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Inhibition of Pparγ, Adipogenesis and Insulin Sensitivity by MAGED1

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Inhibition of Pparγ, Adipogenesis and Insulin Sensitivity by MAGED1 239 2 Journal of Q Wang, J Tang et al. MAGED1 inhibits adipogenesis 239:2 167–180 Endocrinology RESEARCH Inhibition of PPARγ, adipogenesis and insulin sensitivity by MAGED1 Qinghua Wang1,2,*, Jing Tang1,*, Shujun Jiang1,3, Zan Huang1,4, Anying Song1, Siyuan Hou1, Xiang Gao1 and Hai-Bin Ruan5 1State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, Jiangsu, China 2Laboratory Animal Center, Nantong University, Nantong, Jiangsu, China 3School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing, Jiangsu, China 4Laboratory of Gastrointestinal Microbiology, Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, China 5Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA Correspondence should be addressed to X Gao or H-B Ruan: [email protected] or [email protected] *(Q Wang and J Tang contributed equally to this work) Abstract Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator of Key Words adipogenesis and a target of the thiazolidinedione (TZD) class of antidiabetic drugs; f obesity therefore, identifying novel regulators of PPARγ action in adipocytes is essential for the f insulin sensitivity future development of therapeutics for diabetes. MAGE family member D1 (MAGED1), f PPARγ stability by acting as an adaptor for ubiquitin-dependent degradation pathways and a co-factor f energy expenditure for transcription, plays an important role in neural development, cell differentiation f MAGE gene family and circadian rhythm. Here, we showed that MAGED1 expression was downregulated during adipogenesis and loss of MAGED1 promoted preadipocyte proliferation and differentiation in vitro. MAGED1 bound to PPARγ and suppressed the stability and transcriptional activity of PPARγ. Compared to WT littermates, MAGED1-deficient mice showed increased levels of PPARγ protein and its target genes, more CD29+CD34+Sca-1+ adipocyte precursors and hyperplasia of white adipose tissues (WATs). Moreover, MAGED1-deficient mice developed late-onset obesity as a result of decreased energy expenditure and physical activity. However, these mice were metabolically healthy as shown by improved glucose clearance and insulin sensitivity, normal levels of serum lipids and enhanced secretion of adipokines such as leptin and adiponectin. Taken together, our data identify MAGED1 as a novel negative regulator of PPARγ activity, adipogenesis and insulin sensitivity in mice. MAGED1 might therefore serve as a novel Journal of Endocrinology pharmaceutical target to treat obesity-associated insulin resistance. (2018) 239, 167–180 Introduction The obesity epidemic continues to rise as a global health balance and glucose homeostasis by storing excess energy challenge. Obesity is a major risk factor for high blood to prevent ectopic fat accumulation in non-adipose pressure, hyperlipidemia, diabetes, heart disease, cancers, tissues and by secreting various adipokines to modulate etc. (Ng et al. 2014, Ogden et al. 2014). The white adipose whole-body metabolism (Ouchi et al. 2011). Adipocyte tissue (WAT) serves as a critical integrator of energy hyperplasia, or adipogenesis, is the process of adipocyte https://joe.bioscientifica.com © 2018 Society for Endocrinology https://doi.org/10.1530/JOE-18-0349 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/30/2021 10:44:40PM via free access -18-0349 Journal of Q Wang, J Tang et al. MAGED1 inhibits adipogenesis 239:2 168 Endocrinology differentiation from pre-adipocytes, which reside in MAGED1 leads to enhanced preadipocyte proliferation the stromal vascular fraction (SVF) of WAT (Rosen & and adipogenesis. MAGED1-deficient mice develop obesity MacDougald 2006, Gesta et al. 2007, Tran & Kahn 2010). but lack metabolic abnormalities including dyslipidemia, The nuclear receptor peroxisome proliferator-activated impaired glucose tolerance and insulin resistance. receptor-γ (PPARγ) is a master regulator of adipogenesis and most pro- and anti-adipogenic factors seem to function at least in part by regulating PPARγ expression Materials and methods and/or activity (Farmer 2006, Rosen & MacDougald 2006). The thiazolidinedione (TZD) class of antidiabetic Animals drugs agonize PPAR to activate adipogenesis and γ C57BL/6J mice from Jackson Laboratory and Maged1- sensitize insulin action (Spiegelman 1998). The PPAR γ knockout (KO) mice (Wang et al. 2010) were maintained protein is short-lived as it can be ubiquitinated and in an AAALAC-accredited, specific pathogen-free facility degraded through the proteasome (Floyd & Stephens in Model Animal Research Center of Nanjing University. 2002, van Beekum et al. 2009, Kim et al. 2014). Activation All animal protocols were approved by Institutional by TZDs further accelerates PPAR degradation; therefore, γ Animal Care and Use Committee of Nanjing University, understanding how PPAR stability is controlled will shed γ in accordance with published guidelines in the light on new strategies to fine-tune this metabolic master Principles of Laboratory Animal and the Care and Use of regulator to treat type 2 diabetes. Laboratory Animals (National Research Council 1996). Melanoma antigen (MAGE) family member D1 All mice were maintained under 12-h light/darkness (MAGED1), also known as neurotrophin receptor- cycles (light on at 08:00 h and off at 20:00 h) and fed interacting MAGE (NRAGE) and DLXIN-1, plays an with acidified water and standard chowad libitum. important role in transcription, cell signaling and protein Maged1-KO mice had been backcrossed to C57BL/6J stability (Di Certo et al. 2007, Doyle et al. 2010, Wang et al. mice for at least 15 generations. 2010, Mouri et al. 2012). Growing evidence demonstrates that MAGED1 interacts with proteins such as DLX5 (Masuda et al. 2001), p75NTR (Salehi et al. 2000), ITA Serum biochemistry parameters measurements and XIAP (Jordan et al. 2001), UNC5H1 (Williams et al. Mice were fasted for overnight or 6 h or refed for 2 h as 2003), ROR2 (Matsuda et al. 2003) and RORα (Wang et al. indicated. Whole blood was collected from the eye socket 2010) to regulate neural development, cell apoptosis and vein. Serum was collected after a centrifugation at 3000 g proliferation and circadian rhythm. We and others have for 15 min. Lipid metabolites in plasma including total showed that MAGED1-knockout mice exhibit a shortened cholesterol, triglyceride (TG), high-density lipoprotein circadian period and bouts (Wang et al. 2010), impaired and low-density lipoprotein (VLDL) were quantified learning and memory (Yang et al. 2015) and an osteoporotic by colorimetric assays with a 7020 automatic analyzer phenotype (Liu et al. 2015). However, the role of MAGED1 (Hatachi High Technology, Japan). in energy and glucose metabolism has not been studied. Recently, MAGE family proteins were shown to bind Glucose and insulin tolerance tests really interesting new gene (RING) domain proteins to form active E3 ubiquitin ligases (Doyle et al. 2010). For For oral glucose tolerance test, mice were fasted from example, the MAGEC2-TRIM28 complex targets p53 17:00 to 09:00 h and injected with D(+)-glucose (Sigma, for proteasomal degradation (Doyle et al. 2010). The 2 g/kg body weight). For insulin tolerance test (ITT), MAGEL2-TRIM27 promotes K63-linked ubiquitination of mice were fasted from 09:00 to 15:00 h and injected with WASH, thus facilitating endosomal F-actin nucleation and 0.75 U insulin (Novo Nordisk) per 1 kg body weight. Blood retromer-mediated transport (Hao et al. 2013). Likewise, glucose concentrations were determined by GLUCOCARD MAGED1 interacts with RING proteins including PRAJA-1 II Test Meters (Arkray, Minneapolis, MN, USA). (Sasaki et al. 2002), and TRIM28 to a less extent (Doyle et al. 2010). In addition, MAGED1 controls the degradation of Metabolic characterization and body composition the anti-apoptotic factor CHE-1 (Di Certo et al. 2007), and analyses the activity and ubiquitylation of the serotonin transporter (Mouri et al. 2012). In this study, we report that MAGED1 The Comprehensive Lab Animal Monitoring System was interacts with PPARγ and decreases its stability. Loss of used to determine food intake, physical activity, oxygen https://joe.bioscientifica.com © 2018 Society for Endocrinology https://doi.org/10.1530/JOE-18-0349 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/30/2021 10:44:40PM via free access Research Journal of Q Wang, J Tang et al. MAGED1 inhibits adipogenesis 239:2 169 Endocrinology consumption and carbon dioxide production. All mice containing the adipogenic cocktail (850 nM Insulin, were maintained in a 12:12 light/darkness cycle at 22°C 0.5 μM dexamethasone, 250 μM isobutylmethylxanthine and acclimated for 2 days. Data were recorded for at least (IBMX) and 1 μM rosiglitazone) for 48 h. Cells were then 72 h for analyses. kept in basic preadipocyte culture medium with 850 nM A dual-energy X-ray absorptiometry system (GE Medical insulin till analyses. System Lunar, Madison, WI, USA) was used to determine To make Oil-Red O stock, 500 mg of Oil-Red O was body composition. Mice were anesthetized with Avertin dissolved in 100 mL of isopropanol and kept stirring (Sigma Aldrich) and kept on a heat plate for
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