title: 2-Methylcitric acid impairs glutamate metabolism and induces permeability transition in brain mitochondria.
abstract: Accumulation of 2-methylcitric acid (2MCA) is observed in methylmalonic and propionic acidemias, which are clinically characterized by severe neurological symptoms. The exact pathogenetic mechanisms of brain abnormalities in these diseases are poorly established and very little has been reported on the role of 2MCA. In the present work we found that 2MCA markedly inhibited ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate, with a less significant inhibition in pyruvate plus malate respiring mitochondria. However, no alterations occurred when α-ketoglutarate or succinate was used as respiratory substrates, suggesting a defect on glutamate oxidative metabolism. It was also observed that 2MCA decreased ATP formation in glutamate plus malate or pyruvate plus malate-supported mitochondria. Furthermore, 2MCA inhibited glutamate dehydrogenase activity at concentrations as low as 0.5 mM. Kinetic studies revealed that this inhibitory effect was competitive in relation to glutamate. In contrast, assays of osmotic swelling in non-respiring mitochondria suggested that 2MCA did not significantly impair mitochondrial glutamate transport. Finally, 2MCA provoked a significant decrease in mitochondrial membrane potential and induced swelling in Ca(2+)-loaded mitochondria supported by different substrates. These effects were totally prevented by cyclosporine A plus ADP or ruthenium red, indicating induction of mitochondrial permeability transition. Taken together, our data strongly indicate that 2MCA behaves as a potent inhibitor of glutamate oxidation by inhibiting glutamate dehydrogenase activity and as a permeability transition inducer, disturbing mitochondrial energy homeostasis. We presume that 2MCA-induced mitochondrial deleterious effects may contribute to the pathogenesis of brain damage in patients affected by methylmalonic and propionic acidemias. We propose that brain glutamate oxidation is disturbed by 2-methylcitric acid (2MCA), which accumulates in tissues from patients with propionic and methylmalonic acidemias because of a competitive inhibition of glutamate dehydrogenase (GDH) activity. 2MCA also induced mitochondrial permeability transition (PT) and decreased ATP generation in brain mitochondria. We believe that these pathomechanisms may be involved in the neurological dysfunction of these diseases.
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal de Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Alexandre Umpierrez
authors.initials: AU
authors.lastname: Amaral
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal de Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Cristiane
authors.initials: C
authors.lastname: Cecatto
authors.affiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil.
authors.forename: Roger Frigério
authors.initials: RF
authors.lastname: Castilho
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal de Rio Grande do Sul, Porto Alegre, RS, Brazil.|Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil.
authors.forename: Moacir
authors.initials: M
authors.lastname: Wajner
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000592:Amino Acid Metabolism, Inborn Errors / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002951:Citrates / Q000494:pharmacology*; D016572:Cyclosporine / Q000494:pharmacology; D004734:Energy Metabolism / Q000187:drug effects; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007656:Ketoglutaric Acids / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D010085:Oxidative Phosphorylation / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects; D056693:Propionic Acidemia / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
chemical_list: D002951:Citrates; D007656:Ketoglutaric Acids; D008293:Malates; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D018698:Glutamic Acid; C031605:2-methylcitric acid; D000244:Adenosine Diphosphate; C030298:malic acid; D016572:Cyclosporine; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D005969:Glutamate Dehydrogenase; D002118:Calcium
keywords: 2-methylcitric acid; glutamate dehydrogenase; glutamate oxidative metabolism; mitochondrial permeability transition; mitochondrial respiration