Perturbation of human coronary artery endothelial cell redox state and NADPH generation by methylglyoxal.
Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH). We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC) were incubated with high glucose (25 mM, 24 h, 37°C), or methylglyoxal (MGO), glyoxal, or glycolaldehyde (100-500 µM, 1 h, 37°C), before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05) decreased total thiols (∼35%), further experiments with MGO showed significant losses of GSH (∼40%) and NADPH (∼10%); these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10%) NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE) formed; lower levels of N(ε)-(carboxyethyl)lysine (CEL) and N(ε)-(carboxymethyl)lysine (CML) were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis.
Free Radical Group, The Heart Research Institute, Sydney, New South Wales, Australia ; Faculty of Medicine, University of Sydney, Sydney, New South Wales, Australia.
Philip E
PE
Morgan
Free Radical Group, The Heart Research Institute, Sydney, New South Wales, Australia ; Faculty of Medicine, University of Sydney, Sydney, New South Wales, Australia.
Pamela J
PJ
Sheahan
Free Radical Group, The Heart Research Institute, Sydney, New South Wales, Australia ; Faculty of Medicine, University of Sydney, Sydney, New South Wales, Australia.
Michael J
MJ
Davies
D000447:Aldehydes / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000187:drug effects* / Q000382:microbiology / Q000502:physiology; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism; D006801:Humans; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects*; D011765:Pyruvaldehyde / Q000494:pharmacology*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000447:Aldehydes; D017127:Glycation End Products, Advanced; D013438:Sulfhydryl Compounds; C015785:2-deoxyglucose-6-phosphate; D009249:NADP; D019298:Glucose-6-Phosphate; D011765:Pyruvaldehyde; D001120:Arginine; D005978:Glutathione