Exendin-4 protects oxidative stress-induced β-cell apoptosis through reduced JNK and GSK3β activity

Abstract

Oxidative stress induced by chronic hyperglycemia in type 2 diabetes plays a crucial role in progressive loss of β-cell mass through β-cell apoptosis. Glucagon like peptide-1 (GLP-1) has effects on preservation of β-cell mass and its insulin secretory function. GLP-1 possibly increases islet cell mass through stimulated proliferation from β-cell and differentiation to β-cell from progenitor cells. Also, it probably has an antiapoptotic effect on β-cell, but detailed mechanisms are not proven. Therefore, we examined the protective mechanism of GLP-1 in β-cell after induction of oxidative stress. The cell apoptosis decreased to ~50% when cells were treated with 100 µM H(2)O(2) for up to 2 hr. After pretreatment of Ex-4, GLP-1 receptor agonist, flow cytometric analysis shows 41.7% reduction of β-cell apoptosis. This data suggested that pretreatment of Ex-4 protect from oxidative stress-induced apoptosis. Also, Ex-4 treatment decreased GSK3β activation, JNK phosphorylation and caspase-9, -3 activation and recovered the expression of insulin2 mRNA in β-cell lines and secretion of insulin in human islet. These results suggest that Ex-4 may protect β-cell apoptosis by blocking the JNK and GSK3β mediated apoptotic pathway.

Keywords: Apoptosis; Exendin-4; Glucagon-Like Peptide 1; Insulin-Secreting Cells; Oxidative Stress.

Fig. 1

Effects of the NAC or Ex-4 on oxidative stress-induced apoptosis. HIT-T15 cells were pretreated NAC (5 mM) or Ex-4 (25 nM), for 1 hr before stress induction. (A) After exposure to 100 µM H₂O₂, HIT-T15 cells apoptosis increased by time. Cells viability was measured with the MTT reduction assay. (B) After treatment of H₂O₂ (100 µM, 2 hr), effects of the NAC or Ex-4 on cell viability were measured by MTT reduction assay. (C) H₂O₂ (100 µM, 4 hr)-induced apoptotic nuclei reduced via NAC or Ex-4. Photographs were taken using a blue filter at a magnification of ×400. (D) Flow cytometric analysis of apoptosis of HIT-T15 cells exposed to H₂O₂ (100 µM, 8 hr). Apoptotic cells were measured by FACS analysis after Annexin V/PI staining. FL1: Annexin V-FITC, FL2: PI. Data are shown as the means±SE of six independent experiments. ^*P<0.001 vs control cells; ^†P<0.001 vs H₂O₂ alone.

Fig. 2

Protective effect of Ex-4 on oxidative stress-induced apoptosis via PI3-kinase/Akt/GSK3β and JNK signaling pathway in human islet. Human islet was pretreated with H89 (PKA inhibitor, 10 µM) or SP600125 (JNK inhibitor, 20 µM). After 30 min, human islet was treated Ex-4 (25 nM), NAC (5 mM) or forskolin (20 µM), for 1 hr before stress induction. After treatment of H₂O₂ (100 µM, 2 hr), (A) In oxidative stress condition, expression change of total Akt, GSK3β, JNK, phospho-Akt (Ser 473), phospho-GSK3β (Ser-9) and phospho-JNK were assessed by western blot. (B) Total JNK, GSK3β, phospho-GSK3β (Ser-9) and phospho-JNK were detected in the absence or presence of SP600125. These results are representative of three independent experiments.

Fig. 3

Protective effect of Ex-4 on caspase-8, 9, and -3 activity in oxidative stress-induced apoptosis. HIT-T15 cells were pretreated with H89 (PKA inhibitor, 10 µM). After 30 min, HIT-T15 cells were treated Ex-4 (25 nM) or NAC (5 mM), for 1 hr before stress induction. After treatment of H₂O₂ (100 µM, 2 hr), (A) Cell lysates were incubated with IETD-pNA, LEHD-pNA and DEVD-pNA for in vitro caspase-8, -9, and -3, respectively. The caspase-8, -9, and -3 activity were measured by the colorimetric assay. (B) Active form of capase-9 and -3 were detected using western blot analysis. Data are shown as the means±S.E. of three independent experiments. ^*P<0.01 vs control cells; ^†P<0.01 vs H₂O₂ alone; ^‡P<0.01 vs Ex-4 or NAC in treated H₂O₂.

Fig. 4

Effects of the Ex-4 on insulin secretion. Human islet was pretreated with H89 (10 µM). After 30 min, HIT-T15 cells or human islet was treated Ex-4 (25 nM) and NAC (5 mM) for 1 hr before stress induction. After treatment of H₂O₂ (100 µM, 2 hr), (A) Expression levels of insulin2 mRNA were examined by real time RT-PCR in HIT-T15 cells. Data were expressed as the rates to the expression levels to GAPDH in the same sample. GAPDH used for loading control. (B) Insulin secretion in human islet was detected by Insulin ELISA kit. Data are shown as the means±S.E. of four independent experiments. ^*P<0.01 vs H₂O₂ alone; ^†P<0.01 vs Ex-4 in treated H₂O₂.