Autophagy suppression is associated with enhanced preneoplastic hepatocellular lesions in rats fed a lard-containing high-fat diet.

Metabolic dysfunction-associated steatotic liver disease is a major risk factor for hepatocellular carcinoma, yet the role of autophagy in early hepatocarcinogenesis remains unclear. We evaluated autophagy dynamics in a rat model of lard-containing high-fat diet-induced fatty liver with or without partial hepatectomy. Combined high-fat diet and partial hepatectomy significantly increased plasma alanine aminotransferase levels, hepatic steatosis, and preneoplastic lesion development. To analyze these preneoplastic lesions, k-means cluster analysis was performed using the expression levels of LC3, p62, and LAMP2. Initial clustering based on lesion area and LC3 rates identified five clusters, from which three clusters with large areas or high LC3 expression were selected. These were further categorized into two clusters based on their LC3 and p62 levels. The autophagy suppression cluster (high p62 and low LC3 expression) exhibited significantly larger preneoplastic lesions than the autophagy induction cluster (low p62 and high LC3 expression). These findings were further supported by LC3 and LAMP2 clustering and correlated gene expression in the autophagy-lysosome-endosome, lipid metabolism, oxidative stress, and ER stress. Although functional autophagic flux was not directly measured within preneoplastic lesions, cluster analysis of static immunohistochemical markers suggested that autophagy suppression is closely associated with early-stage, steatosis-related hepatocarcinogenesis.