B: MIN6 cells were transfected with an unrelated or an IGF-2 shRNA and then treated as described for A. C: MIN6 cells were incubated in the presence or absence of GLP-1 and in the presence of a nonspecific IgG fraction or an IGF-2 blocking antibody for 4 h before addition of cytokines (see above). D: Islets from β-cell–specific Igf-1R^−/− mice or from their control littermates were incubated in the presence or absence of GLP-1 for 4 h before addition of cytokines (see above). E: Control islets were incubated for 4 h in the presence or absence of GLP-1 and with a nonspecific or an IGF-2 blocking antibody before cytokines treatment. F: Islets from GipR^−/−;Glp-1R^−/− mice were infected with a control adenovirus (Ad-GFP) or an IGF-1R–expressing adenovirus (Ad-IGF1-R) and 1 day later exposed to cytokines, as described in A. A–F: Data are means ± SD, n = 3 independent experiments. **P < 0.01; ***P < 0.001.