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Objective: Mitochondrial pyruvate is a critical intermediary metabolite in gluconeogenesis, lipogenesis, and NADH production. As a result, the mitochondrial pyruvate carrier (MPC) complex has emerged as a promising therapeutic target in metabolic diseases. Clinical trials are currently underway. However, recent in vitro data indicate that MPC inhibition diverts glutamine/glutamate away from glutathione synthesis and toward glutaminolysis to compensate for loss of pyruvate oxidation, possibly sensitizing cells to oxidative insult. Here, we explored this in vivo using the clinically relevant acetaminophen (APAP) overdose model of acute liver injury, which is driven by oxidative stress., Methods: We used pharmacological and genetic approaches to inhibit MPC2 and alanine aminotransferase 2 (ALT2), individually and concomitantly, in mice and cell culture models and determined the effects on APAP hepatotoxicity., Results: We found that MPC inhibition sensitizes the liver to APAP-induced injury in vivo only with concomitant loss of alanine aminotransferase 2 (ALT2). Pharmacological and genetic manipulation of neither MPC2 nor ALT2 alone affected APAP toxicity, but liver-specific double knockout (DKO) significantly worsened APAP-induced liver damage. Further investigation indicated that DKO impaired glutathione synthesis and increased urea cycle flux, consistent with increased glutaminolysis, and these results were reproducible in vitro. Finally, induction of ALT2 and post-treatment with dichloroacetate both reduced APAP-induced liver injury, suggesting new therapeutic avenues., Conclusions: Increased susceptibility to APAP toxicity requires loss of both the MPC and ALT2 in vivo, indicating that MPC inhibition alone is insufficient to disrupt redox balance. Furthermore, the results from ALT2 induction and dichloroacetate in the APAP model suggest new metabolic approaches to the treatment of liver damage., Competing Interests: Declaration of Competing Interest LPJ is Chief Medical Officer of Acetaminophen Toxicity Diagnostics (ATD), LLC, which is developing a diagnostic test for APAP overdose. MR McGill is a consultant for ATD and has received research funding from GlaxoSmithKline which sells APAP. BNF is a shareholder and member of the scientific advisory board of Cirius Therapeutics, which is developing an MPC inhibitor to treat nonalcoholic steatohepatitis. The remaining authors declare that they have no conflicts related to the content of this article. These companies had no role whatsoever in the funding, conceptualization, design, execution, or reporting of the study., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The standard circulating biomarker of liver injury in both clinical settings and drug safety testing is alanine aminotransferase (ALT). However, ALT elevations sometimes lack specificity for tissue damage. To identify novel serum biomarkers with greater specificity for injury, we combined unique animal models with untargeted proteomics, followed by confirmation with immunoblotting. Using proteomics, we identified 109 proteins in serum from mice with acetaminophen (APAP)-induced liver injury that were not detectable in serum from mice with benign ALT elevations due to high-dose dexamethasone (Dex). We selected 4 (alcohol dehydrogenase 1A1 [Aldh1a1], aldehyde dehydrogenase 1 [Adh1], argininosuccinate synthetase 1 [Ass1], and adenosylhomocysteinase [Ahcy]) with high levels for further evaluation. Importantly, all 4 were specific for injury when using immunoblots to compare serum from Dex-treated mice and mice with similar lower ALT elevations due to milder models of APAP or bromobenzene-induced liver injury. Immunoblotting for ALDH1A1, ADH1, and ASS1 in serum from APAP overdose patients without liver injury and APAP overdose patients with mild liver injury revealed that these candidate biomarkers can be detected in humans with moderate liver injury as well. Interestingly, further experiments with serum from rats with bile duct ligation-induced liver disease indicated that Aldh1a1 and Adh1 are not detectable in serum in cholestasis and may therefore be specific for hepatocellular injury and possibly even drug-induced liver injury, in particular. Overall, our results strongly indicate that ALDH1A1, ADH1, and ASS1 are promising specific biomarkers for liver injury. Adoption of these biomarkers could improve preapproval drug safety assessment., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)