Regulation of Drug Metabolism by the Interplay of Inflammatory Signaling, Steatosis, and Xeno-Sensing Receptors in HepaRG Cells

Abstract

Nonalcoholic fatty liver disease (NAFLD), which is characterized by triglyceride deposition in hepatocytes resulting from imbalanced lipid homeostasis, is of increasing concern in Western countries, along with progression to nonalcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. Previous studies suggest a complex, mutual influence of hepatic fat accumulation, NASH-related inflammatory mediators, and drug-sensing receptors regulating xenobiotic metabolism. Here, we investigated the suitability of human HepaRG hepatocarcinoma cells as a model for NAFLD and NASH. Cells were incubated for up to 14 days with an oleate/palmitate mixture (125 M each) and/or with 10 ng/ml of the inflammatory mediator interleukin-6 (IL-6). Effects of these conditions on the regulation of drug metabolism were studied using xenobiotic agonists of the aryl hydrocarbon receptor (AHR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), nuclear factor (erythroid-derived 2)-like 2, and peroxisome proliferator-activated receptor (PPAR). Results underpin the suitability of HepaRG cells for NAFLD- and NASH-related research and constitute a broad-based analysis of the impact of hepatic fatty acid accumulation and inflammation on drug metabolism and its inducibility by xenobiotics. IL-6 exerted pronounced negative regulatory effects on basal as well as on PXR-, CAR-, and PPAR-, but not AHR-dependent induction of drug-metabolizing enzymes. This inhibition was related to diminished transactivation potential of the respective receptors rather than to reduced transcription of nuclear receptor-encoding mRNAs. The most striking effects of IL-6 and/or fatty acid treatment were observed in HepaRG cells after 14 days of treatment, making these cultures appear a suitable model for studying the relationship of fatty acid accumulation, inflammation, and xenobiotic-induced drug metabolism.

Distinct Effects of Inflammation on Cytochrome P450 Regulation and Drug Metabolism: Lessons from Experimental Models and a Potential Role for Pharmacogenetics

“…Treatment with IL-6 downregulated mRNA levels for all isoforms studied, but simultaneously revealed profound differences in the magnitude of downregulation, as the expression of CYP3A4 was markedly more reduced than that of CYP2C9 or CYP2C19. A similar observation was made by Dickmann et al [19] and Klein et al [15] in PHHs and by Tanner et al [23] in the HepaRG cell line, who all reported that IL-6 exerted the strongest downregulation on CYP3A4, whereas the effects of IL-6 on other CYPs, most notably CYP2D6, seemed to be more limited. It should be noted from the work of Klein et al that IL-6 may also induce expression of CYP2E1 in PHHs, which could be relevant for the metabolism of certain anesthetics [15].…”

“…In HepG2 cells, treatment with IL-6 and IL-1β resulted in a 10% decrease of HNF-4α activity as a result of an altered phosphorylation status [61]. Acute and prolonged treatment with IL-6 reduced mRNA levels of HNF-4a in HepaRG cells, but this effect was not seen for HNF-1a and the changes shrink into insignificance compared to the observed downregulation of, e.g., PXR [23]. In contrast, Klein et al found that mRNA levels of the HNF-4α were downregulated (≈40%) by IL-6 only at the early time point of 8 h and seemed to have normalized after 24 h [15].…”

“…This p65 subunit of the NF-κB complex interfered with the distal glucocorticoid response element present in the CAR promotor, leading to repressed transcription of CAR. In contrast, the AhR is not substantially affected by IL-6 treatment [15,23]. As such, it appears that the response to inflammation is substantial for PXR and CAR and their dimerization partner RXR, but not for AhR.…”

“…This highlights that the model is capable of capturing physiological adaptation to inflammation, since CYP3A4 desuppression occurred. Tanner et al collected data on the long-term effects of IL-6 treatment (14 days) in HepaRG cells, which resulted in more pronounced downregulation of P450 expression as compared to short-term treatment [23]. Still, current studies do not address the impact of long-term low concentrations of cytokines as compared to single high-dose treatment, which leaves an open question.…”

The Roles of Xenobiotic Receptors: Beyond Chemical Disposition

“…Metabolic benefits were observed in wild-type (WT) but not CAR 2/2 mice treated with 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a potent mouse CAR agonist (Dong et al, 2009;Gao et al, 2009). Additional studies have shown that CAR is involved in liver regeneration, inflammation, hepatocarcinogenesis, and renal ischemia-reperfusioninduced kidney injury (Huang et al, 2005;Tschuor et al, 2016;Tanner et al, 2018). Here, we will concentrate on the recent findings regarding the role of CAR in energy homeostasis and cell proliferation.…”