BCL-2 expression or antioxidants prevent hyperglycemia-induced formation of intracellular advanced glycation endproducts in bovine endothelial cells.
Hyperglycemia rapidly induces an increase in intracellular advanced glycation end products (AGEs) in bovine endothelial cells, causing an alteration in bFGF activity (Giardino, I., D. Edelstein, and M. Brownlee. 1994. J. Clin. Invest. 94:110-117). Because sugar or sugar-adduct autoxidation is critical for AGE formation in vitro, we evaluated the role of reactive oxygen species (ROS) in intracellular AGE formation, using bovine aortic endothelial cells. 30 mM glucose increased intracellular ROS formation by 250% and lipid peroxidation by 330%, while not affecting ROS in the media. In cells depleted of glutathione, intracellular AGE accumulation increased linearly with ROS generation as measured by immunoblotting and the fluorescent probe DCFH (AGE 0.258-3.531 AU* mm/5x10(4) cells, DCF 57-149 mean AU, r = .998, P < .002). Deferoxamine, alpha-tocopherol, and dimethylsulfoxide each inhibited hyperglycemia-induced formation of both ROS and AGE. To differentiate an effect of ROS generation on AGE formation from an effect of more distal oxidative processes, GM7373 endothelial cell lines were generated that stably expressed the peroxidation-suppressing proto-oncogene bcl-2. bcl-2 had no effect on hyperglycemia-induced intracellular ROS formation. In contrast, bcl-2 expression decreased both lipid peroxidation (100% at 3 h and 29% at 168 h) and AGE formation (55% at 168 h). These data show that a ROS-dependent process plays a central role in the generation of intracellular AGEs, and that inhibition of oxidant pathways prevents intracellular AGE formation.
Giardino
I
I
Department of Medicine, Albert Einstein College of Medicine, New York, USA.
Edelstein
D
D
Brownlee
M
M
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D004336:Drug Antagonism; D004730:Endothelium, Vascular / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D017127:Glycation End Products, Advanced / Q000096:biosynthesis*; D006943:Hyperglycemia / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2
D000975:Antioxidants; D017127:Glycation End Products, Advanced; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D005947:Glucose