Steatotic hepatocytes release mature VLDL via methionine and tyrosine metabolism in a Keap1-Nrf2 dependent manner.

Nonalcoholic fatty liver disease (NAFLD) is a lipotoxic disease wherein hepatic steatosis and oxidative stress are key pathogenic features. However, whether free amino acids (FAAs) are associated with the oxidative stress response against lipotoxicity has yet to be determined. We hypothesized that an imbalance of FAAs aggravates hepatic steatosis by interfering with the oxidative stress sensor. C57BL/6 mouse immortalized hepatocytes, primary hepatocytes and organoids were employed. Steatotic hepatocytes treated with oleic acid (OA) were cultured under FAAs-modifying media based on the concentrations of FAAs in the hepatic portal blood (HPB) of wild type mice (WT). As in vivo experiments, WT, Hepatocyte-specific Keap1 knock out (KO) mice (Keap1∆hepa ) and Cre- control mice (Keap1fx/fx ) were fed high fat (HF) diets with modified amino acid content. The correlations were analyzed between the areas of lipid droplets (LDs) around central vein and plasma OA / FAAs ratio in 61 patients with NAFLD. Mice fed a HF, Met restricted and Tyr deficient diet showed the NAFLD-like phenotype in which the nuclear translocation of Nrf2, triglyceride-rich VLDL and fumarate were decreased in liver but Keap1∆hepa ameliorated these phenomena. Reactive oxygen species and LDs induced by the deprivation of Met and Tyr were prevented in hepatic organoids generated from Keap1∆hepa . Dimethyl fumarate, a Nrf2 inducer ameliorated the steatosis and increased the hepatic fumarate reduced by the deprivation of Met and Tyr in vitro. OA / Met or Tyr ratio in peripheral blood were associated with the hepatic steatosis in NAFLD patients. CONCLUSION: An imbalance between free fatty acids and Met and Tyr induces hepatic steatosis by disturbing the VLDL assembling via the Keap1-Nrf2 system.