Glucagon-like peptide-1 treatment delays the onset of diabetes in 8 week-old db/db mice

Abstract

Aims/hypothesis. Glucagon-like peptide-1 ameliorates the symptoms of diabetes through stimulation of insulin secretion and enhancement of beta-cell mass. We have therefore investigated the effects of glucagonlike peptide-1 on the development of diabetes, using db/db mice as a model of Type II diabetes. Methods. The potent glucagon-like peptide-1 analogue Exendin-4 or vehicle (control) was administered (i.p.; 1 nmol/kg) to obese 6-week old db/db mice daily for 14 days (n=10). Results. By 8 weeks of age, control db/db mice developed hyperglycaemia (fasting: 10.4±0.5 mmol/l), hyperinsulinaemia and impaired glucose tolerance. However, Exendin-4 treatment prevented hyperglycaemia (fasting: 6.1±1.0 mmol/l, p<0.01), with reduced plasma insulin concentrations (p<0.001) and improved glucose tolerance (p<0.05). Peripheral insulin sensitivity was not affected. However, insulin release in vivo and in vitro from the perfused pancreas was improved by Exendin-4, as were pancreatic insulin concentrations (0.54±0.02 vs 0.32±0.01 µg/mg protein, p<0.05). These changes occurred in conjunction with increased beta-cell mass (3.01±0.31 vs 2.22±0.22 mg, p<0.05) and proliferation (BrdU + beta-cells: 1.08±0.20 vs 0.47±0.11%, p<0.05), as well as decreased apoptosis (Tunel + beta-cells: 0.37±0.06 vs 1.20±0.21%). Western blot demonstrated increased expression of Akt1 (by fivefold, p<0.01) and p44 MAP kinase (by sixfold, p<0.01), and decreased activation of caspase-3 (by 30%, p<0.05). Conclusion/interpretation. Our results suggest that Ex4 treatment delays the onset of diabetes in 6-8 week old db/db mice, through a mechanism involving Akt1 and expansion of the functional beta-cell mass.

Mechanisms of action of glucagon-like peptide 1 in the pancreas

“…In the pancreata of the GLP-1-treated animals there were two distinct patterns of Ki-67 immunostaining; a) individual cells alone that co-stained for insulin and Ki-67 located within the islets as well as individual insulin-and Ki-67-positive cells throughout the pancreatic tissue, and, b) aggregates of islet-like but insulin-negative proliferating cells of about 10 cells in size were observed in the exocrine pancreas and not in untreated controls. At about the same time the Brubaker laboratory (Wang and Brubaker, 2002) also observed an increase in β cell area (1.35-fold) and proliferation (2.3-fold) in mice exhibiting a defect in the leptin receptor following treatment with Ex-4 (1 nmol/kg, ip, daily for 2 weeks).…”

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].…”

“…These include stimulation of glucose-dependent insulin secretion [10,11], and inhibition of glucagon release [11,12], gastric emptying [12,13] and food intake [14,15]. 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].…”