Aflatoxin B1 Exacerbates Ketogenic Diet‐Induced Hepatic Oxidative Stress and Glucose and Lipid Metabolic Disturbances in Mice

ABSTRACT Aflatoxin B1 (AFB1) is a widespread mycotoxin with severe hepatotoxicity. While the ketogenic diet (KD) is increasingly adopted, long‐term KD feeding can induce hepatic metabolic disturbances. However, the effects of AFB1 exposure under KD conditions remain unclear. We hypothesized that long‐term KD‐induced metabolic stress increases hepatic susceptibility to AFB1 toxicity, thereby aggravating liver injury. In this study, we investigated the effects of AFB1 on liver injury in mice fed a KD for 10 weeks. The results suggested that compared with the AFB1 and KD groups, co‐exposure with KD+AFB1 significantly increased serum ALT levels by 108.21% and 29.85%, (all p < 0.01), and AST levels by 35.57% ( p < 0.01) and 25.31% ( p < 0.05), respectively. Co‐exposure also aggravated hepatic lipid accumulation, with increased TG, T‐CHO, LDLC, and NEFA levels (all p < 0.01) and decreased HDLC levels ( p < 0.01). Oil Red O staining and TEM further confirmed pronounced hepatic lipid accumulation and ultrastructural damage. Co‐exposure also increased oxidative stress, as indicated by elevated ROS (by 34.23% and 24.77%, respectively) and MDA levels (by 14.96% and 2.14%, respectively), and enhanced hepatic inflammation, with increased TNF‐α, IL‐6, and COX‐2 and decreased IL‐10 levels. Untargeted metabolomics revealed marked hepatic metabolic disturbances, particularly in glycerophospholipid, fatty acid, glutathione, and TCA cycle metabolism. Protein expression analysis further supported these metabolic alterations, showing suppressed p‐AMPK, increased mTOR phosphorylation, and upregulated PPAR‐γ expression following KD+AFB1 co‐exposure. In conclusion, AFB1 aggravates long‐term KD‐associated hepatic oxidative stress, inflammation, and glucose and lipid metabolic disturbances. These findings highlight dietary safety during long‐term KD‐induced metabolic disturbances.