Functional perturbation reveals context-dependent contributions of nuclear receptors to drug-induced hepatic steatosis.

Abstract

Drug-induced hepatic steatosis is mediated by diverse molecular mechanisms, yet several nuclear receptors have been proposed as molecular initiating events or early key events within adverse outcome pathways for hepatic steatosis. However, direct functional evidence supporting these mechanistic roles in human-relevant experimental systems remains limited. The present study evaluated the contribution of selected nuclear receptors to drug-induced hepatic steatosis using complementary human hepatic in vitro models. Stable short hairpin RNA-mediated knockdown of individual nuclear receptors was established in HepG2 and differentiated HepaRG cells, followed by exposure to representative steatogenic drugs, including valproic acid, amiodarone, tamoxifen, and rifampicin. In parallel, primary human hepatocyte spheroids were used to compare drug-induced lipid accumulation with direct pharmacological activation of individual nuclear receptor pathways. While depletion of multiple nuclear receptors markedly affected oleic acid-induced lipid accumulation, drug-induced steatogenic responses exhibited predominantly selective and compound-specific receptor dependencies. In differentiated HepaRG cells, nuclear receptor depletion influenced basal lipid homeostasis more strongly than valproic acid-induced lipid accumulation. Conversely, direct activation of liver X receptor and peroxisome proliferator-activated receptors α and γ in primary human hepatocyte spheroids induced robust lipid accumulation, whereas most steatogenic drugs produced comparatively modest responses. These findings demonstrate that the contribution of individual nuclear receptors to drug-induced hepatic steatosis is highly compound- and context-dependent and cannot be explained by a single conserved receptor pathway. This study provides functional evidence from complementary human-relevant hepatic models that supports refinement of hepatic steatosis adverse outcome pathways and highlights the value of targeted perturbation strategies for mechanistic toxicology.