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Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis

Tamara Heintze1,2, Denise Wilhelm1, Thierry Schmidlin1,2,3

  • 1Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.

Frontiers in Pharmacology
|December 6, 2021
PubMed

Insights

NADPH:cytochrome P450 oxidoreductase (POR) is crucial for drug and steroid metabolism. POR knockdown in human liver cells impacts ADME gene expression and deregulates bile acid and cholesterol biosynthesis.

Area of Science:

  • Biochemistry
  • Pharmacology
  • Genetics

Background:

  • NADPH:cytochrome P450 oxidoreductase (POR) is essential for cytochrome P450 (CYP) enzyme function in endogenous substance biosynthesis and xenobiotic metabolism.
  • POR also supports other redox enzymes in fatty acid and cholesterol pathways.
  • Previous work established CRISPR/Cas9-mediated POR knockdown in HepaRG cells, showing differential effects on CYP activity.

Purpose of the Study:

  • To systematically investigate the impact of POR knockdown on ADME gene expression and related regulators in a human hepatic cell model.
  • To assess functional consequences on bile acid and cholesterol metabolism using quantitative mass spectrometry.
  • To analyze global protein expression changes and deduce the resulting protein network.

Main Methods:

  • CRISPR/Cas9-mediated POR knockdown in HepaRG cells.
  • Quantitative mass spectrometry for targeted metabolomics (bile acids, cholesterol) and untargeted proteomics.
  • Analysis of RNA expression for ADME genes and transcriptional regulators.

Main Results:

  • Significant downregulation of drug metabolism and transport regulators (CAR, PXR, FXR, LXRα) following POR knockdown.
  • Deregulated bile acid and cholesterol biosynthesis with altered levels of specific derivatives.
  • Downregulation of lipid metabolism and biological oxidation pathways indicated by global protein expression changes.

Conclusions:

  • POR knockdown significantly impacts the expression of key ADME regulators and endogenous metabolic pathways in human liver cells.
  • Altered bile acid and cholesterol homeostasis are significant consequences of diminished POR expression.
  • These findings highlight a widespread role for POR in metabolism and provide human data on the effects of reduced POR expression in a genome-edited HepaRG model.
Keywords:
CRISPR/Cas9HepaRGNADPH cytochrome P450 reductasebile acid metabolismcholesterol biosynthesisproteomics

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