Nobiletin suppresses adipocyte differentiation of 3T3-L1 cells by an insulin and IBMX mixture induction Kota Kanda a , Kosuke Nishi a , Ayumu Kadota b , Sogo Nishimoto a , Ming-Cheh Liu c , Takuya Sugahara a, d, ⁎ a Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790-8566, Japan b Ikata Service Co. Ltd., Nishiuwa, Ehime 769-0421, Japan c Department of Pharmacology, The University of Toledo, Toledo, OH 43606, USA d South Ehime Fisheries Research Center, Ehime University, Ainan, Ehime 798-4292, Japan abstract article info Article history: Received 26 May 2011 Received in revised form 25 November 2011 Accepted 28 November 2011 Available online 8 December 2011 Keywords: Nobiletin PPARγ STAT5 3T3-L1 cAMP CREB Background: Nobiletin is a citrus flavonoid which possesses the flavone structure with six methoxy groups. Although nobiletin has been reported to display anti-inflammatory, anti-tumor, and anti-diabetes activities, its effect on adipocyte differentiation remained unclear. In the present study, we investigated the effect of nobiletin on the differentiation of 3T3-L1 preadipocytes into adipocytes. Methods: 3T3-L1 preadipocytes were treated with nobiletin under various differentiation conditions. The ef- fect of nobiletin on adipocyte differentiation was evaluated by oil red O staining, real-time RT-PCR, and West- ern blotting. Results: Nobiletin significantly suppressed the differentiation of 3T3-L1 preadipocytes into adipocytes, upon in- duction with insulin together with a cAMP elevator such as 3-isobutyl-1-methylxanthine (IBMX), by downregu- lating the expression of the gene encoding peroxisome proliferator-activated receptor (PPAR) γ2. In addition, nobiletin decreased the phosphorylation of cAMP-response element-binding protein (CREB) and strongly en- hanced the phophorylation of signal transducer and activator of transcription (STAT) 5. General significance: Nobiletin has a suppressive effect on the differentiation of preadipocytes into adipocytes when cells were induced with a general differentiation cocktail such as insulin, IBMX, and dexamethasone. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Obesity is an etiologic factor for type 2 diabetes, cardiovascular disease, and hypertension. Obesity results from an increase in both the number and size of adipocytes [1]. Adipocytes represent a major energy reservoir in the body, storing excess energy as lipids and re- leasing it on demand. In addition, adipocytes constitute an endocrine system by secreting adipose-derived hormones known as adipokines, which regulate tissues such as muscles and adipose tissues [2]. Thus, the regulation of adipocyte differentiation is crucial for therapies aimed to prevent and improve obesity and the related metabolic syndrome. 3T3-L1 cells are derived from the 3T3 Swiss albino mouse cell line, and are used as an in vitro model of adipocytes to study insulin path- ways, obesity, and cardiovascular diseases [3]. 3T3-L1 preadipocytes differentiate upon exposure to inducers such as insulin, 3-isobutyl- 1-methylxanthine (IBMX), and dexamethasone (DEX) [4]. These in- ducers activate the signaling cascade, thereby facilitate the expres- sion of peroxisome proliferators-activated receptor (PPAR) γ or the production of PPARγ ligands. PPARγ is a master regulator of adipo- cyte differentiation. Forced expression of PPARγ gene is sufficient to induce the adipogenesis in fibroblasts [5], and no factor has been dis- covered that promotes adipocyte differentiation in the absence of PPARγ [6]. In the signaling cascade, the expression of PPARγ gene is induced by CCAAT/enhancer-binding protein (C/EBP) β [7]. In addition, cyclic adenosine monophosphate (cAMP)-response element- binding protein (CREB) is known to be involved in adipocyte differenti- ation [8,9]. CREB is activated through the phosphorylation by protein kinase A (PKA), and may subsequently promote the expression of C/EBPβ gene. Moreover, signal transducer and activator of transcrip- tion (STAT) 5 has been shown to regulate the adipogenesis [10]. Ac- tivation of STAT5 typically induces adipocyte differentiation [11], Biochimica et Biophysica Acta 1820 (2012) 461–468 Abbreviations: cAMP, cyclic adenosine monophosphate; C/EBP, CCAAT/enhancer- binding protein; CREB, cAMP-response element-binding protein; DEX, dexamethasone; DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; IBMX, 3-isobutyl-1-methylxanthine; HRP, horseradish peroxidase; MEF, mice embryonic fibroblast; MEK, mitogen-activated protein kinase/extracellular signaling-regulated protein kinase; PBS, phosphate-buffered saline; PKA, protein kinase A; PPAR, peroxisome proliferators-activated receptor; SD, standard deviation; STAT, signal transducer and activator of transcription; TBS-T, Tris-buffered saline with 0.1% Tween 20 ⁎ Corresponding author at: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan. Tel./fax: + 81 89 946 9863. E-mail address: mars95@agr.ehime-u.ac.jp (T. Sugahara). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.11.015 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen