Glutathione has a diversity of crucial physiological roles, but it principally serves as an endogenous antioxidant. It functions as a cofactor for GPx, the major defense mechanism

against potential toxic hydrogen peroxide and other peroxides [12]. During the detoxification process of peroxides, GSSG is formed. GSH is regenerated from GSSG by the NADPH-dependent enzyme glutathione reductase (GR) [13]. Additionally, glutathione S-transferase (GST) uses GSH as a substrate to form conjugates with electrophiles, resulting in more water soluble metabolites which are more readily excreted. Glutaredoxin (Grx) utilizes the reducing power of glutathione to catalyze disulfide reductions in the presence of NADPH and GR. Grx is involved in regulation of various cellular functions, including electron transport and protein folding [14]. Because little is known about the effects of AGEs on pancreatic beta cells, we

investigated Selleck Fluorouracil the effect of CML on pancreatic cell viability and determined the activity and expression of components belonging to the glutathione system. All chemicals were purchased from Sigma-Aldrich (Steinheim, Germany) unless stated otherwise. CML was obtained from SyMO-Chem BV (Eindhoven, Netherlands). Roswell Park Memorial Institute (RPMI) compound screening assay 1640 medium, Hank’s Balanced Salt Solution (HBSS), trypsin-EDTA (1x), non-heat inactivated fetal calf serum (FCS), and L-Glutamine were obtained from Gibco (Breda, Etomidate The Netherlands). The human pancreatic beta cell line 1.1E7 [15] was obtained from HPA Culture Collections. Cells were cultured in RPMI 1640 medium with 10% non-heat inactivated FCS and 2 mM L-glutamine. Cells were maintained in T75 flasks at 37 °C in a 5% CO2 atmosphere. Cells were seeded at a density of 5000 cells per well in a 96-well plate and after overnight attaching, medium was removed and cells were washed with HBSS. CML was added to the plate in different concentrations (0–1 mM). Subsequently, cells were incubated for 24 hours. After treatment, supernatant was removed and cells were washed with PBS. Next,

100 μl of MTT solution (0.5 mg/ml in culture medium) was added and cells were incubated for 1 hour at 37 °C. After incubation, the plate was washed with PBS and the formazan crystals were dissolved in 200 μl DMSO. Cells were incubated for 30 minutes after which the absorbance at 540 nm was measured spectrophotometrically using a microplate reader. Relative viability is expressed as a percentage relative to untreated cells. The production of intracellular reactive oxygen species was measured using 2,7-dichlorofluorescein diacetate (DCFH-DA) as described previously ([16] and [17]). Cells were seeded at a density of 5000 cells per well in a 96 well plate and after overnight attaching, medium was removed and cells were washed with HBSS. Cells were then incubated with 0.5 mM CML in the presence of 10 μM DCFH-DA.