Transcriptomic and Proteomics Analysis of a Lipid-Loaded HepaRG Model for Steatosis Reveals Altered Regulation in Lipid and Xenobiotic Metabolism

Introduction: Hepatic lipid accumulation (steatosis) is an early indicator of non-alcoholic fatty liver disease (NAFLD), preceding fibrosis and cirrhosis. Understanding its effects on drug-me-tabolizing enzymes (DMEs) and transporters is crucial for assessing potential alterations in drug dis-position among NAFLD patients. This study aimed to replicate steatosis in an in vitro HepaRG cell model and analyze its impact on DMEs and transporters. Methods: Differentiated HepaRG cells were treated with a mixture of saturated (palmitate) and unsatu-rated (oleate) fatty acids (in a 1:2 ratio at 0.5 mM), complexed with BSA for 72 hours to induce lipid accumulation. Confirmation of steatosis was performed using Oil Red O staining and triglyceride (TG) quantification, while cell viability was assessed via the WST-1 assay. RNA sequencing and SWATH-MS proteomic analysis were employed to identify differentially expressed transcripts and proteins in lipid-loaded cells compared to controls. Results: Lipid loading resulted in a ~6-fold increase in TG concentration without compromising cell viability. Transcriptomic analysis identified 393 differentially expressed transcripts (89 upregulated, 304 downregulated), while proteomic analysis detected 165 differentially expressed proteins (127 up-regulated, 38 downregulated). Notably, key mRNA transcripts related to transcription factors (NR1I2, HNF4α), phase 1 DMEs (CYP1A2, 2B6, 2C8, 2C9, 2C19, 3A4), phase 2 DMEs (UGT1A6, 2B7, SULT2A1, 1E1), and transporters (ABCC11, ABCG5, SLCO2B1, SLC10A1) exhibited significant downregulation. Discussion: The observed alterations in DMEs and transporters suggest a potential shift in drug me-tabolism pathways under NAFLD conditions. Downregulation of transcription factors and metabolic enzymes could impact drug efficacy and toxicity, necessitating further research into the pharmacoki-netic implications. Conclusion: The in vitro hepatic steatosis model demonstrated significant changes in the expression of clinically relevant DMEs and transporters. These findings highlight the importance of considering NAFLD-induced metabolic alterations when assessing drug disposition in affected patients