Antioxidant effect of angiotensin (1-7) in the protection of pancreatic ß cell function

Zhang, Fen; Liu, Chang; Wang, Lei; Cao, Xi; Wang, Ying Ying; Yang, Jin Kui

Cell culture. INS-1 insulinoma cells were a gift from Professor Liu Yong (Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai, China), which were originally supplied by Dr Claes Wollheim, University Medical Center (Geneva, Switzerland). The culture medium consisted of RPMI-1640 (Hyclone, Logan, UT, USA) with 11.1 mmol/l D-glucose supplemented with 10% fetal bovine serum (Hyclone), 100 U/ml penicillin, 100 µg/ml streptomycin (both from Invitrogen; Thermo Fisher Technology, Inc., Waltham, MA, USA), 10 mmol/l HEPES, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate and 50 µmol/l mercaptoethanol, in 5% CO 2 at 37˚C. To determine the effect of H 2 O 2 , INS-1 cells were grown for 48 h and subsequently incubated under basal conditions or in the presence of either 10 -8 mol/l Ang (1-7) (Sigma-Aldrich), 10 -6 mol/l A779 (Sigma-Aldrich) or Ang (1-7) and A779 together, for 2 h. H 2 O 2 at a final concentration of 250 µM was added in the final 15 min. Untreated cells served as a control. The cell supernatant was rapidly removed and rinsed twice with phosphate-buffered saline (PBS; Hyclone). Insulin secretion was determined at 16.7 mM glucose KRBB and insulin levels were measured using an insulin enzyme-linked immunosorbent assay kit (EMD Millipore, Billerica, MA, USA).