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

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.