Molecular mechanisms of GLP-1-induced ERK1/2 phosphorylation. A, after a 2-h stabilization period in KRB buffer, MIN6 cells were treated with 10 nm GLP-1 for various times as indicated. Identical amounts of lysate proteins (25 μg) were subjected to 10% SDS-PAGE and immunoblotted (IB) with the anti-phospho-ERK1/2 antibody. ERK1/2 content within total cell lysates is shown as a control. Typical autoradiographs representative of five independent experiments are shown. B, graph representing temporal pattern of ERK2 phosphorylation levels (P-ERK2) deduced from quantitative results of five representative experiments obtained from analyses using a Java-based image processing program (Image J), developed by Wayne Rasband (National Institutes of Health, Bethesda, MD). C, ⁴⁵Ca²⁺ uptake, cAMP production, and insulin secretion from MIN6 cells were measured after incubation with various glucose concentrations with or without 10 nm GLP-1, as described under “Experimental Procedures.” Data are means of 3–5 independent experiments. D, F, and H, Western blot assays of phospho-ERK1/2 in MIN6 cells following 10 nm GLP-1 stimulation for 0, 5, or 20 min, with or without inhibitor treatment. The VDCC antagonist nifedipine (2 μm) was not present during the 2-h KRB stabilization period but was added simultaneously with GLP-1 (D). The PKA inhibitor H89 (2 μm) or the PI 3-kinase inhibitor wortmannin (100 nm) were added during the 2-h KRB stabilization period and maintained during the 5- or 20-min GLP-1 stimulation (F and H). 25 μg of protein lysates were then subjected to 10% SDS-PAGE and immunoblotted with the anti-phospho-ERK1/2 antibody. As a control, total ERK1/2 is shown. Typical autoradiographs representative of 3–5 independent experiments are shown. E, G, and I, graphs representing levels of ERK2 phosphorylation deduced from quantitative results of 3–5 representative experiments.

β-Arrestin 1 interacts with ERK1/2 upon GLP-1 stimulation restraining activated ERK1/2 in the cytoplasm. A, after a 2-h stabilization period in KRB buffer, cells were stimulated with 10 nm GLP-1 for various times and lysed, and cytosolic versus nuclear fractions were separated by subcellular fractionation. Cytosolic fraction was then subjected to immunoprecipitation (IP) in reduced conditions with the mouse monoclonal anti-β-arrestin 1 antibody. Immunoprecipitated proteins were resolved by 10% SDS-PAGE and blotted (IB) with anti-total ERK1/2 antibody. Relative quantities of β-arrestin 1 determined by reblots in these immunoprecipitation experiments using the goat polyclonal anti-β-arrestin 1 antibody are also shown. ERK1/2 content within total cell lysates is shown as a control. B, the graph represents the temporal pattern of β-arrestin 1 and ERK1/2 association deduced from quantitative results of three representative β-arrestin 1 immunoprecipitation experiments. C, 2-h KRB-stabilized cells were incubated with 10 nm GLP-1 for various times, as indicated, and nuclear versus cytoplasm fractions were prepared. Immunoblots with the active phosphorylated form of ERK1/2 are shown. Tubulin and CREB were also detected as loading control and to show cross-contamination in the cytoplasmic and nuclear fractions, if any. Typical autoradiographs representative of three independent experiments are shown. D, the graph represents levels of ERK2 phosphorylation deduced from quantitative results of three independent experiments.

β-Arrestin 1 is required for GLP-1-induced Bad phosphorylation (Ser-112) in MIN6 cells. A, after a 2-h stabilization period in KRB buffer, MIN6 cells were treated with 10 nm GLP-1 for various times as indicated. Identical amounts of lysate proteins (25 μg) were subjected to 10% SDS-PAGE and immunoblotted (IB) with the anti-phospho-Bad antibodies that recognize the Ser-112- or the Ser-155-phosphorylated form of the protein. Typical autoradiographs representative of 3–5 independent experiments are shown. Bad content within total cell lysates is shown as internal and loading control. B, the graph represents levels of phosphorylation of Bad (Ser-112) deduced from quantitative results of 3–5 representative experiments. C, after a 2-h stabilization period in KRB buffer, cells were stimulated with 10 nm GLP-1 for 20 min. The U0126 (10 μm) was added during the 2-h KRB stabilization period and maintained during GLP-1 stimulation. 25 μg of protein lysates were subjected to 10% SDS-PAGE and immunoblotted with anti-phospho-Bad (Ser-112) antibody. Bad content within total cell lysates is shown as internal and loading control. Typical autoradiographs representative of three independent experiments are shown. D, the graph represents levels of phosphorylation of Bad (Ser-112) deduced from quantitative results of three representative experiments. E, MIN6 cells were transfected with control or β-arrestin 1 siRNA for 6 h. 72 h after transfection, cells were stabilized in KRB buffer for 2 h and then stimulated for 20 min with 10 nm GLP-1. 25 μg of protein lysates were then subjected to 10% SDS-PAGE and immunoblotted with the anti-phospho-Bad (Ser-112) antibody. Bad content within total cell lysates is shown as a loading control. Typical autoradiographs representative of three independent experiments are shown. F, the graph represents levels of phosphorylation of Bad (Ser-112) deduced from quantitative results of three independent experiments. CTL, control.

β-Arrestin 1 knockdown prevents the interaction of BAD with the 14-3-3 protein induced by GLP-1 in MIN6 cells. A, MIN6 cells were transfected with control or β-arrestin 1 siRNA for 6 h. 72 h after transfection, cells were stabilized in KRB buffer for 2 h and then stimulated for 20 min with 10 nm GLP-1. Cell lysates were then subjected to immunoprecipitation (IP) in reduced conditions with the mouse monoclonal anti-Bad (C7) antibody. Immunoprecipitated proteins were resolved by 10% SDS-PAGE and blotted (IB) with anti-total 14-3-3 β (C-20) protein antibody. 14-3-3 protein content within total cell lysates is shown as control. B, the graph represents the association of Bad with 14-3-3 protein deduced from quantitative results of three representative Bad immunoprecipitation experiments. CTL, control.

Glucose-stimulated insulin secretion from WT or β-arrestin 1 KO mice. A, islets were preincubated for 30 min in KRB buffer supplemented with 1.4 mm glucose. Following preincubation, islets were further stimulated with 1.4 or 16.7 mm glucose for 20 min. Data are means of five independent experiments. B and C, insulin and protein content determination as described under “Experimental Procedures.”

Knockdown of β-arrestin 1 impairs the GLP-1-induced ERK1/2 phosphorylation kinetic pattern in MIN6 cells. A, MIN6 cells were transfected with control or two different β-arrestin 1 siRNA duplexes tested at 25, 50, or 100 nm (final concentrations) for 6 h. 72 h after transfection, cells were lysed, and 35–45 μg of protein lysates were subjected to 10% SDS-PAGE and immunoblotted (IB) with anti-β-arrestin 1 and anti-β-arrestin 2 antibodies. The graph representing relative expression of β-arrestins following β-arrestin 1 siRNA is deduced from quantitative results of 10 representative experiments. B, MIN6 cells were transfected with control or β-arrestin 1 siRNA (β-arr1 siRNA#2). Following transfection, cells were incubated in KRB buffer for a 2-h stabilization period and stimulated with 10 nm GLP-1 for various times as indicated. 25 μg of protein lysates were subjected to 10% SDS-PAGE and immunoblotted with the anti-phosphorylated ERK1/2 antibody. ERK1/2 content within total cell lysates is shown as a loading control. Typical autoradiographs representative of eight independent experiments are shown. C, the graph represents levels of phosphorylation of ERK2 deduced from quantitative results of eight representative experiments. CTL, control.

GLP-1 induced p90RSK phosphorylation through a β-arrestin 1-ERK1/2-dependent pathway in MIN6 cells. A, after a 2-h stabilization period in KRB buffer, MIN6 cells were treated with 10 nm GLP-1 for various times as indicated. Identical amounts of lysate proteins (25 μg) were subjected to 10% SDS-PAGE and immunoblotted (IB) with the anti-phospho-p90RSK antibody that recognizes the phosphorylated Thr-573 form of this kinase. Tubulin content within total cell lysates is shown as an internal and loading control. Typical autoradiographs representative of five independent experiments are shown. B, the graph represents levels of phosphorylated p90RSK (P-p90RSK) deduced from quantitative results of five representative experiments. C, after a 2-h stabilization period in KRB buffer, cells were stimulated with 10 nm GLP-1 for 20 min. The U0126 (10 μm), was added during the 2-h KRB stabilization period and maintained during GLP-1 stimulation. 25 μg of protein lysates were subjected to 10% SDS-PAGE and immunoblotted with anti-phospho-p90RSK or anti-phospho-ERK1/2 antibodies. Tubulin and ERK1/2 contents within total cell lysates are shown as internal and loading control. Typical autoradiographs representative of three independent experiments are shown. D, the graphs represent levels of phospho-p90RSK or phospho-ERK2 deduced from quantitative results of three representative experiments. E, MIN6 cells were transfected with control or β-arrestin 1 siRNA for 6 h. 72 h after transfection, cells were stabilized in KRB buffer for 2 h and then stimulated for 20 min with 10 nm GLP-1. 25 μg of protein lysates were then subjected to 10% SDS-PAGE and immunoblotted with the anti-phospho-p90RSK antibody. Tubulin content within total cell lysates is shown as a loading control. Typical autoradiographs representative of three independent experiments are shown. F, the graph represents levels of phospho-p90RSK deduced from quantitative results of three independent experiments. CTL, control.

β-Arrestin 1 is required for GLP-1-induced ERK1/2 and Bad phosphorylation (Ser-112) in mouse pancreatic islets. A, islets isolated from normal control mice were stabilized in KRB buffer supplemented with 1.4 mm glucose for 2 h and then incubated with 1.4 mm glucose plus 100 nm GLP-1 for 20 min. The U0126 (10 μm) was added during the 2-h KRB stabilization period and maintained during GLP-1 stimulation. 25 μg of protein lysates were subjected to 10% SDS-PAGE and immunoblotted (IB) with the anti-phospho-ERK1/2, anti-phospho-p90RSK, anti-phospho-Bad (Ser-112), or anti-tubulin antibodies. The most representative blots obtained from three independent experiments are shown. C, islets isolated from WT or β-arrestin 1 KO mice were stabilized in KRB buffer supplemented with 1.4 mm glucose for 2 h and then incubated with 1.4 mm glucose plus 100 nm GLP-1 for 20 min. 15–25 μg of protein lysates were subjected to 10% SDS-PAGE and immunoblotted with the anti-phospho-ERK1/2, anti-phospho-Bad (Ser-112), anti-β-arrestin 1, or anti-tubulin antibodies. The most representative blots obtained from three independent experiments performed with 10–12 WT or β-arrestin 1 KO mice are shown. B and D, the graphs represent levels of phosphorylation of ERK2, p90RSK, and phosphorylation of Bad (Ser-112) deduced from quantitative results of three independent experiments.

Role of β-arrestin 1 in GLP-1-stimulated protection from apoptotic β-cell death. A, MIN6 cells were transfected with control or β-arrestin 1 siRNA. Following transfection, cells were cultured in serum free-DMEM containing 10 or 25 mm glucose in the presence or absence of 10 nm GLP-1 for 72 h. 45 μg protein lysates were subjected to 12.5% SDS-PAGE and immunoblotted (IB) with the anti-cleaved caspase-3 or anti-tubulin antibodies. Tubulin content within total cell lysates is shown as a loading control. Typical autoradiographs representative of 5 independent experiments are shown. B, the graph represents levels of cleaved caspase-3 deduced from quantitative results of five representative experiments. C, following isolation, islets were cultured overnight in RPMI containing 10 mm glucose and 5% bovine serum albumin. DNA fragmentation was measured in islets cultured in RPMI containing 10 or 30 mm glucose in the presence or absence of 100 nm GLP-1 for an additional 120-h period. The graph represents levels of DNA fragmentation deduced from quantitative results of 3–5 representative experiments. CTL, control.

p90RSK phosphorylation of Bad is required for GLP-1 antiapoptotic effect. A, MIN6 cells were transfected with control pcDNA3 vector, WT Bad, or mutant Bad S112A for 6 h. Following transfection, cells were cultured in serum-free DMEM containing 10 mm glucose in the presence or absence of 10 nm GLP-1 for 72 h. Levels of cleaved caspase-3 and tubulin were visualized by immunoblotting (IB). Tubulin content within total cell lysates is shown as loading control. Typical autoradiographs representative of three independent experiments are shown. B, the graph represents levels of cleaved caspase-3 deduced from quantitative results of three independent experiments. CTL, control.