A Combination of Transcriptomics and Metabolomics Uncovers Enhanced Bile Acid Biosynthesis in HepG2 Cells Expressing CCAAT/Enhancer-Binding Protein β (C/EBPβ), Hepatocyte Nuclear Factor 4α (HNF4α), and Constitutive Androstane Receptor (CAR)

Abstract

The development of hepatoma-based in vitro models to study hepatocyte physiology is an invaluable tool for both industry and academia. Here, we develop an in vitro model based on the HepG2 cell line that produces chenodeoxycholic acid, the main bile acid in humans, in amounts comparable to human hepatocytes. A combination of adenoviral transfections for CCAAT/enhancer-binding protein β (C/EBPβ), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor (CAR) decreased intracellular glutamate, succinate, leucine, and valine levels in HepG2 cells, suggestive of a switch to catabolism to increase lipogenic acetyl CoA and increased anaplerosis to replenish the tricarboxylic acid cycle. Transcripts of key genes involved in bile acid synthesis were significantly induced by approximately 160-fold. Consistently, chenodeoxycholic acid production rate was increased by more than 20-fold. Comparison between mRNA and bile acid levels suggest that 12-alpha hydroxylation of 7-alpha-hydroxy-4-cholesten-3-one is the limiting step in cholic acid synthesis in HepG2 cells. These data reveal that introduction of three hepatocyte-related transcription factors enhance anabolic reactions in HepG2 cells and provide a suitable model to study bile acid biosynthesis under pathophysiological conditions.

Genome-wide expression profiling reveals increased stability and mitochondrial energy metabolism of the human liver cell line HepaRG-CAR

“…CAR is implicated not only in biotransformation through regulating the transcript level of a large array of genes involved in biotransformation, however, it also is involved, among others, in bile acid synthesis and in energy homeostasis by governing lipogenesis and gluconeogenesis (Bechmann et al 2012;Chiang 2009;Cui and Klaassen 2016). The upregulation of bile acid synthesis is in line with our previous findings that several CYP enzymes are induced at transcript and/or activity level in HepaRG-CAR cells (van der Mark et al 2017), as CYP enzymes are involved in the synthesis of bile acids from cholesterol (Cui and Klaassen 2016;Petrick and Klaassen 2007;Blazquez et al 2013).…”

Design and fabrication of a scalable liver-lobule-on-a-chip microphysiological platform

“…In HepG2 cultures no significant network of bile canaliculi was detected. Previously, HepG2 has been reported to secrete bile acid in much lower levels than human hepatocytes, but improved with supplementation of media with human serum albumin (Pramfalk et al 2016) or adenoviral transfection with CCAAT/ enhancer-binding protein β (C/EBPβ), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor (CAR) (Blazquez et al 2013).…”