Dose-dependent cysteine-mediated protection of insulin-producing cells from damage by hydrogen peroxide

• Published in: Biochemical pharmacology Vol. 63; no. 7; pp. 1297 - 1304
• Main Authors: Rasilainen, Suvi, Nieminen, Jenni M., Levonen, Anna-Liisa, Otonkoski, Timo, Lapatto, Risto
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.04.2002; Elsevier Science
• Subjects: Animals; Anti-oxidant; Antioxidants - pharmacology; Apoptosis; Biological and medical sciences; Cell death; Cell Survival - drug effects; Coloring Agents - metabolism; Cysteine; Cysteine - pharmacology; DNA - drug effects; DNA - metabolism; Dose-Response Relationship, Drug; Drug Interactions; General and cellular metabolism. Vitamins; Glutathione - metabolism; Hydrogen Peroxide - pharmacology; Insulin - metabolism; Islets of Langerhans - drug effects; Islets of Langerhans - metabolism; Islets of Langerhans - pathology; Islets of Langerhans - physiology; Medical sciences; Oxidative stress; Pancreatic beta cell; Pathogenesis; Pharmacology. Drug treatments; Protective Agents - pharmacology; Rats; Tetrazolium Salts - metabolism; Thiazoles - metabolism; Tumor Cells, Cultured; ADP, adenosinediphosphate; Oxidative stress; Cysteine; RINm5F cells, rat insulinoma cell line; EDTA, ethylenediaminetetraacetic acid; GSH, glutathione; PI, propidium iodide; Anti-oxidant; Pathogenesis; Pancreatic beta cell; NAC, N-acetylcysteine; KRB, Krebs–Ringer buffer; PBS, phosphate-buffered saline; MTT, C, N-diphenyl- N′-4,5-dimethyl thiazol-2-yl tetrazolium bromide; Cell death; H 2O 2, hydrogen peroxide; HO, Hoechst 33342; IDDM, insulin dependent diabetes mellitus; INS-1, cells mouse insulinoma cell line; ROS, reactive oxygen species; HEPES, ( N[2-hydroxyethyl]piperazine- N′-[2-ethanesulfonic acid]); ATP, adenosinetriphosphate; RT, room temperature; Pancreatic hormone; Antioxidant; Metabolism; Hydrogen Peroxides; In vitro; Insulin; Protein hormone; Cytoprotector; Aminoacid; β Cell; Glutathione
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/S0006-2952(02)00864-X

Aims/Hypothesis: Oxidative damage is believed to play a key role in the process of pancreatic beta cell destruction leading to type 1 diabetes. The beta cells are sensitive to oxidative stress because their intracellular anti-oxidative defence mechanisms are weak. The defence mechanisms depend heavily on glutathione, the synthesis of which is dependent on the availability of cysteine. We investigated whether an increased amount of cysteine available could protect beta cells from oxidative damage. Methods: Rat insulinoma cells (RINm5F) were exposed to 50 or 100 μM hydrogen peroxide in the presence of three different cysteine concentrations (0.1, 1 and 5 mM). Cell viability was analyzed by vital staining and the cellular metabolic status by C, N-diphenyl- N′-4,5-dimethyl thiazol-2-yl tetrazolium bromide (MTT) analysis. Intracellular insulin, DNA and glutathione contents were measured. The mechanism of death was further clarified by gel electrophoretic DNA fragmentation analysis. Results: Hydrogen peroxide decreased cell viability and induced functional impairment. Vital staining indicated that 1 mM cysteine effectively protected the cells. The protective effect was confirmed by the MTT assay showing preserved metabolic integrity, and by measurements of intact intracellular insulin and DNA content. Cysteine increased intracellular glutathione. Gel electrophoretic analysis of DNA revealed hydrogen peroxide-induced apoptotic fragmentation. This was also abolished by 1 mM cysteine. The therapeutic window of cysteine was narrow: 0.1 mM cysteine provided inadequate protection, and 5 mM cysteine was already toxic in this setting. Conclusion: A proper dose of cysteine could provide a safe and effective means to protect beta cells from oxidative damage.