Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis
NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports oth...
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2021
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oai:doaj.org-article:34b2326905204693ab01efe6622b88fe2021-11-15T04:38:48ZEffects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis1663-981210.3389/fphar.2021.769703https://doaj.org/article/34b2326905204693ab01efe6622b88fe2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fphar.2021.769703/fullhttps://doaj.org/toc/1663-9812NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with POR gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model.Tamara HeintzeTamara HeintzeDenise WilhelmThierry SchmidlinThierry SchmidlinThierry SchmidlinUte HofmannUte HofmannUlrich M. ZangerUlrich M. ZangerMatthias SchwabMatthias SchwabMatthias SchwabKathrin KleinKathrin KleinFrontiers Media S.A.articleHepaRGCRISPR/Cas9NADPH cytochrome P450 reductasebile acid metabolismcholesterol biosynthesisproteomicsTherapeutics. PharmacologyRM1-950ENFrontiers in Pharmacology, Vol 12 (2021) |
| institution |
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DOAJ |
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EN |
| topic |
HepaRG CRISPR/Cas9 NADPH cytochrome P450 reductase bile acid metabolism cholesterol biosynthesis proteomics Therapeutics. Pharmacology RM1-950 |
| spellingShingle |
HepaRG CRISPR/Cas9 NADPH cytochrome P450 reductase bile acid metabolism cholesterol biosynthesis proteomics Therapeutics. Pharmacology RM1-950 Tamara Heintze Tamara Heintze Denise Wilhelm Thierry Schmidlin Thierry Schmidlin Thierry Schmidlin Ute Hofmann Ute Hofmann Ulrich M. Zanger Ulrich M. Zanger Matthias Schwab Matthias Schwab Matthias Schwab Kathrin Klein Kathrin Klein Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis |
| description |
NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with POR gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model. |
| format |
article |
| author |
Tamara Heintze Tamara Heintze Denise Wilhelm Thierry Schmidlin Thierry Schmidlin Thierry Schmidlin Ute Hofmann Ute Hofmann Ulrich M. Zanger Ulrich M. Zanger Matthias Schwab Matthias Schwab Matthias Schwab Kathrin Klein Kathrin Klein |
| author_facet |
Tamara Heintze Tamara Heintze Denise Wilhelm Thierry Schmidlin Thierry Schmidlin Thierry Schmidlin Ute Hofmann Ute Hofmann Ulrich M. Zanger Ulrich M. Zanger Matthias Schwab Matthias Schwab Matthias Schwab Kathrin Klein Kathrin Klein |
| author_sort |
Tamara Heintze |
| title |
Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis |
| title_short |
Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis |
| title_full |
Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis |
| title_fullStr |
Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis |
| title_full_unstemmed |
Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis |
| title_sort |
effects of diminished nadph:cytochrome p450 reductase in human hepatocytes on lipid and bile acid homeostasis |
| publisher |
Frontiers Media S.A. |
| publishDate |
2021 |
| url |
https://doaj.org/article/34b2326905204693ab01efe6622b88fe |
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