Original article Bioenergetic cues shift FXR splicing towards FXRa2 to modulate hepatic lipolysis and fatty acid metabolism Jorge C. Correia 1,2, Julie Massart 3, Jan Freark de Boer 4, Margareta Porsmyr-Palmertz 1, Vicente Martínez-Redondo 1, Leandro Z. Agudelo 1, Indranil Sinha 5, David Meierhofer 6, Vera Ribeiro 2, Marie Björnholm 3, Sascha Sauer 6, Karin Dahlman-Wright 5, Juleen R. Zierath 3, Albert K. Groen 4, Jorge L. Ruas 1,* ABSTRACT Objective: Farnesoid X receptor (FXR) plays a prominent role in hepatic lipid metabolism. The FXR gene encodes four proteins with structural differences suggestive of discrete biological functions about which little is known. Methods: We expressed each FXR variant in primary hepatocytes and evaluated global gene expression, lipid profile, and metabolic fluxes. Gene À À delivery of FXR variants to Fxr / mouse liver was performed to evaluate their role in vivo. The effects of fasting and physical exercise on hepatic Fxr splicing were determined. Results: We show that FXR splice isoforms regulate largely different gene sets and have specific effects on hepatic metabolism. FXRa2 (but not a1) activates a broad transcriptional program in hepatocytes conducive to lipolysis, fatty acid oxidation, and ketogenesis. Consequently, FXRa2 À À decreases cellular lipid accumulation and improves cellular insulin signaling to AKT. FXRa2 expression in Fxr / mouse liver activates a similar gene program and robustly decreases hepatic triglyceride levels. On the other hand, FXRa1 reduces hepatic triglyceride content to a lesser extent and does so through regulation of lipogenic gene expression. Bioenergetic cues, such as fasting and exercise, dynamically regulate Fxr splicing in mouse liver to increase Fxra2 expression. Conclusions: Our results show that the main FXR variants in human liver (a1 and a2) reduce hepatic lipid accumulation through distinct mechanisms and to different degrees. Taking this novel mechanism into account could greatly improve the pharmacological targeting and therapeutic efficacy of FXR agonists. Ó 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords FXR isoforms; Splicing; NAFLD; Insulin resistance; Energy metabolism 1. INTRODUCTION and circulating triacylglycerol (TAG) levels and improve glucose ho- meostasis [9,10]. The regulation of hepatic energy storage and metabolism is essential Four biologically active variants are expressed from the FXR gene for an appropriate adaptation to fluctuating metabolic demands. (FXRa1 through 4), resulting from alternative promoter usage and Impaired control over hepatic lipid homeostasis can lead to excessive mRNA splicing (Figure 1A) [11,12]. FXR isoforms display distinct lipid accumulation, a prominent feature of non-alcoholic fatty liver developmental and tissue-specific expression patterns and are disease (NAFLD), the most prevalent liver disease in Western societies differentially regulated by metabolic cues [11,13,14]. FXRa3 and a4 [1]. NAFLD is considered the hepatic hallmark of the metabolic syn- possess extended N-termini with an A/B domain different from the one drome and is strongly associated with hepatic insulin resistance and in FXRa1 and a2. In addition, FXRa1 and a3 have a four amino acid type 2 diabetes. Farnesoid X receptor (FXR) is a bile acid-activated insert (MYTG) in the hinge region. The structural disparity between FXR nuclear receptor abundantly expressed in liver [2,3]. FXR expression variants has functional implications that may influence the therapeutic is markedly decreased in obese rodents and NAFLD patients [4e6]. outcome of pan-FXR agonists. Indeed, FXR agonists promote distinct Genetic FXR ablation promotes hepatic steatosis and hyperlipidemia gene expression profiles in hepatocytes, although the contribution of [7,8], whereas hepatic FXR expression and activation decrease local FXR isoform-specificity remains unknown [15]. Structural differences 1Department of Physiology and Pharmacology, Molecular & Cellular Exercise Physiology Unit, Karolinska Institutet, Stockholm, Sweden 2Center for Biomedical Research, University of Algarve, Faro, Portugal 3Department of Molecular Medicine and Surgery, Section for Integrative Physiology, Karolinska Institutet, Stockholm, Sweden 4Department of Pediatrics and Laboratory Medicine, University of Groningen, University Medical Center Groningen, The Netherlands 5Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Stockholm, Sweden 6Max Planck Institute for Molecular Genetics, Berlin, Germany *Corresponding author. Karolinska Institutet, Department of Physiology and Pharmacology, von Eulers väg 8, SE-171 77, Stockholm, Sweden. Tel.: þ46 8 524 87261. E-mail: [email protected] (J.L. Ruas). Received September 4, 2015 Revision received September 13, 2015 Accepted September 16, 2015 Available online 26 September 2015 http://dx.doi.org/10.1016/j.molmet.2015.09.005 MOLECULAR METABOLISM 4 (2015) 891e902 Ó 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 891 www.molecularmetabolism.com Original article Figure 1: FXR isoform-specific gene regulation. (A) Human FXR isoforms. The different N-termini (orange in FXRa1/a2; pink in FXRa3/a4) and the MYTG insert in FXRa1/a3 are indicated. DBD, DNA-binding domain; LBD, ligand-binding domain. (BeC) FXR mRNA (B) and protein (C) levels in hepatocytes expressing each FXR variant or GFP alone (control). (D) Gene expression analysis by qRT-PCR. (E) Heatmap of gene expression profiles in hepatocytes expressing each FXR isoform or GFP (F) Bioinformatic pathway analysis of gene expression profiling. Represented are biological functions significantly associated with one or more FXR isoforms. (G) Predicted effects on hepatic metabolism for each FXR isoform. Z-score values indicate induction (orange) or repression (blue). Bars depict mean values and error bars represent SEM (n 3). *p < 0.05 vs. control. Up < 0.05 vs. FXRa1/a2. xp < 0.05 vs. FXRa1/a3. 892 MOLECULAR METABOLISM 4 (2015) 891e902 Ó 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com in the A/B domain of FXR isoforms modulate coactivator interaction against FXR (Santa Cruz Biotechnologies), AKT (Cell Signaling), and [16], whereas the MYTG insert affects DNA-binding and target gene phospho-AKT (Ser473) (Cell Signaling), diluted in 0.1% BSA. Quanti- specificity [11,14,17,18]. For example, the human bile salt export fication was performed using ImageJ [21]. pump (BSEP) is regulated by FXRs in an isoform-dependent manner, predominantly by FXRa2 [14,18]. Notably, hepatic FXRa2/a1 ratios 2.5. Neutral lipid staining and BSEP levels are decreased in hepatocellular carcinoma patients Hepatocytes were fixed with 4% paraformaldehyde for 10 min at room and by pro-inflammatory stimulation [14]. To date, the regulation of temperature. After 3 washes with PBS, cells were incubated for 2 min FXR splicing and its implications on hepatic energy metabolism remain in 60% isopropanol and stained with freshly prepared Oil Red-O so- unknown. lution (0.4% in 60% isopropanol) for 30 min at room temperature. Cells Here, we show that FXR variants regulate hepatic lipid metabolism in an were washed with PBS, incubated for 2 min with 300 nM 40,6- isoform-dependent manner. This constitutesa novel mechanism by which diamidino-2-phenylindole, dihydrochloride (DAPI) (Life Technologies), alternative FXR splicing in the liver integrates systemic energetic demands and washed with PBS again. Images were taken at a 200Â magni- with a gene program of enhanced lipid handling/utilization and ketogen- fication. Total red and blue pixels were quantified using the Color esis that positively impacts hepatic steatosis and insulin sensitivity. Range Select tool in Adobe Photoshop CS5.1. 2. MATERIALS AND METHODS 2.6. Lipid profiling Hepatocytes were incubated with 0.2 mCi/mL [U-14C] palmitate (Perkin 2.1. Generation of recombinant adenoviruses Elmer, USA) and 25 nM non-radioactive palmitate for 4 h. Lipids were Human FXRa1, a2, a3, and a4 were amplified by PCR and cloned into extracted, separated by thin layer chromatography, detected by the NotI/XhoI sites of a pcDNA3.1 plasmid (Life Technologies) con- autoradiography, and quantified as previously described [22]. taining a FLAG tag between the BamHI and NotI sites. The cDNAs encoding each FLAG-tagged FXR were subcloned into the pAdTrack- 2.7. FA oxidation assay CMV vector (Stratagene). Adenoviruses expressing each FXR variant Hepatocytes were incubated in DMEM supplemented with 1 g/L were generated using the AdEasy adenoviral vector system (Stra- glucose, 12.5 mM BSA-conjugated palmitic acid (Sigma Aldrich), and tagene), amplified in Ad-293 cells and purified by CsCl gradient 2.5 mCi palmitic acid [9,10-3H(N)] (Perkin Elmer, USA). Four hours centrifugation as previously described [19]. All adenoviruses generated later, 0.2 mL of media were collected to 2 mL tubes containing 0.8 mL also express GFP from an independent CMV promoter. An adenovirus of activated charcoal slurry. Samples incubated at room temperature expressing only GFP was used as a control in all experiments. for 30 min with frequent mixing. After centrifugation at 13,000 rpm for 15 min, 0.2 mL of supernatant were transferred to 4 mL scintillation 2.2. Cell culture, transduction,