The Role of Peroxisomal ACOT4 and ACOT8 in Modulating Lipid Metabolism and Stress Pathways in Steatotic Hepatocytes

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by excessive lipid accumulation in hepatocytes and the development of lipotoxic stress that disrupts cellular homeostasis. Peroxisomal lipid metabolism plays an important role in maintaining acyl-CoA balance during fatty acid overload, yet the contribution of peroxisomal acyl-CoA thioesterases to early lipotoxic responses remains insufficiently understood. The aim of this study was to investigate the functional involvement of the peroxisomal enzymes acyl-CoA thioesterase 4 (ACOT4) and acyl-CoA thioesterase 8 (ACOT8) in hepatocyte lipid handling under acute lipotoxic conditions. An in vitro model of steatosis was established in AML12 cells through exposure to a mixture of palmitate and oleate (1:2). Lipid accumulation, metabolic stress markers, and gene expression profiles were assessed following individual silencing of Acot4 or Acot8. Fatty acids treatment induced steatosis and increased the expression of Acot4 and Acot8, alongside genes associated with lipid storage and endoplasmic reticulum (ER) stress. Silencing of either Acot4 or Acot8 reduced intracellular lipid accumulation and attenuated stress-related gene expression, indicating that the upregulation of these enzymes during lipid overload may represent a maladaptive response that promotes lipotoxicity rather than preventing it. These findings suggest that peroxisomal acyl-CoA metabolism contributes to early lipotoxic stress and highlights ACOT4 and ACOT8 as potential druggable nodes to mitigate hepatocellular dysfunction during the onset of MASLD. Advisor: Tomasz K. Bednarski