Glucagon-like peptide-1 increases beta-cell glucose competence and proliferation by translational induction of insulin-like growth factor-1 receptor expression

Abstract

Glucagon-like peptide-1 (GLP-1) protects beta-cells against apoptosis, increases their glucose competence, and induces their proliferation. We previously demonstrated that the anti-apoptotic effect was mediated by an increase in insulin-like growth factor-1 receptor (IGF-1R) expression and signaling, which was dependent on autocrine secretion of insulin-like growth factor 2 (IGF-2). Here, we further investigated how GLP-1 induces IGF-1R expression and whether the IGF-2/IGF-1R autocrine loop is also involved in mediating GLP-1-increase in glucose competence and proliferation. We show that GLP-1 up-regulated IGF-1R expression by a protein kinase A-dependent translational control mechanism, whereas isobutylmethylxanthine, which led to higher intracellular accumulation of cAMP than GLP-1, increased both IGF-1R transcription and translation. We then demonstrated, using MIN6 cells and primary islets, that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF-2 secretion. We showed that GLP-1-induced primary beta-cell proliferation was suppressed by Igf-1r gene inactivation and by IGF-2 immunoneutralization or knockdown. Together our data show that regulation of beta-cell number and function by GLP-1 depends on the cAMP/protein kinase A mediated-induction of IGF-1R expression and the increased activity of an IGF-2/IGF-1R autocrine loop.

FIGURE 1.

GLP-1 increases IGF-1R protein but not mRNA expression in MIN6 cells and primary mouse islets. MIN6 cells were exposed to GLP-1 (100 nm) for the indicated periods of time and IGF-1R mRNA (A) as well as protein (B) expressions were quantitated by qRT-PCR and by Western blot analyses, respectively. Mouse control islets were exposed to GLP-1 (100 nm) for 18 h, and IGF-1R mRNA (C) as well as protein (D) expressions were quantitated by qRT-PCR and by Western blot analyses, respectively. The levels are expressed as arbitrary units (a.u.). The data are the means ± S.D. from three independent experiments. **, p < 0.01; ***, p < 0.001.

FIGURE 2.

IBMX induces a strong increase in both IGF-1R mRNA and protein expression in MIN6 cells and primary mouse islets. MIN6 cells were exposed to IBMX (10 μm) for the indicated periods of time, and IGF-1R mRNA (A) as well as protein (B) expressions were quantitated by qRT-PCR and by Western blot analyses, respectively. Mouse control islets were exposed to IBMX (10 μm) for 18 h and IGF-1R mRNA (C) as well as protein (D) expressions were quantitated by qRT-PCR and by Western blot analyses, respectively. The levels are expressed as arbitrary units (a.u.). The data are the means ± S.D. from three independent experiments. **, p < 0.01; ***, p < 0.001. DMSO, dimethyl sulfoxide; wt, wild type.

FIGURE 3.

GLP-1 and IBMX differentially increase cAMP levels in MIN6 cells. MIN6 cells were incubated in the presence or absence of GLP-1 (100 nm) or IBMX (10 μm) for 30 s or 2 min, and intracellular cAMP contents were measured by radioimmunoassay. The data are the means ± S.D., n = 3 independent experiments. **, p < 0.01 (treatments compared with control); §§, p < 0.01 (IBMX treatment compared with GLP-1 treatment).

FIGURE 4.

IBMX and GLP-1 regulate IGF-1R expression by transcriptional and post-transcriptional mechanisms. A and B, MIN6 cells were exposed to either IBMX (10 μm) or GLP-1 (100 nm) for 18 h and in the presence or absence of actinomycin D (1 μg/ml). IGF1-R mRNA (A) as well as protein (B) expression was quantitated by qRT-PCR and Western blot analysis, respectively. C, MIN6 cells (left panel) or mouse islets (right panel) were biosynthetically labeled for 1 h with [³⁵S]methionine and [³⁵S]cysteine in the presence or absence of GLP-1 (100 nm). IGF-1R was immunoprecipitated from the cell lysates and separated by gel electrophoresis; the amount of newly synthesized precursor receptor was quantitated by densitometric analysis of the autoradiograms. The data are the means ± S.D. from three independent experiments. **, p < 0.01; ***, p < 0.001. Ctrl, control.

FIGURE 5.

GLP-1 and IBMX induce IGF-1R expression through activation of the cAMP/PKA pathway. A, MIN6 cells were exposed for 18 h to GLP-1 (100 nm) to induce IGF-1R expression. This induction was blocked in the presence of the protein kinase A inhibitor H-89 (10 μm) or the cAMP antagonist Rp-CPT-cAMP (100 μm). Activation of Epac2 by 8-CPT-2′-O-Me-cAMP (50 μm) did not induce IGF-1R expression in the absence of GLP-1. B, induction of the IGF-1R by GLP-1 in MIN6 cells expression, which was not suppressed by the MAP kinase inhibitor PD98059 (PD, 50 μm), the PI 3-kinase inhibitors LY294002 (LY, 50 μm), or wortmannin (50 nm) nor by the Ca²⁺-channel blocker nimodipine (Nimo, 1 μm). C, induction of IGF-1R by IBMX (10 μm) was mimicked by exposing the cells to forskolin (FSK, 10 μm), or the protein kinase A activator SP-CPT-cAMP (100 μm). However, induction of IGF-1R expression was not increased by Epac2 activator 8-CPT-2′-O-Me-cAMP (50 μm) in the absence of IBMX. D, induction of IGF-1R expression by IBMX was not suppressed by PD98059, by LY294002, wortmannin (50 nm), nor by nimodipine. The levels are expressed as arbitrary units (a.u.). The data are the means ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant.

FIGURE 6.

MIN6 glucose competence is modulated by IGF-1R and IGF-2 expression. A, MIN6 cells were co-transfected with a control (pcDNA3) or IGF-1R (pcDNA3-IGF1-R) expression plasmid together with a plasmid encoding hGH. Two days later the cells were exposed to 2 or 20 mm glucose for 1 h, and secreted and intracellular hGH were measured. Secretion was calculated as the ratio of secreted over intracellular hGH, and this ratio was set at 1 for cells treated in control conditions exposed to 2 mm glucose. B, MIN6 cells were co-transfected with an unrelated or an IGF-1R-specific siRNA together with hGH-expressing plasmid. Secretion experiments were performed and expressed as above. C, MIN6 cells were co-transfected with an unrelated or an IGF-2-specific shRNA together with hGH-expressing plasmid. Secretion experiments were performed and expressed as above. The data are the means ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01.

FIGURE 7.

Mouse islet glucose competence is modulated by IGF-1R and IGF-2 expression. A, mouse islets were infected with a control adenovirus (Ad GFP) or an IGF-1R-expressing adenovirus (AdIGF1-R) and 2 days later exposed to either 2 or 20 mm glucose, and insulin secretion was measured and expressed as in Fig. 6. B, mouse islets were infected as in A and 2 days later exposed to either 2 or 20 mm glucose in the presence of a control IgG or an IGF-2 blocking antibody. Insulin secretion is reported relative to secretion at 2 mm glucose (stimulatory index). C, mouse islets were infected with an adenovirus expressing a unrelated (Luc) or an IGF-2-specific shRNA, and the islets were then processed as above for measurement of glucose-stimulated insulin secretion. The Western blot in the right panel shows the reduction in ProIGF-2 expression in AdshIGF-2-infected cells. IGF-1R, IGF-2, and β-actin expression was detected by Western blot analysis. The data are the means ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 8.

GLP-1-mediated β-cell proliferation depends on the IGF-2/IGF-1R autocrine loop. A, islets from Igf-1R^lox/lox mice were plated on extracellular matrix-coated dishes for 7 days to form monolayers. They were then infected with Ad-GFP or an Ad-CRE. Two days later they were exposed to GLP-1 (100 nm) for 48 h and then with BrdUrd for the last 24 h. BrdUrd-positive cells were then recorded and are expressed as percentages of BrdUrd-positive cells. B, mouse islets monolayers were exposed to GLP-1 for 48 h in the presence of nonspecific IgG fraction or a neutralizing IGF-2 antibody, and BrdUrd was added for the last 24 h. Proliferation was recorded as BrdUrd-positive cells. C, mouse islet monolayers were infected with an adenovirus expressing an unrelated (Luc) or an IGF-2-specific shRNA. GLP-1-induced proliferation was measured as above. The data are the means ± S.D. from three independent experiments. ***, p < 0.001.