Dose-dependent cysteine-mediated protection of insulin-producing cells from damage by hydrogen peroxide.
OBJECTIVE
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 microM hydrogen peroxide in the presence of three different cysteine concentrations (0.1, 1 and 5mM). 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 1mM 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 1mM cysteine. The therapeutic window of cysteine was narrow: 0.1mM cysteine provided inadequate protection, and 5mM cysteine was already toxic in this setting.


CONCLUSIONS
A proper dose of cysteine could provide a safe and effective means to protect beta cells from oxidative damage.
Rasilainen
Suvi
S
Haartman Institute, Transplantation Laboratory, University of Helsinki, Haartmaninkatu 8, 00014 Helsinki, Finland. suvi.rasilainen@helsinki.fi
Nieminen
Jenni M
JM
Levonen
Anna-Liisa
AL
Otonkoski
Timo
T
Lapatto
Risto
R
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents / Q000378:metabolism; D003545:Cysteine / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000502:physiology; D020011:Protective Agents / Q000494:pharmacology*; D051381:Rats; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D004396:Coloring Agents; D007328:Insulin; D020011:Protective Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D004247:DNA; D006861:Hydrogen Peroxide; C022616:thiazolyl blue; D005978:Glutathione; D003545:Cysteine