Effects of obestatin on lipotoxicity-induced apoptosis in INS-1 cells

Abstract

Objective To investigate the effect of obestatin (OB) on lipotoxicity-induced apoptosis in pancreatic β-cell line INS-1. Methods The INS-1 cells were divided into the following groups: 5% bovine serum albumin (BSA) group, 5% BSA+ 1 nmol/L OB group, 5% BSA+ 10 nmol/L OB group, 5% BSA+ 100 nmol/L OB group, 5% BSA+ 500 nmol/L OB group, 0.5 mmol/L palmitic acid (PA) group, 0.5 mmol/L PA+ 1 nmol/L OB group, 0.5 mmol/L PA+ 10 nmol/L OB group, 0.5 mmol/L PA+ 100 nmol/L OB group, 0.5 mmol/L PA+ 500 nmol/L OB group. Cell survival rate was evaluated by using CCK-8. To investigate cell apoptosis, the INS-1 cells were then divided into 7 groups, including 5% BSA group, 100 nmol/L OB group, 50 μmol/L LY294002 group, 0.5 mmol/L PA group, 0.5 mmol/L PA+ 50 μmol/L LY294002 group, 0.5 mmol/L PA+ 100 nmol/L OB group, and 0.5 mmol/L PA+ 100 nmol/L OB+ 50 μmol/L LY294002 group. The INS-1 cells were finally divided into 5% BSA group, 0.5 mmol/L PA+ 100 nmol/L OB 0 min group, 0.5 mmol/L PA+ 100 nmol/L OB 15 min group, 0.5 mmol/L PA+ 100 nmol/L OB 30 min group, and 0.5 mmol/L PA+ 100 nmol/L OB 60 min group to assess total PKB and p-PKB (Ser 473) levels. Data were analyzed by using One-way ANOVA. Results OB alone dose-dependently promoted cell growth, and 100 nmol/L OB produced best result when compared with 5% BSA alone. Cell viability was decreased by 35% after PA treatment. Treatment with OB dose-dependently prevented PA-induced toxicity, most effectively by 35% at 100 nmol/L, with 10 nmol/L being the lowest active concentration by 23% when compared with PA alone. The percentage of apoptosis reached to 26% after PA treatment for 24 h, which was significantly increased when compared with BSA alone. Treatment with OB at 100 nmol/L reversed apoptosis rate by 15%. Obestatin at 100 nmol/L induced rapid activation of PKB and reached the maximal effect at 30 min. Obestatin-induced activation of PKB phosphorylation was markedly blocked by LY294002. The percentage of apoptosis increased to 23% after LY294002 treatment. Conclusions Obestatin inhibits lipotoxicity-induced apoptosis in pancreatic β-cell line INS-1 through PI3K/PKB signal pathway and the peptide may have potential in treating diabetes. Key words: Obestatin; Pancreatic beta-cells; Apoptosis