Hepatic pyruvate and alanine metabolism are critical and complementary for maintenance of antioxidant capacity and resistance to oxidative insult in mice

Pyruvate is a critical intermediary metabolite in gluconeogenesis, lipogenesis, as well as NADH production. As a result, there is growing interest in targeting the mitochondrial pyruvate carrier (MPC) complex in liver and metabolic diseases. 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 using the clinically relevant acetaminophen (APAP) overdose model of acute liver injury, which is driven by oxidative stress. We report that MPC inhibition does indeed sensitize the liver to APAP-induced injury in vivo, but only with concomitant loss of alanine aminotransferase 2 (ALT2). Pharmacologic and genetic manipulation of neither MPC2 nor ALT2 alone affected APAP toxicity, but liver-specific double knockout (DKO) of these proteins significantly worsened the liver damage. Further investigation confirmed that DKO impaired glutathione synthesis and increased urea cycle flux, consistent with increased glutaminolysis. Furthermore, APAP toxicity was exacerbated by inhibition of both the MPC and ALT in vitro. Thus, increased glutaminolysis and susceptibility to oxidative stress requires loss of both the MPC and ALT2 in vivo and exacerbates them in vitro. Finally, induction of ALT2 reduced APAP-induced injury. ### 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. * ALF : acute liver failure ALT : alanine aminotransferase APAP : acetaminophen CKO : combined ALT2/MPC knockout GSH : reduced glutathione GSSG : oxidized glutathione 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