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

George D Mcilroy,Seshu R Tammireddy,Benjamin H Maskrey,Louise Grant,Mary K Doherty,David G Watson,Mirela Delibegović,Phillip D Whitfield,Nimesh Mody

doi:10.1016/j.bcp.2015.11.017

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.

Highlights

Retinol and the retinoid metabolism pathway play an important role in body mass regulation and adipocyte biology [1,2,3,4,5,6]
FEN completely inhibited adipocyte differentiation in the 3T3L1 cell line [6] and in C3H10T1/2 pluripotent stem cells indicating that inhibition of adipogenesis is a common characteristic of FEN treatment (Fig. 1a)
RARa antagonism prevented inhibition of adipocyte genes (Fig. 3b) and inhibited Crbp1 and Raldh1 by !50% (Fig. 3c). These findings strongly demonstrate that the mechanism of FEN action to inhibit 3T3-L1 adipogenesis is mediated by ligandinduced activation of RARa signalling and genes involved in retinoid metabolism

Summary

Introduction

Retinol (vitamin A) and the retinoid metabolism pathway play an important role in body mass regulation and adipocyte biology [1,2,3,4,5,6]. Targeting retinoid homeostasis may offer a therapeutic approach for obesity and type-2 diabetes. All-trans-retinoic acid (RA), which is the most active metabolite of retinol, has long been known to inhibit adipogenesis through the prevention of C/EBPb mediated transcription [1,8], More recently, RA has been shown to improve obesity and glucose homeostasis in vivo [4].

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Results

Conclusion