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Downregulation of Max Dimerization Protein 3 Is Involved in Decreased Visceral Adipose Tissue by Inhibiting Adipocyte Differentiation in Zebrafish and Mice

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Downregulation of Max Dimerization Protein 3 Is Involved in Decreased Visceral Adipose Tissue by Inhibiting Adipocyte Differentiation in Zebrafish and Mice International Journal of Obesity (2014) 38, 1053–1060 & 2014 Macmillan Publishers Limited All rights reserved 0307-0565/14 www.nature.com/ijo ORIGINAL ARTICLE Downregulation of Max dimerization protein 3 is involved in decreased visceral adipose tissue by inhibiting adipocyte differentiation in zebrafish and mice Y Shimada1,2,3,4,5, J Kuroyanagi1, B Zhang1, M Ariyoshi1,2, N Umemoto1,2, Y Nishimura1,2,3,4,5 and T Tanaka1,2,3,4,5 BACKGROUND: The diet-induced obesity model of zebrafish (DIO-zebrafish) share a common pathophysiological pathway with mammalian obesity. OBJECTIVES: We aimed to investigate the role of Max dimerization protein 3 (MXD3) in visceral fat accumulation and adipocyte differentiation, by conducting knockdown experiments using zebrafish and mouse preadipocytes. METHODS: To identify genes related to visceral adiposity, we conducted transcriptome analyses of human and zebrafish obese populations using the Gene Expression Omnibus and DNA microarray. We then intraperitoneally injected morpholino antisense oligonucleotides (MO-mxd3) to knockdown mxd3 gene expression in DIO-zebrafish and measured several parameters, which reflected human obesity and associated metabolic diseases. Finally, lentiviral Mxd3 shRNA knockdown in mouse 3T3-L1 preadipocytes was conducted. Quantitative PCR analyses of several differentiation markers were conducted during these gene knockdown experiments. RESULTS: We found that MXD3 expression was increased in the obese population in humans and zebrafish. Intraperitoneal MO-mxd3 administration to DIO-zebrafish suppressed the increase in body weight, visceral fat accumulation and the size of mature adipocytes. Subsequently, dyslipidemia and liver steatosis were also ameliorated by MO-mxd3. In mouse adipocytes, Mxd3 expression was drastically increased in the early differentiation stage. Mxd3 shRNA inhibited preadipocyte proliferation and adipocyte maturation. Quantitative PCR analyses showed that the early differentiation marker, CCAAT/enhancer-binding protein delta (Cebpd) and late differentiation markers (CCAAT/enhancer-binding protein, alpha and peroxisome proliferator-activated receptor gamma) were downregulated by Mxd3 knockdown in 3T3-L1 cells and DIO-zebrafish. Subsequently, mature adipocyte markers (adiponectin and caveolin 1 for zebrafish, and fatty acid binding protein 4 and stearoyl-coenzyme A desaturase 1 for mouse adipocytes) were also decreased. CONCLUSION: Mxd3 regulates preadipocyte proliferation and early adipocyte differentiation via Cebpd downregulation in vitro and in vivo. Integrated analysis of human and zebrafish transcriptomes allows identification of a novel therapeutic target against human obesity and further associated metabolic disease. International Journal of Obesity (2014) 38, 1053–1060; doi:10.1038/ijo.2013.217 Keywords: visceral fat; overfeeding; transcriptome; preadipocyte; Danio rerio INTRODUCTION 2 diabetes mellitus, dyslipidemias, non-alcoholic fatty liver 7 Recent studies have shown that a large increase in the occurrence and gallstones and cardiovascular diseases. Regulation of pre- of obesity is a severe problem in developed countries.1,2 Obesity is adipocyte proliferation and adipocyte maturation to hypertrophy characterized by an increase in body weight and visceral adipose is now emerging as one of the most important processes in tissue hyperplasia and hypertrophy with excessive fat storage. In developing obesity. In fact, the removal of visceral fat of obese general, obesity can be classified into visceral and subcutaneous subjects improves dyslipidemia and insulin resistance in humans8 types according to fat distribution. Visceral obesity accompanies and in rodent models.9,10 On the basis of these observations, or precedes components of metabolic syndrome, such as chemicals and drug targets capable of restoring or preventing hyperinsulinemia and insulin resistance.3 A number of studies of the structural and functional alteration of adipose tissue may human and animal models have indicated that an increase in represent an effective approach against obesity and its associated preadipocyte cell number and adipocyte cell size is associated metabolic diseases. with mortality due to visceral obesity.4–6 The development of The zebrafish (Danio rerio), a small vertebrate, have recently hyperplastic adipose tissues is currently associated with the most become the focus of attention as a genetically tractable model severe form of obesity and its associated diseases, including type animal for human diseases.11 Zebrafish have multiple advantages, 1Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, Mie, Japan; 2Department of Systems Pharmacology, Mie University Graduate School of Medicine, Mie, Japan; 3Mie University Medical Zebrafish Research Center, Mie, Japan; 4Department of Bioinformatics, Mie University Life Science Research Center, Mie, Japan and 5Department of Omics Medicine, Mie University Industrial Technology Innovation, Mie, Japan. Correspondence: Dr T Tanaka, Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan. E-mail: [email protected] Received 10 June 2013; revised 16 October 2013; accepted 5 November 2013; accepted article preview online 20 November 2013; advance online publication, 24 December 2013 MXD3 knockdown decreases visceral fat in zebrafish Y Shimada et al 1054 such as a high degree of genetic conservation compared with described previously.18 Sections were also counterstained with Mayer’s mammals, their ease of genetic manipulation, availability for hematoxylin (Wako Pure Chemicals, Osaka, Japan) to visualize the nuclei high-throughput screening and live imaging. In addition, lipid according to the manufacturer’s protocol. metabolism in zebrafish has demonstrated similarity to that in humans in terms of absorption through the intestine with the aid Nile red staining 12 of bile produced in the liver, transport of fat and cholesterol by For in vivo staining of visceral fat of zebrafish, we conduced Nile red lipoproteins,13 use by b-oxidation,14 and storage as triacylglycerols staining according to our previous study.17 In brief, zebrafish were in visceral, subcutaneous and intramuscular adipocyte depots.15 transferred into 1000 ml of swimming water containing 100 mg of Nile red In light of these similarities and advantages, the zebrafish are used (Wako Pure Chemicals) and incubated overnight in the dark at 28 1C. in the field of lipid metabolism research as a model for studies After incubation, the fish were rinsed in fresh water and observed with an of lipid-related diseases, including atherosclerosis induced by MZ16F fluorescence microscope (Leica Microsystems) equipped with a 16 DP71 digital camera (Olympus, Tokyo, Japan) using a GFP2 filter (Leica high-cholesterol diets and obesity induced by overexpression of Microsystems). the endogenous melanocortin antagonist agouti-related protein.15 We previously constructed a diet-induced obesity model of zebrafish (DIO-zebrafish) overfed with Artemia as a high-fat diet.17 Body fat volume measurement by computed tomography Areas of visceral and subcutaneous fat depots in zebrafish were measured DIO-zebrafish showed an increase in body weight with visceral 21 adiposity, plasma triacylglyceride (TG) elevation and liver steatosis. by computed tomography (CT) as described previously. In brief, a 3D micro-CT scan was performed with an in vivo System R_mCT 3D micro-CT DNA microarray analyses showed that the transcriptome profiles 17 18 scanner (Rigaku Corporation, Tokyo, Japan). The following settings were of visceral adipose tissue and liver steatosis of DIO-zebrafish used: voltage, 90 kV; current, 100 mA; magnification, Â 4; slice thickness were highly consistent with human obesity and rodent models (scanning width), 50 mm; and exposure time, 2 min. Reconstruction and of DIO. We have discovered several candidate genes related to image viewing were performed with the i-View One Volume Viewer visceral adiposity from previous DNA microarray analyses,17 and software (J. Morita Mfg, Kyoto, Japan). The CT images were visualized and the knockdown of max dimerization protein 3 (mxd3) suppresses analyzed using the dedicated software CTAtlas Metabolic Analysis Ver. 2.03 development of visceral adiposity in DIO-zebrafish. (Rigaku Corporation). Statistical analysis MATERIALS AND METHODS All data are shown as mean±s.e.m. For comparison of two means, An expanded Methods section is available in the online-only statistical significance was evaluated by the unpaired Student’s t-test. Supplementary Data. For multiple comparisons, one-way ANOVA followed by the Bonferroni-Dunn multiple-comparison procedure was used. DIO-zebrafish 19 Ethics The zebrafish AB wild-type and rose mutant lines were supplied by the Zebrafish International Resource Center (University of Oregon, Eugene, OR) All animal experiments were conducted according to the Act on Welfare and maintained in our facility according to our established protocols.20 and Management of Animals (Ministry of the Environment of Japan) and Adult zebrafish (3- to 4-month-old females) were used for DIO-zebrafish. complied with international guidelines. Ethics approval from the local DIO-zebrafish were generated by overfeeding (OF) of Artemia (60 mg cysts/ Institutional Animal Care and Use Committee was not sought because this fish per day; Miyako Kagaku, Tokyo, Japan) as described in
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