Sensitivity of the 2-oxoglutarate carrier to alcohol intake contributes to mitochondrial glutathione depletion

Authors

  • Olga Coll,

    1. Liver Unit, Hospital Clínic i Provincial, Institut de Malalties Digestives, Instituto de Investigaciones Biomédicas August Pi i Sunyer and Departamento de Patología Experimental, Instituto Investigaciones Biomédicas Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, Spain
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  • Anna Colell,

    1. Liver Unit, Hospital Clínic i Provincial, Institut de Malalties Digestives, Instituto de Investigaciones Biomédicas August Pi i Sunyer and Departamento de Patología Experimental, Instituto Investigaciones Biomédicas Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, Spain
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  • Carmen García-Ruiz,

    1. Liver Unit, Hospital Clínic i Provincial, Institut de Malalties Digestives, Instituto de Investigaciones Biomédicas August Pi i Sunyer and Departamento de Patología Experimental, Instituto Investigaciones Biomédicas Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, Spain
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  • Neil Kaplowitz,

    1. USC Research Center for Alcoholic Liver and Pancreatic Diseases, Department of Medicine, Keck School of Medicine, Los Angeles, CA
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  • J. C. Fernández-Checa Ph.D.

    Corresponding author
    1. Liver Unit, Hospital Clínic i Provincial, Institut de Malalties Digestives, Instituto de Investigaciones Biomédicas August Pi i Sunyer and Departamento de Patología Experimental, Instituto Investigaciones Biomédicas Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, Spain
    • Liver Unit, Hospital Clinic i Provincial, Villarroel, 170, 08036 Barcelona, Spain. fax: (34) 93-451-5272
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Abstract

The mitochondrial pool of reduced glutathione (mGSH) is known to play a protective role against liver injury and cytokine-mediated cell death. However, the identification of the mitochondrial carriers involved in its transport in hepatocellular mitochondria remains unestablished. In this study, we show that the functional expression of the 2-oxoglutarate carrier from HepG2 cells in mitochondria from Xenopus laevis oocytes conferred a reduced glutathione (GSH) transport activity that was inhibited by phenylsuccinate, a specific inhibitor of the carrier. In addition, the mitochondrial transport of GSH and 2-oxoglutarate in isolated mitochondria from rat liver exhibited mutual competition and sensitivity to glutamate and phenylsuccinate. Interestingly, the kinetics of 2-oxoglutarate transport in rat liver mitochondria displayed a single Michaelis-Menten component with a Michaelis constant of 3.1 ± 0.3 mmol/L and maximum velocity of 1.9 ± 0.1 nmol/mg protein/25 seconds. Furthermore, the initial rate of 2-oxoglutarate was reduced in mitochondria from alcohol-fed rat livers, an effect that was not accompanied by an alcohol-induced decrease in the 2-oxoglutarate messenger RNA levels but rather by changes in mitochondrial membrane dynamics induced by alcohol. The fluidization of mitochondria by the fluidizing agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) (A2C) restored the initial transport rate of both GSH and 2-oxoglutarate. Finally, these changes were reproduced in normal liver mitochondria enriched in cholesterol where the fluidization of cholesterol-enriched mitochondria with A2C restored the order membrane parameter and the mitochondrial 2-oxoglutarate uptake. In conclusion, these findings provide unequivocal evidence for 2-oxoglutarate as a GSH carrier and its sensitivity to membrane dynamics perturbation contributes in part to the alcohol-induced mGSH depletion.

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