ARTICLE pubs.acs.org/JAFC
Nobiletin Suppresses Adipogenesis by Regulating the Expression of Adipogenic Transcription Factors and the Activation of AMP-Activated Protein Kinase (AMPK) Youngmin Choi,† Younghwa Kim,† Hyeonmi Ham,† Yooheon Park,‡ Heon-Sang Jeong,† and Junsoo Lee*,† †
Department of Food Science and Technology, College of Agriculture, Chungbuk National University, Cheongju 361-763, Republic of Korea ‡ Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States ABSTRACT: The objective of this study was to elucidate the effect of nobiletin (5,6,7,8,30 ,40 -hexamethoxyflavone) on adipogenesis in 3T3-L1 cells. To determine the effect of nobiletin on adipogenesis, preadipocyte differentiation was induced in the presence or absence of nobiletin (10100 μM) for 4 days. The results revealed that nobiletin markedly inhibited lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity and blocked the expression of adipogenic transcription factors, including peroxisome proliferator-activated receptors (PPARγ) and CCAAT/enhancer binding proteins (C/EBPα). Moreover, nobiletin significantly increased AMP-activated protein kinase (AMPK), a major regulator of cellular energy balance, phosphorylation, and intracellular reactive oxygen species (ROS) generation. This study also investigated the involvement of AMPK in the expression of a major transcription factor, PPARγ. It was found that pretreatment with compound C, a cell permeable inhibitor of AMPK, abolished the inhibitory effects of nobiletin on PPARγ expression. The results suggest that nobiletin exerts antiadipogenic effects through modulation of the PPARγ and AMPK signaling pathway and, therefore, may be a promising antiobesity agent. KEYWORDS: nobiletin, PPARγ, AMPK, intracellular ROS, 3T3-L1
’ INTRODUCTION Obesity is one of the most serious health problems in both Westernized and developing countries. The prevalence of obesity is closely correlated with those of type 2 diabetes, cardiovascular disease, hypertension, and cancer, which are important causes of morbidity and mortality.1 Obesity is caused not only by hypertrophy of adipose tissue but also by adipose tissue hyperplasia, which triggers the differentiation of preadipocytes into adipocytes.2 Although adipocyte differentiation is a very complex process, many studies have demonstrated that the inhibition of adipogenesis and modulation of adipocyte function might be potential therapeutic approaches for the amelioration of obesity.3,4 A well-established in vitro model to study adipogenesis and adipocyte differentiation is the immortal 3T3-L1 cell line. Under controlled conditions, cells of this type can differentiate into cell types ranging from preadipocytes to mature adipocytes. During adipocyte differentiation, multiple transcription factors, such as CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs), are involved in regulating the expression of many adipogenic proteins, including glucose transport-4, lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (aP2).57 Therefore, over the past two decades, a number of studies have been conducted to identify new phytochemicals that can suppress adipocyte differentiation and down-regulate the expression of PPARγ and C/EBPα in 3T3-L1 cells.4,8 In recent years, AMP-activated protein kinase (AMPK) has emerged as a major regulator of appetite, body weight, and cellular energy balance.9 Previous studies have shown that activation of AMPK in adipocytes leads to decreased fatty acid uptake, decreased triglyceride accumulation, and increased r 2011 American Chemical Society
fatty acid oxidation via inactivation of acetyl-CoA carboxylase (ACC) and suppression of lipogenic genes.10 Moreover, AMPK also regulates PPARγ and C/EBPα, which are critical regulators of adipogenesis and fat accumulation in adipocytes.11 Therefore, it is natural to consider AMPK as a target for the treatment of obesity and its related metabolic disorders.12 Because of the adverse effects of antiobesity medications, a number of studies have been performed to investigate the antiobesity effects of plant-derived components such as quercetin, resveratrol, rutin, and catechin.4,13 Citrus fruit-derived flavonoids and their metabolites have been shown to exert diverse protective biological actions. Nobiletin (5,6,7,8,30 ,40 -hexamethoxyflavone) is abundantly present in the fruit peel of Citrus and has several biological activities. It has been reported to exhibit various biological effects on mouse leukemia cells14 and human cancer cells15 and to suppress lipopolysaccharide-induced nitric oxide production in murine macrophages.16 According to recent studies, nobiletin improves hyperglycemia and insulin resistance in obese diabetic ob/ob mice17 and decreases the secretion of an insulin resistance factor, resistin, in 3T3-L1 adipocytes.18 However, not much is known about the antiadipogenic effects of nobiletin in 3T3-L1 cells. The objective of this study was to evaluate the effect of nobiletin on adipogenesis in 3T3-L1 cells. We also investigated the influence of AMPK cascades on the antiobesity activity of nobiletin.
Received: August 19, 2011 Revised: October 21, 2011 Accepted: November 16, 2011 Published: November 16, 2011 12843
dx.doi.org/10.1021/jf2033208 | J. Agric. Food Chem. 2011, 59, 12843–12849
Journal of Agricultural and Food Chemistry
ARTICLE
Figure 1. Effect of nobiletin on lipid accumulation and GPDH activity during adipocyte differentiation. (A) Cells were treated with different concentrations of nobiletin during days 04 in the differentiation medium and then changed to 10% FBSDMEM without nobiletin for an additional 2days. On day 6, the number of live cells was determined by MTT assay. (B) Cells were treated with nobiletin during days 04 in the differentiation medium; subsequently, the cell culture medium was replaced with 10% FBSDMEM without nobiletin, and the cells were maintained for another 2 days. On day 6, cells were stained with Oil Red O. (C) GPDH activity was measured as described under Materials and Methods. Results are the mean ( SEM of three independent experiments. / indicates a statistically significant difference when compared to the differentiated control group (p < 0.05).
’ MATERIALS AND METHODS Reagents. Nobiletin (5,6,7,8,3 0 ,40 -hexamethoxyflavone) and compound C (6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4ylpyrrazolo[1,5-α]pyrimidine) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Sigma-Aldrich (St. Louis, MO), respectively. Cell culture reagents, including Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), fetal bovine serum (FBS), and penicillin/streptomycin, were purchased from Invitrogen (Carlsbad, CA). Insulin, 3-isobutyl-1methylxanthine (IBMX), and dexamethasone were supplied by Sigma-Aldrich. Primary antibodies against PPARγ and C/EBPα were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and phospho-AMPKα (Thr172), AMPKα, and aP2 antibodies were obtained from Cell Signaling Technology (Danvers, MA). Anti-βactin antibody was obtained from Sigma-Aldrich. Cell Culture and Adipocyte Differentiation. The 3T3-L1 preadipocyte cell line was obtained from American Type Culture Collection (Manassas, VA) and maintained in DMEM containing
10% FCS, 100 units/mL penicillin, and 50 μg/mL streptomycin at 37 °C under a 5% CO2 atmosphere. To induce differentiation of preadipocytes into mature adipocytes, postconfluent cells (designated day 0) were cultured in a differentiation medium containing 10% FBS, 167 nM insulin, 0.5 mM IBMX, and 1 μM dexamethasone. The cell culture medium was replaced with 10% FBSDMEM containing only 167 nM insulin after 48 h (day 2), and the maturing adipocytes were switched to 10% FBSDMEM for an additional 2 days. On day 6, mature adipocytes were subjected to Oil Red O staining and glycerol3-phosphate dehydrogenase (GPDH) assay and harvested for immunoblot analysis. To determine the effect of nobiletin on adipogenesis, differentiation of postconfluent preadipocytes was induced in the presence or absence of nobiletin (10100 μM) for 4 days. A stock solution of nobiletin was prepared in methanol at a concentration of 20 mM. Cell Viability Assay. MTT assay was employed to determine the effect of nobiletin on the viability of 3T3-L1 adipocyte cells. Cells (1 104 per 48 wells) were incubated in the presence or absence of nobiletin (10, 25, 50, and 100 μM) in the differentiation medium for 4 days and 12844
dx.doi.org/10.1021/jf2033208 |J. Agric. Food Chem. 2011, 59, 12843–12849
Journal of Agricultural and Food Chemistry
ARTICLE
Figure 2. Effect of nobiletin on adipocyte-specific protein expression. Cells were treated with nobiletin during days 04 in the differentiation medium; subsequently, the cell culture medium was replaced with 10% FBSDMEM without nobiletin, and the cells were maintained for another 2 days. On day 6, cells were harvested. Protein levels of PPARγ (A), C/EBPα (B), and aP2 (C) were determined by Western blot analysis and expressed as percent of control. Results are the mean ( SEM of three independent experiments. / indicates a statistically significant difference when compared to the differentiated control group (p < 0.05). then changed to 10% FBSDMEM for an additional 2 days without nobiletin. On day 6, 20 μL of MTT reagent (5 mg/mL) was added, and the cells were incubated for an additional 4 h at 37 °C. The violet formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was quantified using a microplate reader (BioTek, Inc., Winooski, VT) at 550 nm. The cell viability (%) was obtained by comparing the absorbances of the samples and a control. Oil Red O Staining. To monitor the intracellular lipid accumulation, Oil Red O staining was performed on day 6. In brief, cells were
washed twice with phosphate-buffered saline (PBS), fixed in 10% formalin for at least 15 min at room temperature, and then washed with PBS twice. The lipid droplets in cells were stained with 0.3% Oil Red O solution at room temperature and washed exhaustively with distilled water. Stained cells were photographed using a phasecontrast Olympus CKX41 microscope (Tokyo, Japan) in combination with a digital camera at 100 magnification. The stained lipid droplets were dissolved in isopropanol and quantified at 490 nm by using a microplate reader. 12845
dx.doi.org/10.1021/jf2033208 |J. Agric. Food Chem. 2011, 59, 12843–12849
Journal of Agricultural and Food Chemistry
ARTICLE
Glycerol-3-phosphate Dehydrogenase (GPDH) Assay. Nobiletin-treated 3T3-L1 adipocytes were harvested on day 6, washed twice carefully with cold PBS, and then collected in cold buffer containing 0.25 M sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM Tris-base, and 1 mM dithiothreitol at pH 7.4. Harvested cells were lysed for 10 s by using a Vibra-Cell VCX 750 sonicator (Sonics & Materials, Inc., Newtown, CT). Lysates were centrifuged at 10000g for 10 min at 4 °C, and the supernatants were immediately used for the protein and GPDH activity assays. GPDH activity was determined according to the procedure of Wise and Green.19 The protein concentration of each sample was determined using the BCA protein assay reagent (Pierce, Rockford, IL). Enzyme activity was expressed as units per milligram of protein. Western Blot Analysis. On day 6, 3T3-L1 adipocytes were immediately washed with PBS buffer and lysed using a PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, South Korea). The protein concentration of each sample was determined using BCA protein assay reagent. Samples were resolved in 812% SDSpolyacrylamide gels, followed by electrophoretic transfer to a nitrocellulose membrane. Primary antibodies against PPARγ, C/EBPα, phosphoAMPKα, AMPKα, aP2, and β-actin were applied overnight at 4 °C at a 1:1000 dilution. After incubating with the secondary antibody at room temperature for 2 h, the proteins were detected using the ECL Western blotting analysis system (Pierce). Determination of Reactive Oxygen Species (ROS). ROS were quantified with a fluorescent probe, 20 ,70 -dichlorofluorescin diacetate (DCFH-DA), as previously described.20 Differentiating 3T3-L1 adipocytes were treated with nobiletin (10, 25, 50, and 100 μM) in differentiation medium for 4 days. Then, the cells were washed with PBS, and DCFH-DA (20 μM) in DMEM was added to the wells. After 3 h, the cells were washed with PBS, and the fluorescence intensity, corresponding to intracellular ROS concentration, was measured using a fluorescent spectrophotometer (Perkin-Elmer, Norwalk, CT) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Statistical Analysis. Results are reported as the mean ( SEM. Significant differences among treatment means were determined using a one-way analysis of variance (ANOVA) followed by Tukey’s test using SAS version 9.1 (SAS Institute, Cary, NC). A p value of
