Glucagon-like peptide-1 regulates proliferation and apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells

Abstract

Aims/hypothesis. The incretin hormone glucagon-like peptide-1 augments islet cell mass in vivo by increasing proliferation and decreasing apoptosis of the beta cells. However, the signalling pathways that mediate these effects are mostly unknown. Using a clonal rat pancreatic beta cell line (INS-1), we examined the role of protein kinase B in mediating beta-cell growth and survival stimulated by glucagon-like peptide-1. Methods. Immunoblot analysis was used to detect active (phospho-) and total protein kinase B. Proliferation was assessed using 3 H-thymidine incorporation, while apoptosis was quantitated using 4′-6-diamidino-2-phenylindole staining and APO percentage apoptosis assay. Kinase-dead and wild-type protein kinase B was introduced into cells using adenoviral vectors. Results. Glucagon-like peptide-1 rapidly activated protein kinase B in INS-1 cells (by 2.7±0.7-fold, p<0.05). This effect was completely abrogated by inhibition, with wortmannin, of the upstream activator of protein kinase B, phosphatidylinositol-3-kinase. Glucagon-like peptide-1 also stimulated INS-1 cell proliferation in a dose-dependent manner (by 1.8±0.5-fold at 10 −7 mol/l, p<0.01), and inhibited staurosporine-induced apoptosis (by 69±12%, p<0.05). Both of these effects were also prevented by wortmannin treatment. Ablation of protein kinase B by adenovirus-mediated overexpression of the kinase-dead form of protein kinase Bα prevented protein kinase B phosphorylation and completely abrogated both cellular proliferation (p<0.05) and protection from drug-induced cellular death (p<0.01) induced by glucagon-like peptide-1. Conclusions/interpretation. These results identify protein kinase B as an essential mediator linking the glucagon-like peptide-1 signal to the intracellular machinery that modulates beta-cell growth and survival. [Diabetologia (2004) 47:478-487]

Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B

“…Immunoblotting Cells were grown in six-well plates to 80-85% confluence and then pre-treated with or without 10 nmol/l exendin-4 for 18 h, followed by incubation with a mixture of cytokines in the absence or presence of exendin-4 for a further 18 h. Cells were subsequently lysed in buffer containing 1% Triton X-100 and a mixture of protease and phosphatase inhibitors, as previously described [19,22]. Protein content was measured by the Bradford assay (Bio-Rad, Hercules, CA, USA), and protein samples (50 μg) were separated on an 8 or 10% SDS-PAGE gel and electrotransferred onto polyvinylidene difluoride filters (Bio-Rad).…”

“…The proteins were probed with the following specific primary antibodies: rabbit antibodies directed against phosphorylated Ser 473 -phosphorylated PKB, total PKB, phosphorylated GSK3β (glycogen synthase kinase 3β), total GSK3, phosphorylated and total CREB (cAMP-response-element-binding protein), cleaved caspase-3 (each at 1:1,000 dilution; New England BioLabs, Mississauga, ON, Canada), iNOS (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), MnSOD (manganese superoxide dismutase; 1:1,000 dilution; Santa Cruz Biotechnology), catalase (1:1,000 dilution) and β-actin (1:4,000 dilution) (both from Sigma Chemical Company). The immunoreactive bands were then visualised with horseradish peroxidase-conjugated sheep anti-rabbit IgG using an ECL detection system (Amersham Pharmacia Biotech, Baie d'Urfe, QC, Canada), as described previously [19,22].…”

“…Indeed, PKB is increasingly implicated as a key player in the regulation of beta cell growth and survival [27]. Furthermore, GLP-1 has been demonstrated to increase PKB levels in beta cells, both in vivo in db/db mice and in vitro in INS-1 cells [19,22,28]. We have recently demonstrated that the adenovirus-mediated ablation of PKB abrogates the effect of GLP-1 in the prevention of staurosporine-induced apoptosis in INS-1 cells [22].…”

“…Recent data show that the actions of GLP-1 that protect against diabetes also include the enhancement of beta cell mass in rodents, through inhibition of beta cell apoptosis, and stimulation of beta cell proliferation and islet neogenesis [16][17][18][19]. Furthermore, GLP-1 and its agonists increase the survival of immortalised rodent beta cell lines when challenged with various apoptotic stimulators, including hydrogen peroxide, fatty acids, streptozotocin and staurosporine [17,[20][21][22]. Importantly, exendin-4 treatment also reduces cytokine-induced apoptosis in purified rat beta cells [17].…”

“…Furthermore, GLP-1 has been demonstrated to increase PKB levels in beta cells, both in vivo in db/db mice and in vitro in INS-1 cells [19,22,28]. We have recently demonstrated that the adenovirus-mediated ablation of PKB abrogates the effect of GLP-1 in the prevention of staurosporine-induced apoptosis in INS-1 cells [22]. These findings suggest an important role for PKB as a mediator of the anti-apoptotic actions of GLP-1 in the beta cell.…”