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Updated: Jan 17, 2026
Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells
Published on: July 18, 2019
Transcriptomic and Proteomics Analysis of a Lipid-Loaded HepaRG Model for Steatosis Reveals Altered Regulation in
Anitha Saravanakumar1, Cassandra A Tierney2, Wen He2
1Clinical Pharmacokinetics Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI, USA.
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-metabolizing enzymes (DMEs) and transporters is crucial for assessing potential alterations in drug disposition 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 unsaturated (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 SWATHMS 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 upregulated, 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 metabolism pathways under NAFLD conditions. Downregulation of transcription factors and metabolic enzymes could impact drug efficacy and toxicity, necessitating further research into the pharmacokinetic 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.
