title: Fenretinide mediated retinoic acid receptor signalling and inhibition of ceramide biosynthesis regulates adipogenesis, lipid accumulation, mitochondrial function and nutrient stress signalling in adipocytes and adipose tissue.
abstract: Fenretinide (FEN) is a synthetic retinoid that inhibits obesity and insulin resistance in high-fat diet (HFD)-fed mice and completely prevents 3T3-L1 pre-adipocyte differentiation. The aim of this study was to determine the mechanism(s) of FEN action in 3T3-L1 adipocytes and in mice. We used the 3T3-L1 model of adipogenesis, fully differentiated 3T3-L1 adipocytes and adipose tissue from HFD-induced obese mice to investigate the mechanisms of FEN action. We measured expression of adipogenic and retinoid genes by qPCR and activation of nutrient-signalling pathways by western blotting. Global lipid and metabolite analysis was performed and specific ceramide lipid species measured by liquid chromatography-mass spectrometry. We provide direct evidence that FEN inhibits 3T3-L1 adipogenesis via RA-receptor (RAR)-dependent signaling. However, RARα antagonism did not prevent FEN-induced decreases in lipid levels in mature 3T3-L1 adipocytes, suggesting an RAR-independent mechanism. Lipidomics analysis revealed that FEN increased dihydroceramide lipid species 5- to 16-fold in adipocytes, indicating an inhibition of the final step of ceramide biosynthesis. A similar blockade in adipose tissue from FEN-treated obese mice was associated with a complete normalisation of impaired mitochondrial β-oxidation and tricarboxylic acid cycle flux. The FEN catabolite, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-OXO), also decreased lipid accumulation without affecting adipogenesis. FEN and 4-OXO (but not RA) treatment additionally led to the activation of p38-MAPK, peIF2α and autophagy markers in adipocytes. Overall our data reveals FEN utilises both RAR-dependent and -independent pathways to regulate adipocyte biology, both of which may be required for FEN to prevent obesity and insulin resistance in vivo.
authors.affiliation: Institute of Medical Sciences, College of Life Sciences & Medicine, University of Aberdeen, Aberdeen, UK. Electronic address: g.mcilroy@abdn.ac.uk.
authors.forename: George D
authors.initials: GD
authors.lastname: Mcilroy
authors.affiliation: Lipidomics Research Facility, Department of Diabetes and Cardiovascular Science, University of the Highlands and Islands, Inverness, UK. Electronic address: seshu.tammireddy@uhi.ac.uk.
authors.forename: Seshu R
authors.initials: SR
authors.lastname: Tammireddy
authors.affiliation: Lipidomics Research Facility, Department of Diabetes and Cardiovascular Science, University of the Highlands and Islands, Inverness, UK. Electronic address: ben.maskrey@uhi.ac.uk.
authors.forename: Benjamin H
authors.initials: BH
authors.lastname: Maskrey
authors.affiliation: Institute of Medical Sciences, College of Life Sciences & Medicine, University of Aberdeen, Aberdeen, UK. Electronic address: louisegrant@abdn.ac.uk.
authors.forename: Louise
authors.initials: L
authors.lastname: Grant
authors.affiliation: Lipidomics Research Facility, Department of Diabetes and Cardiovascular Science, University of the Highlands and Islands, Inverness, UK. Electronic address: mary.doherty@uhi.ac.uk.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Doherty
authors.affiliation: Metabolomics Group, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK. Electronic address: D.G.Watson@strath.ac.uk.
authors.forename: David G
authors.initials: DG
authors.lastname: Watson
authors.affiliation: Institute of Medical Sciences, College of Life Sciences & Medicine, University of Aberdeen, Aberdeen, UK. Electronic address: m.delibegovic@abdn.ac.uk.
authors.forename: Mirela
authors.initials: M
authors.lastname: Delibegović
authors.affiliation: Lipidomics Research Facility, Department of Diabetes and Cardiovascular Science, University of the Highlands and Islands, Inverness, UK. Electronic address: phil.whitfield@uhi.ac.uk.
authors.forename: Phillip D
authors.initials: PD
authors.lastname: Whitfield
authors.affiliation: Institute of Medical Sciences, College of Life Sciences & Medicine, University of Aberdeen, Aberdeen, UK. Electronic address: n.mody@abdn.ac.uk.
authors.forename: Nimesh
authors.initials: N
authors.lastname: Mody
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D050156:Adipogenesis / Q000187:drug effects / Q000502:physiology*; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002518:Ceramides / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D059305:Diet, High-Fat / Q000009:adverse effects; D017313:Fenretinide / Q000494:pharmacology*; D050155:Lipogenesis / Q000187:drug effects / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008820:Mice, Obese; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D002518:Ceramides; D018168:Receptors, Retinoic Acid; D017313:Fenretinide
keywords: 4-oxo-N-(4-Hydroxyphenyl)retinamide (PubChem CID: 11258242); Adipocytes; Dihydroceramide; Fenretinide; Fenretinide (PubChem CID: 5288209); Lipidomics; Metabolomics; Retinoic acid; Retinoic acid (PubChem CID: 444795); Rosiglitazone (PubChem CID: 77999)