Glucagon-like peptide-1 protects the apoptosis of pancreatic β cells induced by endoplasmic reticulum stress

Abstract

Objective To explore the potential protective mechanism of glucagon-like peptide-1(GLP-1) on thapsigargin (TG)induced pancreatic β cell apoptosis. Methods The rat pancreatic β cell line-r9 cells cultured in vitro were used to compare the pancreatic β cell apoptosis with the presence of GLP-1 and GLP-1 receptor agonists exendin-4 while endoplasmic reticulum (ER)stress was induced by thapsigargin. Further, we investigated the effects of GLP-1 and exendin-4 on the release of cytochrome c and generation of reactive oxygen species (ROS) during ER stress. This experiment was divided into four groups, normal group, TG group (100 nmol/L), GLP-1(10 nmol/L)+ TG(100 nmol/L)group and Exendin-4(100 nmol/L)+ TG(100 nmol/L)group, and apoptosis rate of the four groups was detected by DNA fragmentation, TUNEL staining, AnnexinV-PI double dye flow cytometric analysis. We used the Western Blot to test the release of cytochrome c, used fluorescence probe DCFH-DA to test the reactive oxygen species(ROS) level and used rhodamine-110(Z-DEVD-R110) dyeing to detect the cells Caspase3's activity. We used t-testing to compare the difference between two groups and used one way analysis of variance to test the differences among the four groups. Results The apoptosis of the latter two groups which were tested by DNA fragmentation and TUNEL staining was significantly reduced compared with TG group (t=6.13 or 6.73, both P<0.01). The apoptosis rate of β cells tested by AnnexinV-PI double dye flow cytometry was 26%, which was reduced to 19.7% and 19.2% in the existence of GLP-1 and exendin-4, respectively. Further experiments showed that GLP-1 and exendin-4 reduced cytochrome c release, ROS generation and caspase3 activation. The Western blot of cytoplasm albumen showed that GLP-1 and exendin-4 can reduce the release of cytochrome c in ER stress (t=5.37 or 7.26, both P<0.01). The detection of ROS in mitochondria showed that ROS, a certain amount of which in cytoplasm could be detected under normal circumstances, was significantly increased in the existence of TG. However, GLP-1 and exendin-4 could reduced the release of ROS. In addition, GLP-1 and exendin-4 could inhibit the activation and release of Caspase-3 (t=3.46 or 4.52, both P<0.01). Conclusions GLP-1 could decrease the TG-induced pancreatic β cell apoptosis, which was probably through the machanisms of inhibiting cytochrome c release, Caspase-3 activation and ROS generation in ER stress. Key words: Glucagon-like peptide-1; β cell; ER stress; Apoptosis; Oxidative stress