Elsevier

Biochemical Pharmacology

Volume 77, Issue 11, 1 June 2009, Pages 1709-1716
Biochemical Pharmacology

Methylglyoxal-induced mitochondrial dysfunction in vascular smooth muscle cells

https://doi.org/10.1016/j.bcp.2009.02.024Get rights and content

Abstract

The effects of methylglyoxal (MG) on mitochondria with specific foci on peroxynitrite (ONOO) production, manganese superoxide dismutase (MnSOD) activity, and mitochondrial functions in vascular smooth muscle A-10 cells were investigated. Mitochondrial MG content was significantly increased after A-10 cells were treated with exogenous MG, and so did advanced glycated endproducts (AGEs) formation, indicated by the appearance of Nɛ-(carboxyethyl) lysine, in A-10 cells. The levels of mitochondrial reactive oxygen species (mtROS) and ONOO were significantly increased by MG treatment. Application of ONOO specific scavenger uric acid lowered the level of mtROS. MG significantly enhanced the production of mitochondrial superoxide (O2radical dot) and nitric oxide (NO), which were inhibited by SOD mimic 4-hydroxy-tempo and mitochondrial nitric oxide synthase (mtNOS) specific inhibitor 7-nitroindazole, respectively. The activity of MnSOD was decreased by MG treatment. Furthermore, MG decreased respiratory complex III activity and ATP synthesis in mitochondria, indicating an impaired mitochondrial respiratory chain. AGEs cross-link breaker alagebrium reversed all aforementioned mitochondrial effects of MG. Our data demonstrated that mitochondrial function is under the control of MG. By inhibiting Complex III activity, MG induces mitochondrial oxidative stress and reduces ATP production. These discoveries will help unmask molecular mechanisms for various MG-induced mitochondrial dysfunction-related cellular disorders.

Graphical abstract

Methylglyoxal significantly inhibited mitochondrial complex III activity in rat aortic smooth muscle cells.

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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 (O2radical dot) in mitochondria, which accounts for about 85% of total intracellular O2radical dot[1], [2]. Electron leakage most often occurs at complex I and complex III of the ETC, and the amount of O2radical dot increases dramatically if these complexes are inhibited [3]. Under physiological condition, O2radical dot 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 O2radical dot 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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