Biochimica et Biophysica Acta 1820 (2012) 461–468 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen 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 Ikata Service Co. Ltd., Nishiuwa, Ehime 769-0421, Japan Department of Pharmacology, The University of Toledo, Toledo, OH 43606, USA d South Ehime Fisheries Research Center, Ehime University, Ainan, Ehime 798-4292, Japan b c a r t i c l e i n f o 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 a b s t r a c t 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 effect of nobiletin on adipocyte differentiation was evaluated by oil red O staining, real-time RT-PCR, and Western blotting. Results: Nobiletin significantly suppressed the differentiation of 3T3-L1 preadipocytes into adipocytes, upon induction with insulin together with a cAMP elevator such as 3-isobutyl-1-methylxanthine (IBMX), by downregulating 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 enhanced 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 releasing 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 Abbreviations: cAMP, cyclic adenosine monophosphate; C/EBP, CCAAT/enhancerbinding 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 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 pathways, obesity, and cardiovascular diseases [3]. 3T3-L1 preadipocytes differentiate upon exposure to inducers such as insulin, 3-isobutyl1-methylxanthine (IBMX), and dexamethasone (DEX) [4]. These inducers activate the signaling cascade, thereby facilitate the expression of peroxisome proliferators-activated receptor (PPAR) γ or the production of PPARγ ligands. PPARγ is a master regulator of adipocyte differentiation. Forced expression of PPARγ gene is sufficient to induce the adipogenesis in fibroblasts [5], and no factor has been discovered 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 elementbinding protein (CREB) is known to be involved in adipocyte differentiation [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 transcription (STAT) 5 has been shown to regulate the adipogenesis [10]. Activation of STAT5 typically induces adipocyte differentiation [11], 462 K. Kanda et al. / Biochimica et Biophysica Acta 1820 (2012) 461–468 Committee and were performed in accordance with applicable guidelines and regulations. while excessive activation of STAT5 has been reported to inhibit the adipogenesis in 3T3-L1 cells [12]. Nobiletin, a citrus flavonoid which has a typical flavonoid structure with six methoxy groups, is a major component in citrus fruits [13]. It has been reported that nobiletin displays anti-inflammatory [14], anti-tumor [15], and anti-diabetes activities [16]. In addition, it has been reported that nobiletin may stimulate the differentiation of preadipocytes into adipocytes by the induction with insulin alone [17], and enhance the expression of adiponectin gene at mRNA and protein levels [17–19]. On the other hand, Miyamoyo et al. [20] reported a suppressive effect of nobiletin on adipocyte differentiation of 3T3-L1 cells and proposed that nobiletin may inhibit the mitogenactivated protein kinase/extracellular signaling-regulated protein kinase (MEK) signaling pathway, thereby leading to the suppression of adipocyte differentiation. Therefore, the effect of nobiletin on adipocyte differentiation remained unclear. In this study, we investigated the effect of nobiletin on the differentiation of 3T3-L1 preadipocytes into adipocytes under various inducing conditions. Oil red O staining was performed using a lipid assay kit (Primary Cell, Hokkaido, Japan) according to the manufacturer's instructions. Briefly, cells were washed with phosphate-buffered saline (PBS) and fixed for at least 15 min with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (Nacalai Tesque, Kyoto, Japan). The cells were washed twice with PBS, and then stained for 15 min using an oil red O staining solution. After washing with distilled water, stained cells were observed under a microscope. Following the microscopic observation, an extraction solution was added to cells and the absorbance of the extracted dye was spectrophotometrically measured at 540 nm using a microplate reader (SH-9000, Corona Electric, Ibaraki, Japan). 2. Materials and methods 2.5. Real-time RT-PCR 2.1. Reagents Total RNA was isolated from 3T3-L1 cells using Sepasol-RNA I Super G (Nacalai Tesque) according to the manufacturer's instructions. Total RNA was used as a template for cDNA synthesis performed using MMLVreverse transcriptase (Promega, Madison, WI, USA) and oligo-(dT)20 primer. The real-time PCR mixture, with a final volume of 20 μl, consisted of Fast SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1 μM of a forward primer, 1 μM of a reverse primer, and 0.1 μg of a cDNA sample. The thermal cycling conditions were: 20 s at 95 °C, and 40 cycles of 3 s at 95 °C and 30 s at 60 °C. PCR products were measured with a StepOnePlus Real-time PCR System (Applied Biosystems), and the relative gene expression was calculated based on the comparative CT method using a StepOne Software v2.1 (Applied Biosystems). The expression of β-actin mRNA was used as an endogenous control. Primer pairs used here for the amplification were: 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ and 5′-ATGGAGCCACCGATCCACA-3′ for mouse β-actin; 5′-AACTCTGGGAGATTCTCCTGTTGA-3′ and 5′-GAAGTGCTCATAGGCAGTGCAT-3′ for mouse PPARγ2, 5′-CATGGCCAAGCCCAACAT-3′ and 5′-CGCCCAGTTTGAAGGAAATC-3′ for mouse aP2; 5′-GGGGTTGTTGATGTTTTTGG-3′ and 5′-CGAAACGAAAAGGTTCTCA-3′ for mouse C/EBPβ. Data were shown as the fold difference normalized to the β-actin. Nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone; >95% purity) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Nobiletin was dissolved in DMSO to make a 0.1 M solution and used for the experiments described below. Insulin, DEX, and IBMX were products of Sigma (St. Louis, MO, USA). Antibodies against β-actin (sc-1616), PPARγ (sc7196), and STAT5 (sc-835) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against CREB (#9197), phosphorylated CREB (#9198), and phosphorylated STAT5 (#9351) were purchased from Cell Signaling Technology (Danvers, MA, USA). 2.2. Cells and cell culture 3T3-L1 preadipocytes obtained from American Type Culture Collection (Rockville, MD, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma) containing 10% fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS, USA) at 37 °C in a humidified atmosphere of 5% CO2. Differentiation of 3T3-L1 preadipocytes into mature adipocytes was conducted as reported previously [21]. Briefly, 2 days after reaching confluency, 3T3-L1 preadipocytes grown in individual wells of collagen-coated 24-well cell culture plates (BD Falcon, Franklin Lakes, NJ, USA) were subjected to induction by replacing the culture medium with the induction DMEM, and the induction proceeded for 2 days. Subsequently, culture media were changed to DMEM containing 10% FBS and 5 μg/ml of insulin every 2 days unless otherwise noted. The complete induction medium consisted of DMEM medium containing 10% FBS, 5 μg/ml of insulin, 0.5 mM IBMX, and 10 μM DEX. Incomplete induction media lacking one or two of the inducing reagents from the complete induction medium were indicated in the “Results” section and in the figure legends. 2.3. Isolation and cell culture of mouse embryonic fibroblasts (MEFs) MEFs were isolated from BALB/c mouse embryos on day 14.5 of gestation. Briefly, embryos were minced and treated with trypsin and ethylenediaminetetraacetic acid (EDTA) at 37 °C for 10 min, and were then added DNase I (Takara Bio, Shiga, Japan) and incubated for 5 min. Cells were dispersed by pipetting, passed through a 40 μm cell strainer (BD Falcon), and washed with DMEM containing 10% FBS. Methods for cell culture and the induction into adipocytes were the same as those described above for 3T3-L1 cells. All animal experiments described here were carried out in accordance with a protocol approved by the Ehime University Animal Care and Use 2.4. Oil red O staining 2.6. Western blot analysis Total cell lysate was prepared using a lysis buffer consisting of 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 2 μg/ml of aprotinin (Roche Applied Science, Basel, Switzerland), pefabloc SC (Roche Applied Science), and a protease inhibitor cocktail (Roche Applied Science). Proteins in the total cell lysate were separated by a polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Hybond-P, GE Healthcare, Buckinghamshire, UK). The membrane was incubated with Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% skim milk for 1 h at room temperature. After washing with TBS-T, the membrane was incubated with a primary antibody in 5% bovine serum albumin-TBS-T at 4 °C overnight. After washing with TBS-T, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin antibody (DakoCytomation, Glostrup, Denmark) or HRP-conjugated anti-goat IgG antibody (Santa Cruz Biotechnology) in 5% skim milk-TBS-T at room temperature for 1 h. After washing with TBS-T, blots were developed by an ECL Western Blotting Detection Reagent (GE Healthcare) or ImmunoStar LD (Wako Pure Chemical Industries). Bands were visualized using a ChemiDoc XRS Plus apparatus (Bio-Rad Laboratories, Hercules, CA, USA), and the chemiluminescent intensity was quantified using the Quantity One software (Bio-Rad Laboratories). 463 K. Kanda et al. / Biochimica et Biophysica Acta 1820 (2012) 461–468 2.7. Statistical analysis Data obtained were expressed as the mean ± standard deviation (SD). The student's t test was used to assess the statistical significance of the difference against the control. Values with *p b 0.05 or **p b 0.01 were considered to be statistically significant. 3. Results 3.1. Effect of nobiletin on lipid accumulation in 3T3-L1 cells In an initial study, 3T3-L1 preadipocytes were treated with the complete induction medium containing insulin, IBMX, and DEX to induce their differentiation into mature adipocytes. Nobiletin was added to the complete induction medium for 2 days. On day 2 after induction, culture medium was changed to DMEM supplemented with 10% FBS and 5 μg/ml of insulin, and the cells were cultured for another 5 days. Lipid accumulation in the adipocytes was measured by oil red O-staining on day 7. As shown in Fig. 1A and B, nobiletin significantly suppressed the lipid accumulation in 3T3-L1 adipocytes in a dose-dependent manner with statistical significances, suggesting that nobiletin inhibits adipogenesis in 3T3-L1 cells when the adipocyte differentiation was induced with insulin, IBMX, and DEX. and DEX. In the experiments described below, we used 3T3-L1 cells to analyze further the mechanism of the suppressive effect of nobiletin on adipogenesis, because nobiletin exerted a stronger effect on 3T3-L1 cells than on MEFs. 3.3. Evaluation of the optimal effect of nobiletin in suppressing the lipid accumulation To clarify the mechanism of the suppressive effect of nobiletin on adipogenesis, we first examined the time course of the effect of nobiletin on adipocyte differentiation in regard to the suppression of lipid accumulation. 3T3-L1 cells were treated with nobiletin at different times (from 0 to 6 days) following induction, and thereafter were monitored for different durations (from 1 to 7 days). Results shown in Fig. 2A indicated that when nobiletin treatment was administered between day 0 and day 2 upon induction, the lipid accumulation was significantly suppressed (Fig. 2B). Interestingly, treatment of nobiletin after 2 days of induction exerted virtually no effect on lipid accumulation in differentiated 3T3-L1 adipocytes. It was also observed that no differences in lipid accumulation were observed irrespective of the duration of treatment with nobiletin. These results indicated that nobiletin can affect only 3T3-L1 preadipocytes within the first 2 days following the induction treatment to suppress the lipid accumulation. 3.2. Effect of nobiletin on lipid accumulation in MEFs 3.4. Effect of nobiletin on the translational level of PPARγ To investigate whether the suppressive effect of nobiletin on lipid accumulation is specific only for 3T3-L1 cells, primary preadipocytes, MEFs were used. Adipocyte differentiation of MEFs was induced with the complete induction medium supplemented with different concentrations of nobiletin. On day 2 after induction, culture media were changed to DMEM supplemented with 10% FBS and insulin, and the cells were cultured for another 5 days. Subsequent oil red O-staining revealed that nobiletin significantly suppressed lipid accumulation in MEFs (Fig. 1C, D), indicating that nobiletin inhibits the adipogenesis not only in 3T3-L1 cells, but also in primary preadipocytes when their differentiation was induced with insulin, IBMX, A The adipogenesis is known to be promoted by increased expression of PPARγ gene. Therefore, we decided to investigate PPARγ expression level in 3T3-L1 cells by Western blot analysis. Nobiletin did not affect the protein expression of PPARγ1 in 3T3-L1 cells (Fig. 3A, B), but significantly suppressed the PPARγ2 protein expression (Fig. 3A, C). It has been reported that the expression of PPARγ2 promotes the adipogenesis in 3T3-L1 cells, whereas PPARγ1 may not [22]. These results therefore suggest that nobiletin may inhibit the adipocyte differentiation by down-regulating PPARγ2 expression level in 3T3-L1 cells. C Pre 0 1 10 100 Pre 0 Nobiletin (µM) 10 100 D B 1.0 0.8 0.8 ** ** 0.6 ** 0.4 0.2 0.0 Pre 0 1 10 Nobiletin (µM) 100 Absorbance (540 nm) Absorbance (540 nm) 1 Nobiletin (µM) ** * 1 10 0.6 ** 0.4 0.2 0.0 Pre 0 100 Nobiletin (µM) Fig. 1. Effect of nobiletin on lipid accumulation in 3T3-L1 cells and MEFs. (A) Micrographs of 3T3-L1 cells. (B) Lipid accumulation in 3T3-L1 cells assessed by the spectrophotometric measurement of oil red O-stained 3T3-L1 cells. (C) Micrographs of MEFs. (D) Lipid accumulation in MEFs was assessed by the spectrophotometric measurement of oil red O-stained cells. 3T3-L1 cells and MEFs were induced to differentiate with 10% FBS-DMEM medium containing insulin, DEX, IBMX, and 0, 1, 10, or 100 μM of nobiletin for 2 days, and then culture media were replaced with 10% FBS-DMEM medium containing insulin for the following 5 days. Control cells were treated with DMSO instead of nobiletin. Preadipocytes were cultured in 10% FBS-DMEM medium. Data were represented as the mean ± SD (n = 3). *p b 0.05 and **p b 0.01 against control. 464 K. Kanda et al. / Biochimica et Biophysica Acta 1820 (2012) 461–468 A B Day 0 1 2 3 a: Preadipocyte b: Control 4 5 6 7 Absorbance (540 nm) 0.0 0.4 0.8 * c: 1.2 d: f: ** Nobiletin (100 µM) ** e: g: h: ** i: Fig. 2. Suppressive effect of nobiletin on the lipid accumulation by a various treating period. (A) 3T3-L1 preadipocytes were induced to differentiate with 10% FBS-DMEM medium containing insulin, DEX, and IBMX for 2 days (from day 0 to day 2), and then culture media were replaced with 10% FBS-DMEM medium containing insulin for the following 5 days. Nobiletin (100 μM) was added during the period indicated as an arrow, and oil red O-staining was performed on day 7 from initiating the differentiation. Preadipocytes were cultured in 10% FBS-DMEM medium. Control cells were treated with DMSO instead of nobiletin. (B) Quantitative analysis of lipid accumulation was assessed by the spectrophotometric measurement of oil red O-stained adipocytes. Data were represented as the mean ± SD (n = 2). *p b 0.05 and **p b 0.01 against control. expression at the protein level as shown in Fig. 3C, the transcripts of PPARγ2 and aP2, a target gene of PPARγ, were significantly decreased following treatment with nobiletin on day 1 (Fig. 4A) and on day 3 (Fig. 4B), respectively. Moreover, nobiletin significantly suppressed C/EBPβ expression on day 1 (Fig. 4C). It is well known that in the 3.5. Effect of nobiletin on adipogenic gene expression To clarify the mechanism underlying the regulation of PPARγ2 expression, we investigated the expression of adipogenic genes by realtime RT-PCR. Consistent with the observation of increased PPARγ2 A 2 PPARγ 1 Actin (day) 0 1 2 3 4 Control B 3 4 C PPARγ2 * 10 5 Preadipocytes Relative expression Relative expression 2 Nobiletin PPARγ1 Control 4 Nobiletin 3 2 1 0 1 0 1 2 3 Days after induction 4 ** 5 ** 0 0 1 2 3 4 Days after induction Fig. 3. Western blot analysis of PPARγ protein in 3T3-L1 cells treated with nobiletin. (A) A representative image from two independent experiments. (B) PPARγ1 expression level in each cell sample relative to that in preadipocytes compensated with actin as an internal control. (C) PPARγ2 expression level in each cell sample relative to that in preadipocytes compensated with actin as an internal control. 3T3-L1 cells were induced to differentiate with 10% FBS-DMEM medium containing insulin, DEX, IBMX, and 100 μM of nobiletin for 2 days, and then culture media were replaced with 10% FBS-DMEM medium containing insulin for the following 2 days. Preadipocytes were cultured in 10% FBS-DMEM medium. Control cells were treated with DMSO instead of nobiletin. Cell lysates were prepared at the indicated day after the differentiation induction, and Western blotting was performed. Data were represented as the mean ± SD (n = 2). *p b 0.05 and **p b 0.01 against control. 465 K. Kanda et al. / Biochimica et Biophysica Acta 1820 (2012) 461–468 A Relative expression B PPARγ2 Preadipocytes Control Nobiletin 50 40 30 ** C aP2 1000 60 C/EBPβ 10 * 800 8 600 6 400 4 200 2 * 20 10 0 0 1 2 3 4 Days after induction 0 0 1 2 3 0 4 0 1 2 3 4 Days after induction Days after induction Fig. 4. Effect of nobiletin on the expression of adipogenic genes in 3T3-L1 cells. (A) PPARγ2; (B) aP2; (C) C/EBPβ. 3T3-L1 cells were induced to differentiate with 10% FBS-DMEM medium containing insulin, DEX, IBMX, and 100 μM of nobiletin for 2 days, and then culture media were replaced with 10% FBS-DMEM medium containing insulin for the following 2 days. Preadipocytes were cultured in 10% FBS-DMEM medium. Control cells were treated with DMSO instead of nobiletin. Total RNA was prepared at the indicated day after the differentiation induction and analyzed for the indicated genes. β-Actin mRNA was analyzed as an internal control. Data were represented as a relative expression level ± SD against preadipocytes (n = 3). *p b 0.05 and **p b 0.01 against control. first 2 days during the adipocyte differentiation, the expression of C/EBPβ gene is up-regulated and that C/EBPβ promotes the PPARγ gene expression [23]. Taken together, these findings suggest that nobiletin may suppress the adipocyte differentiation by down-regulating PPARγ2 gene expression via decreased C/EBPβ expression. 3.6. Influence of different combinations of differentiation inducers on the suppressive effect of nobiletin To reveal the interaction of nobiletin with the adipocyte differentiation inducers, the effect of nobiletin on the adipogenesis in 3T3-L1 preadipocytes upon induction with insulin alone, insulin plus IBMX, or insulin plus DEX was examined. As shown in Fig. 5, nobiletin enhanced PPARγ2 expression when 3T3-L1 preadipocytes were induced with insulin alone. In contrast, nobiletin significantly suppressed PPARγ2 expression when differentiation of 3T3-L1 preadipocytes was induced by insulin plus IBMX. The PPARγ2 gene expression was unaffected by inducing when the cells were induced by insulin plus DEX. These results suggest that nobiletin may down-regulate the signaling pathways mediated by IBMX. 3.7. Effect of nobiletin on the phosphorylation of CREB IBMX is known to increase the intracellular cAMP level by inhibiting phosphodiesterase. Accumulated cAMP may activate PKA, which in turn activates CREB through its phosphorylation. Phosphorylated CREB then participates in inducing C/EBPβ expression, and C/EBPβ in turn induces the expression of both PPARγ and C/EBPα. As shown in Fig. 4C, nobiletin reduced C/EBPβ expression. To clarify whether nobiletin exerts its effect upstream of the C/EBPβ signaling pathway, the activation of CREB involved in the signaling pathway stimulated by IBMX was examined. As shown in Fig. 6A, CREB was A p-CREB CREB (min) 0 5 15 30 60 5 Control 15 30 60 Nobiletin B 10 Control Nobiletin Ratio of p-CREB/CREB Relative PPARγ2 expression 4 3 2 * 1 5 0 0 Insulin Insulin Insulin + IBMX + DEX Fig. 5. Effect of nobiletin on the differentiation by various inducer combinations in 3T3L1 cells. 3T3-L1 cells were induced to differentiate with 10% FBS-DMEM medium with insulin only, with insulin and DEX, or with insulin and IBMX and treated with 100 μM of nobiletin. Control cells were treated with DMSO instead of nobiletin. Forty-eight hours after the induction, total RNA samples were prepared and analyzed for PPARγ2 gene expression. β-Actin mRNA was analyzed as an internal control. Results were expressed as the PPARγ2 expression level relative to control. Data were represented as the mean ± SD (n = 3). *p b 0.05 against control. Preadipocytes Control Nobiletin 0 5 15 30 60 Time after induction (min) Fig. 6. Effect of nobiletin on the phosphorylation of CREB in 3T3-L1 cells. (A) A representative image from two independent experiments. (B) The scanned bar graph shows fold the induction of phosphorylated CREB over CREB. 3T3-L1 cells were induced to differentiate with 10% FBS-DMEM medium containing insulin, DEX, and IBMX and treated with 100 μM of nobiletin. Preadipocytes were cultured in 10% FBS-DMEM medium. Control cells were treated with DMSO instead of nobiletin. Cell lysates were prepared at indicated time after the differentiation induction, and Western blotting was performed. p-CREB represents the phosphorylated CREB. Results were expressed as the ratio of p-CREB/CREB in each cell sample relative to that in preadipocytes. Data were represented as the mean±SD (n=2). 466 K. Kanda et al. / Biochimica et Biophysica Acta 1820 (2012) 461–468 A p-STAT5 STAT5 B 4 Ratio of p-STAT5/STAT5 strongly phosphorylated upon treatment with adipocyte differentiation inducers. Interestingly, nobiletin suppressed the phosphorylation of CREB at 60 min after initiation of the induction, although the phosphorylated amount of CREB was not significantly different from that of the control (Fig. 6B). The phosphorylation of CREB has been reported to be induced by the insulin signaling pathway [24,25]. Saito et al. [17] reported that the phosphorylation of CREB was enhanced by nobiletin in 3T3-L1 preadipocytes 1 h following induction of differentiation when the cells were induced to differentiate with insulin only. Thus, decreased phosphorylation of CREB by treating with nobiletin may be neutralized by the activation of the insulin signaling pathway by nobiletin. Therefore, it is possible that there may be another signaling pathway involved in the suppression of PPARγ2 gene expression by nobiletin. 3.8. Effect of nobiletin on the phosphorylation of STAT5 To investigate another suppressive mechanism of adipocyte differentiation by nobiletin, the phosphorylation of STAT5 was examined. STAT5 is known to be involved in the early phase of adipogenesis [11]. Excessive activation of STAT5, however, has been shown to inhibit adipocyte differentiation [12]. As shown in Fig. 7A and B, nobiletin strongly enhanced the phosphorylation of STAT5, suggesting that the suppressive effect of nobiletin on adipocyte differentiation is involved in the excessive activation of STAT5. The effect of nobiletin on STAT5 phosphorylation in the presence or absence of IBMX in 3T3-L1 cells was also examined. Result revealed that the phosphorylation of STAT5 by nobiletin was further enhanced with concomitant treatment with IBMX (Fig. 8A), though there was no statistical significance (Fig. 8B). Taken together, these findings indicate that the excessive activation of STAT5 might have occurred due to the synergic effect of nobiletin and IBMX. A p-STAT5 STAT5 (min) 0 5 15 30 60 5 Control Ratio of p-STAT5/STAT5 B 20 15 30 60 Nobiletin Preadipocytes Control Nobiletin 15 10 5 0 0 5 15 30 60 Time after induction (min) Fig. 7. Effect of nobletin on the phosphorylation of STAT5 in 3T3-L1 cells. (A) A representative image from two independent experiments. (B) The scanned bar graph shows the fold induction of phosphorylated STAT5 over STAT5. 3T3-L1 cells were induced to differentiate with 10% FBS-DMEM medium containing insulin, DEX, and IBMX and treated with 100 μM of nobiletin. Control cells were treated with DMSO instead of nobiletin. Cell lysates were prepared at indicated time after the differentiation induction, and Western blotting was performed. p-STAT5 represents the phosphorylated STAT5. Results were expressed as the ratio of p-STAT5/STAT5 in each cell sample relative to that in preadipocytes. Data were represented as the mean ± SD (n = 2). 3 2 1 0 Nobiletin IBMX – – – + + – + + Fig. 8. Effect of nobletin and IBMX on the phosphorylation of STAT5 in 3T3-L1 cells. (A) A representative image from two independent experiments. (B) The scanned bar graph shows the fold induction of phosphorylated STAT5 over STAT5. 3T3-L1 cells were induced to differentiate with 10% FBS-DMEM medium with insulin only or with insulin and IBMX, and treated with or without 100 μM nobiletin. Control cells were induced with insulin only and treated with DMSO instead of nobiletin. Cell lysates were prepared 15 min after the differentiation induction, and Western blotting was performed. p-STAT5 represents the phosphorylated STAT5. Results were expressed as the ratio of p-STAT5/STAT5 in each cell sample relative to that in the control. Data were represented as the mean ± SD (n = 2). 4. Discussion In this study, we found that nobiletin suppressed the lipid accumulation in both of 3T3-L1 cells and MEFs. The suppressive effect of nobiletin on MEFs was milder compared with that on 3T3-L1 cells (Fig. 1B, D). The absorbance determined for the control MEFs in the oil red O staining experiment was lower than that determined for 3T3-L1 cells, implying that the induction for adipocyte differentiation with insulin, IBMX, and DEX might be insufficient for MEFs or might take longer time than for 3T3-L1 cells. PPARγ is a member of the nuclear receptor superfamily, and is the master regulator of the adipogenesis. In the present study, nobiletin was shown to suppress PPARγ2 expression at both protein and mRNA levels 3T3-L1 cells (Figs. 3C and 4A). It is possible that the suppressive effect of nobiletin on adipocyte differentiation may be due to the down-regulation of PPARγ2 expression. This latter speculation was supported by the observation of the decreased expression of aP2, a target of PPARγ (Fig. 4B). C/EBPβ is known to induce PPARγ expression during adipogenic differentiation [23]. In this study, it was demonstrated that the expression of C/EBPβ gene in 3T3-L1 cells was suppressed by treatment with nobiletin (Fig. 4C). This finding implies that the decreased PPARγ2 expression was caused by the down-regulation of C/EBPβ expression. We also examined the effect of nobiletin on the phosphorylation of CREB in 3T3-L1 cells. Results showed that the phosphorylation of CREB was decreased by treatment with nobiletin 60 min after the induction (Fig. 6A). Because it is well known that phosphorylated CREB regulates C/EBPβ expression [26], down-regulation of the expression of C/EBPβ might be due to the decreased phosphorylation of CREB. On the other hand, it has been reported that nobiletin stimulates the insulin signaling to induce the differentiation of 3T3-L1 preadipocytes into adipocytes [17] and that the insulin signaling also participates in the phosphorylation of CREB [24,25]. Studies have also demonstrated that IBMX may accelerate the phosphorylation of CREB by activating the cAMPdependent PKA signaling pathway [27]. Moreover, nobiletin has been K. Kanda et al. / Biochimica et Biophysica Acta 1820 (2012) 461–468 reported to enhance the activation of PKA [28]. Based on these findings, it is possible that the suppressive effect of nobiletin on the phosphorylation of CREB may be limited. Treatment of 3T3-L1 preadipocytes with nobiletin suppressed adipocyte differentiation when the cells were induced with insulin plus IBMX. The differentiation induced with insulin alone or with insulin and DEX, however, was not inhibited (Fig. 5), suggesting that nobiletin affects the signaling pathways mediated by IBMX. When 3T3-L1 preadipocytes were induced with insulin, DEX, and instead of IBMX, forskolin, an alternative cAMP elevator, PPARγ2 expression was suppressed by nobiletin as well (data not shown), suggesting that the suppressive effect of nobiletin on PPARγ2 expression may involve the cAMP-dependent signaling pathways. Matsuzaki et al. [28] previously reported that nobiletin activates PKA, indicating that the mode of action of nobiletin in adipocyte differentiation resembles that of a cAMP elevator. It was observed that the adipocyte differentiation was enhanced by nobiletin when 3T3-L1 preadipocytes were induced with insulin alone (Fig. 5), implying that, like IBMX, nobiletin facilitates adipocyte differentiation by stimulating the cAMP-dependent signaling pathways. Saito et al. [17] also reported that nobiletin enhanced adipocyte differentiation of 3T3-L1 cells when the cells were induced with insulin alone. As mentioned earlier, cAMP activates PKA, and the resulting phosphorylated PKA activates CREB. If nobiletin indeed accelerates the cAMP/PKA/CREB pathway, adipocyte differentiation should be enhanced. In our study, however, nobiletin depressed adipocyte differentiation when preadipocytes were induced with insulin and a cAMP elevator (IBMX or forskolin). Therefore, it assumes speculated that another key player in the cAMP-dependent adipocyte differentiation may be involved in the suppression of PPARγ2 expression by nobiletin. STAT5 is activated by the growth hormone-mediated signaling and has been shown to participate in the early phase of the adipogenesis by regulating the PPARγ activity [10,11]. STAT5 is activated in multiple pathways, including a cAMP-activated PKA pathway [29]. However, excessive activation of STAT5 may inhibit the adipogenesis through down-regulating PPARγ expression [12]. Therefore, it is possible that STAT5 may be excessively activated by PKA, which is activated by nobiletin and by IBMX-induced cAMP accumulation. Therefore, we decided to examine the effect of nobiletin on the phosphorylation of STAT5 in adipocyte differentiation of 3T3-L1 cells. As expected, the phosphorylation of STAT5 was extensively stimulated, compared with the control (Fig. 7A, B). In addition, the extent of phosphorylation of STAT5 was highest when both nobiletin and IBMX were both used (Fig. 8A, B), indicating that the suppressive effect of nobiletin on PPARγ2 expression in adipocyte differentiation is caused by an excessive activation of STAT5, which controls the PPARγ expression. It can therefore be proposed that STATs regulate signaling pathways in both positive and negative manners to maintain homeostasis [30]. Excessive activation of STAT5 may exert a feedback effect on the signaling system to suppress the adipogenesis through decreased PPARγ2 gene expression. The suppressive effect of nobiletin was partially decreased when H-89, a PKA inhibitor, was co-administered (data not shown), supporting the hypothesis. 5. Conclusion We demonstrated that nobiletin suppresses adipocyte differentiation in 3T3-L1 preadipocytes and in MEFs when these cells were induced with a general inducer cocktail containing insulin, DEX, and IBMX. The results are consistent with a recent report showing that nobiletin decreased the intracellular triglyceride accumulation in 3T3-L1 cells [19]. Nobiletin exerted a suppressive effect within the first 2 days after induction of differentiation through the down-regulation of PPARγ2 expression. The suppressive effect of nobiletin on PPARγ2 expression appeared to be due to decreased phosphorylation of CREB 467 and excessive activation of STAT5 through the cAMP-dependent PKA signaling induced by both IBMX and nobiletin. 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