| Various theories have been proposed to explain the hyperglycemia-induced pathogenesis of vascular complications of diabetes. These include the activation of the polyol pathway, redox potential alterations, a de novo synthesis of diacylglycerol and activation of PKC and the nonenzymatic glycation. Furthermore, hyperglycemia increased the production of reactive oxygen species (ROS) inside the cells. Michael Brownlee and coworkers demonstrated recently that normalizing the glucose-induced increased levels of mitochondrial ROS prevented the activation of PKC, formation of AGEs, sorbitol accumulation and NFkB activation. Therefore, ROS produced by the mitochondrial electron transport chain seems to be a causal link between elevated levels of glucose and the pathways responsible for hyperglycemia-induced vascular damage. However, it is not clear how superoxide anion radicals are produced. The working hypothesis of this study is that increased concentrations of the glycolytic intermediate methylglyoxal (MGO) explain the hyperglycemia-induced alterations in endothelial cells by increasing ROS release followed by a ROS-dependent formation of AGEs, PKC activation and NFkB activation. Methylglyoxal is increased in diabetes mellitus and has been identified as an important intermediate in the non-enzymatic glycation. We have shown in vitro that MGO-modified proteins induced ROS. MGO primarily reacts with arginine residues. A possible second mechanism of MGO-induced oxidative stress is via an inhibition of the adenine nucleotide translocator (ANT) in mitochondria. The ANT is involved in the uptake of ADP and export of ATP. Inhibition of ANT will lead to low ADP and high ATP levels inside the mitochondria and high ADP and low ATP in the cytosol and may stimulate ROS production. Because of the arginine residues involved in the active site of ANT, MGO may modify the ANT and as such may stimulate mitochondrial ROS production. The objectives are: - To determine the accumulation of methylglyoxal and methylglyoxal adducts in different tissues in STZ rats; - To determine in isolated rat liver mitochondria the effect of methylglyoxal on ADT and ATP concentrations and production of ROS; - To study in in vitro experiments whether methylglyoxal and methylglyoxal adduct are increased in glucose-incubated endothelial cells and may lead to an increase in ROS production. With these biochemical studies more insight will be gained into the significance of MGO in the initiation of hyperglycemia-induced endothelial dysfunction. In addition to the present proposal, studies are going on with the aim to test to what extent MGO-adducts in type 1 diabetic patients, as measured with specific monoclonal antibodies in plasma samples, are related to vascular complications. |
