Methylglyoxal-induced mitochondrial dysfunction in vascular smooth muscle cells
Graphical abstract
Methylglyoxal significantly inhibited mitochondrial complex III activity in rat aortic smooth muscle cells.
Introduction
Mitochondria are the powerhouse of mammalian cells. When electrons pass through complexes I–IV of the electron transport chain (ETC), 2–5% of electrons leak out of the ETC and interact with oxygen to form superoxide (O2−) in mitochondria, which accounts for about 85% of total intracellular O2−[1], [2]. Electron leakage most often occurs at complex I and complex III of the ETC, and the amount of O2− increases dramatically if these complexes are inhibited [3]. Under physiological condition, O2− is converted to hydrogen peroxide (H2O2) by manganese superoxide dismutase (MnSOD), which is the primary anti-oxidant defensive enzyme in mitochondria [4]. This anti-oxidant system ensures the clearance of free radicals and protects cells against oxidative damage. Mitochondria also contain specific nitric oxide synthase (mtNOS), which catalyzes the production of nitric oxide (NO) [5]. A considerable amount of NO generated from mtNOS reacts with O2− to form peroxynitrite (ONOO−) [6]. ONOO− is a highly reactive oxidant, damaging proteins, DNA, and lipids [7]. Mitochondrial oxidative stress is tightly related to the pathophysiology of type 2 diabetes and associated complications [8].
Methylglyoxal (MG) is a dicarbonyl compound which readily reacts with certain proteins to form advanced glycated endproducts (AGEs), like N-carboxyethyl-lysine (CEL). This rapid interaction contributes to the pathogenesis of insulin resistance syndrome, such as diabetes and hypertension [9], [10], [11]. We have previously shown that MG induced the generation of reactive oxygen species (ROS) in hypertensive rat vascular smooth muscle cells (VSMCs) and animal tissues [12], [13]. We also found that MG [14] or fructose (a precursor of MG) [15] induced the production of ONOO− in cultured rat thoracic aortic smooth muscle cells (A-10 cells).
To date, the role of MG in the regulation of mitochondrial function is unclear. We hypothesized that MG affects mitochondrial function by interfering with respiratory complexes and altering mitochondrial ONOO− production. In the present study, changes in mitochondrial ROS production, activity of mitochondrial complex, and MnSOD activity in A-10 cells in the presence of exogenous MG were investigated. AGEs cross-link breaker alagebrium and non-specific anti-oxidant n-acetyl-l-cysteine (NAC) were also used in this study.
Section snippets
Chemicals and antibodies
Anti-nitrotyrosine antibody and bovine serum were purchased from Invitrogen Corporation (Burlington, ON, Canada). Anti-CEL antibody was obtained from Novo Nordisk (A/S, Denmark). Alagebrium was from Alteon Inc. (Parsippany, NJ, USA). Cell culture medium, FITC IgG fluorescent antibody, MG, NAC, o-phenylenediamine (o-PD), 2-methylquinoxaline, 5-methylquinoxaline, KCN, 2,6-dichlorophenolindophenol (DCPIP), rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, coenzyme Q1, cytochrome C, NaN3,
Effect of MG on mtROS generation
After A-10 cells were treated with exogenous MG (30 μM) for 18 h, mitochondrial MG content increased by 50.7% (0.205 ± 0.012 nmol/mg vs. 0.136 ± 0.014 nmol/mg mitochondrial protein, p < 0.01, n = 4 for each group). Alagebrium (50 μM) had no effect on basal content of mitochondrial MG but its presence decreased the effect of exogenous MG on mitochondrial MG content (0.14 ± 0.009 nmol/mg vs. 0.205 ± 0.01 nmol/mg mitochondrial protein, p < 0.01, n = 4 for each group). NAC (600 μM) had no effect on mitochondrial MG
Discussion
MG causes cross-link among lysine, cysteine, and arginine residues of selective proteins to form AGEs, like CEL, altering the structure of proteins and their functions [9]. Higher levels of MG have been found in diabetic patients than in healthy controls [21]. In the present study, we observed that mitochondrial MG content was significantly increased after the cells were treated with exogenous MG. It appears that MG can move across plasmalemma and mitochondrial membrane to attack different
Acknowledgements
This work was supported by operating grants from the Canadian Institutes of Health Research (CIHR, MOP-68938) and from Heart and Stroke Foundation of Saskatchewan to L. Wu. H. Wang is supported by a studentship from the GREAT program of CIHR/Heart and Stroke Foundation of Canada.
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