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Blocking mitochondrial alanine and pyruvate metabolism in hepatocytes worsens acetaminophen-induced liver injury in mice

View ORCID ProfileJoel H. Vazquez, Nicole K.H. Yiew, View ORCID ProfileMichael R. Martino, View ORCID ProfileFelicia D. Allard, View ORCID ProfileEric U. Yee, Sandra S. McCullough, View ORCID ProfileLaura P. James, View ORCID ProfileBrian N. Finck, View ORCID ProfileMitchell R. McGill
doi: https://doi.org/10.1101/2022.06.14.495517
Joel H. Vazquez
1Dept. of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR USA
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  • ORCID record for Joel H. Vazquez
Nicole K.H. Yiew
2Dept. of Medicine, Washington University School of Medicine, St. Louis, MO USA
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Michael R. Martino
2Dept. of Medicine, Washington University School of Medicine, St. Louis, MO USA
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Felicia D. Allard
3Dept. of Pathology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR USA
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  • ORCID record for Felicia D. Allard
Eric U. Yee
3Dept. of Pathology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR USA
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Sandra S. McCullough
4Dept. of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR USA
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Laura P. James
4Dept. of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR USA
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Brian N. Finck
2Dept. of Medicine, Washington University School of Medicine, St. Louis, MO USA
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Mitchell R. McGill
1Dept. of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR USA
3Dept. of Pathology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR USA
5Dept. of Environmental Health Sciences, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR USA
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  • For correspondence: mmcgill@uams.edu
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ABSTRACT

Background and Aims Acetaminophen (APAP) overdose causes mitochondrial damage and acute liver injury. Alanine and pyruvate are important substrates for the TCA cycle and increasing evidence points to a protective role for mitochondrial intermediary metabolism in APAP hepatotoxicity. We hypothesized that suppressing hepatic alanine and pyruvate metabolism by blocking alanine transaminase (ALT) and/or the mitochondrial pyruvate carrier (MPC) would impair mitochondrial metabolism in APAP toxicity and therefore exacerbate APAP-induced injury.

Methods We treated wild-type mice with the ALT inhibitor β-chloro-l-alanine (BCLA) before a toxic dose of APAP (300 mg/kg) and measured liver injury (serum ALT and lactate dehydrogenase [LDH], histology) and other parameters. We also generated liver-specific ALT2 knockout (KO) mice, mitochondrial pyruvate carrier 2 (MPC) KO mice, and combined ALT2/MPC KO (CKO) mice, and compared injury, APAP-protein adducts, glutathione, c-Jun N-terminal kinase activation, Cyp2e1 expression, and serum metabolomics among genotypes.

Results BCLA reduced hepatic ALT activity up to 65% but did not affect injury. ALT2 deletion also had no effect on APAP hepatotoxicity. Similarly, MPC deletion alone had no effect. However, combined ALT2 and MPC loss significantly worsened injury (p = 0.005 for LDH for WT vs. CKO; p = 0.07 for % necrosis), increased plasma pyruvate content (p = 0.032), altered the plasma abundance of several amino acids, and decreased basal hepatic glutathione (p = 0.053). Importantly, the exacerbation of injury in CKO mice was not associated with increased APAP-protein adducts.

Conclusions The MPC and ALT systems are critical redundant mechanisms to conserve mitochondrial metabolism and limit APAP toxicity. ALT activity is more than a passive biomarker of injury in this context.

Competing Interest Statement

This study was funded in part by a 2018 Pinnacle Research Award from the AASLD Foundation (MRM); the Arkansas Biosciences Institute (MRM), which is the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000; and the National Institutes of Health grants T32 GM106999 (JHV), R01 DK104735 (BNF), R01 DK117657 (BNF), UL1TR003107 (LPJ), and SB1DK079387 (LPJ). LPJ is the Chief Medical Officer of Acetaminophen Toxicity Diagnostics, LLC, which is developing methods for measurement of APAP-protein adducts; MRM consults for Acetaminophen Toxicity Diagnostics, LLC, and has received research funds from GlaxoSmithKline for unrelated projects; BNF is a shareholder and member of the scientific advisory board of Cirius Therapeutics, which is developing an MPC inhibitor for treating nonalcoholic steatohepatitis. The remaining authors declare that they have no conflicts of interest related to the content of this article.

Footnotes

  • Grant support: This study was funded in part by a 2018 Pinnacle Research Award from the AASLD Foundation (MRM); the Arkansas Biosciences Institute (MRM), which is the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000; and the National Institutes of Health grants T32 GM106999 (JHV), R01 DK104735 (BNF), R01 DK117657 (BNF), UL1TR003107 (LPJ), and SB1DK079387 (LPJ).

  • Disclosures: LPJ is the Chief Medical Officer of Acetaminophen Toxicity Diagnostics, LLC, which is developing methods for measurement of APAP-protein adducts; MRM consults for Acetaminophen Toxicity Diagnostics, LLC, and has received research funds from GlaxoSmithKline for unrelated projects; BNF is a shareholder and member of the scientific advisory board of Cirius Therapeutics, which is developing an MPC inhibitor for treating nonalcoholic steatohepatitis. The remaining authors declare that they have no conflicts of interest related to the content of this article.

  • Abbreviations

    ALF
    acute liver failure
    ALT
    alanine aminotransferase
    APAP
    acetaminophen
    CKO
    combined ALT2/MPC knockout
    JNK
    c-Jun N-terminal kinase
    KO
    knockout
    LDH
    lactate dehydrogenase
    MPC
    mitochondrial pyruvate carrier
    NAPQI
    N-acetyl-p-benzoquinone imine
    P450
    cytochrome P450
    PDH
    pyruvate dehydrogenase
    PDK4
    pyruvate dehydrogenase kinase 4
    TCA
    tricarboxylic acid
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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    Blocking mitochondrial alanine and pyruvate metabolism in hepatocytes worsens acetaminophen-induced liver injury in mice
    Joel H. Vazquez, Nicole K.H. Yiew, Michael R. Martino, Felicia D. Allard, Eric U. Yee, Sandra S. McCullough, Laura P. James, Brian N. Finck, Mitchell R. McGill
    bioRxiv 2022.06.14.495517; doi: https://doi.org/10.1101/2022.06.14.495517
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    Blocking mitochondrial alanine and pyruvate metabolism in hepatocytes worsens acetaminophen-induced liver injury in mice
    Joel H. Vazquez, Nicole K.H. Yiew, Michael R. Martino, Felicia D. Allard, Eric U. Yee, Sandra S. McCullough, Laura P. James, Brian N. Finck, Mitchell R. McGill
    bioRxiv 2022.06.14.495517; doi: https://doi.org/10.1101/2022.06.14.495517

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