Abstract
Fatty acid microsomal ω-oxidation involves cytochrome P450 enzymes. Some of them belonging to the CYP4F3 family are mainly expressed in the liver, making this organ a major player in energy homeostasis and lipid metabolism. To study this important regulation pathway, we used HepaRG cells, which gradually undergo a complete differentiation process. Even at the early stage of the differentiation process, CYP4F3B generated by alternative splicing of the CYP4F3 gene represented the prevalent isoform in HepaRG cells as in the liver. Its increasing expression associated with hepatocyte differentiation status suggested a hepatic-specific control of this isoform. As in liver microsomes, the catalytic hydroxylation of the CYP4F3B substrate [1-14C]Z9(10)-epoxystearic acid led to major production of 18-hydroxy-9(10)-epoxystearic acid. When treated with saturated, monounsaturated, or polyunsaturated fatty acids, CYP4F3B and CYP4A11 expression remained unchanged whereas CYP4F2 and CYP4F12 expression was transiently up-regulated. A 24-h exposure of differentiated HepaRG cells to various polyunsaturated fatty acids and derivatives induced microvesicular steatosis; down-regulation of lipid metabolism gene regulators such as sterol regulatory element-binding protein-1c, fatty acid synthase, peroxisome proliferator-activated receptor γ (PPARγ), PPARα, and decreased expression of glucose-dependent metabolism genes, which could limit de novo lipogenesis. Docosahexaenoic acid seemed to be the most effective compound. These results suggest that a PPARα-independent pathway could participate to limit lipogenesis and emphasize the role of hepatocytes in the fatty acid ω-hydroxylation pathway. They also give insights on the use of HepaRG hepatocytes to open new avenues of investigations on factors mediating the lipid metabolic pathway and finding new hypolipidemic molecules.
Footnotes
This work was supported by l'Agence Nationale de la Recherche [Grant PCV07_184566, μHepaReTox project]; le Conseil Régional de Bretagne [Grants PRIR-A3CBL9, 560408]; and the European Community 6th Framework Program [Grant COMICS, project 037575]. This research was also supported in part by Institut National de la Santé et Recherche Médicale and Centre National de Recherche Scientifique et Technique. S.M. was a recipient of Agence National de la Recherche Technique (ANRT) fellowship, and V.C. was a recipient of ANRT and BIOPREDIC International Company fellowship.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.036848.
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ABBREVIATIONS:
- FA
- fatty acid
- Z(10)-EpSTA
- Z9(10)-epoxystearic acid
- Z9(10)-EpSTA
- [1-14C]Z9(10)-epoxystearic acid
- AA
- arachidonic acid
- C/EBPα
- CCAAT/enhancer binding protein α
- DHA
- docosahexaenoic acid
- DMSO
- dimethyl sulfoxide
- EPA
- eicosapentaenoic acid
- FAS
- fatty acid synthase
- GAPDH
- glyceraldehydes-3-phosphate dehydrogenase
- GC-MS
- gas chromatography-mass spectrometry
- MUFA
- monounsaturated acid
- PBS
- phosphate-buffered saline
- PEPCK-1
- phosphoenolpyruvate carboxykinase 1
- PPAR
- peroxisome proliferator-activated receptor
- PUFA
- polyunsaturated fatty acid
- RP-HPLC
- reverse-phase high-performance liquid chromatography
- SREBP-1c
- sterol regulatory element-binding protein-1
- P450
- cytochrome P450
- HNF4α
- hepatocyte nuclear factor 4α
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- qPCR
- quantitative PCR
- Ct
- cycle threshold.
- Received October 28, 2010.
- Accepted July 21, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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