Article pubs.acs.org/JAFC
Fisetin Up-regulates the Expression of Adiponectin in 3T3-L1 Adipocytes via the Activation of Silent Mating Type Information Regulation 2 Homologue 1 (SIRT1)-Deacetylase and Peroxisome Proliferator-Activated Receptors (PPARs) Taewon Jin,†,‡ Oh Yoen Kim,§ Min-Jeong Shin,⊥ Eun Young Choi,†,‡ Sung Sook Lee,†,‡ Ye Sun Han,⊗ and Ji Hyung Chung*,† †
Department of Applied Bioscience, College of Life Science, CHA University, Gyeonggi-do 463-400, Republic of Korea Graduate Program in Science for Aging, Yonsei University, Seoul 120-749, Republic of Korea § Department of Food Science and Nutrition, Dong-A University, Busan 604-714, Republic of Korea ⊥ Department of Food and Nutrition, Korea University, Seoul 136-704, Republic of Korea ⊗ Department of Advanced Technology Fusion, Konkuk University, Seoul 143-701, Korea ‡
ABSTRACT: Adiponectin, an adipokine, has been described as showing physiological benefits against obesity-related malfunctions and vascular dysfunction. Several natural compounds that promote the expression and secretion of adipokines in adipocytes could be useful for treating metabolic disorders. This study investigated the effect of fisetin, a dietary flavonoid, on the regulation of adiponectin in adipocytes using 3T3-L1 preadipocytes. The expression and secretion of adiponectin increased in 3T3-L1 cells upon treatment with fisetin in a dose-dependent manner. Fisetin-induced adiponectin secretion was inhibited by peroxisome proliferator-activated receptor (PPAR) antagonists. It was also revealed that fisetin increased the activities of PPARs and silent mating type information regulation 2 homologue 1 (SIRT1) in a dose-dependent manner. Furthermore, the upregulation of adiponectin and the activation of PPARs induced by fisetin were prevented by a SIRT1 inhibitor. Fisetin also promoted deacetylation of PPAR γ coactivator 1 (PGC-1) and its interaction with PPARs. SIRT knockdown by siRNA significantly decreased both adiponectin production and PPARs−PGC-1 interaction. These results provide evidence that fisetin promotes the gene expression of adiponectin through the activation of SIRT1 and PPARs in adipocytes. KEYWORDS: fisetin, adiponectin, SIRT1, PPAR, adipocytes
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INTRODUCTION Obesity is a worldwide public health problem closely associated with other chronic diseases or symptoms, such as dyslipidemia, metabolic syndrome, type 2 diabetes mellitus, atherosclerosis, and cardiovascular diseases (CVDs).1,2 It is characterized by increases in the number and size of adipocytes, which are regulated by metabolic, genetic, and nutritional factors.3−5 Adipose tissue is now recognized not only as a storage organ of excess energy in the body but also a major endocrine tissue secreting a variety of hormones and adipokines into the blood.2−4 Many studies have focused on the roles and mechanisms of adipokines regarding obesity and related diseases.3−5 Adipokines are a group of endocrine molecules specifically expressed in the adipocytes. They participate in various functions and regulations in the body system, that is, energy metabolism, insulin sensitivity, and inflammation.3−5 Among the adipokines, adiponectin is well-known for its antiobesity, antidiabetic, and antiatherosclerotic roles.6−9 It was reported that adiponectin transcription is regulated by several transcription factors including peroxisome proliferator-activated receptors (PPARs) and CCAAT/enhancer-binding proteins (C/EBPs), which are involved in antiadipogenesis and adipokine expression.10 The major work of adiponectin is known to increase the activity of AMP-activated protein kinase (AMPK), which regulates energy © 2014 American Chemical Society
expenditure, restores insulin sensitivity, and reduces inflammation response.11 Recently, many studies have focused on revealing candidate molecules that regulate the expression and secretion of adipokines.12−15 Among the candidates, flavonoids abundant in plant foods have attracted public attention due to their multiple biological functions and health beneficial effects, which include enzyme modulation, gene transcription, antioxidative reaction, reduction of obesity and CVDs, and restoration of insulin sensitivity.15−17 Among the flavonoids, fisetin recently became an object of attention for its antioxidative, antiinflammatory, anticancer, and antimetastasis properties.17 It is a yellow flavonol present in vegetables and fruits (mainly in strawberries, persimmons, onions, and mangoes), a flavone, its structure being derived from 3-hydroxy-2-phenylchromen-4one.17 It is also, due to its structural characteristics, predicted as a calorie restriction mimetic compound, which may act in a broad range related to silent mating type information regulation 2 homologue 1 (SIRT1) regulation.18 In addition, very recently, Jung et al. reported that fisetin suppressed p70 ribosomal S6 Received: Revised: Accepted: Published: 10468
June 16, 2014 September 21, 2014 October 6, 2014 October 6, 2014 dx.doi.org/10.1021/jf502849j | J. Agric. Food Chem. 2014, 62, 10468−10474
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Figure 1. Morphology and oil red O staining of differentiated 3T3-L1 cells: (A) morphology of 3T3-L1 preadipocytes before differentiation; (B) morphology of differentiated 3T3-L1 cells at 10 day. (C) On 10 day after differentiation, the cells were stained with oil red O and viewed under a microscope. photographed by using an Olympus CKX41 microscope (Olympus, Japan). Immunoblot Analysis. Fisetin-treated differentiated cells were harvested and washed twice in ice-cold PBS. Samples were placed in ice-cold lysis buffer containing 40 mM HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 1% Triton, and EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) for 30 min. Cell lysates were clarified by centrifugation (13000 rpm for 30 min). Protein concentrations were determined using a Bicinchoninic Acid Protein Assay Kit (Sigma-Aldrich). Proteins were separated in 8% or 12.5% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was blocked in 5% nonfat dry milk dissolved in Tris-buffered saline (TBS) for 1 h. After washing with TBS with 0.05% Tween-20, the membrane was incubated with primary antibodies and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The bands were detected using the enhanced chemiluminescence (ECL) reagent (Santa Cruz Biotechnology). Immunoprecipitation. After cell lysis, cell lysates were clarified by centrifugation. Cell extract (200−500 μg) was precleared with protein A/G agarose (Santa Cruz Biotechnology) on a rotating platform plate at 4 °C for 1 h. Then sample was incubated with antibody on a rotation platform plate at 4 °C for 2 h. After a washing with PBS, the sample was subjected to SDS-PAGE and immunoblot analysis. SIRT1 Activity Assay. SIRT1 activity assay proceeded with a Deacetylase Fluorometric Activity Assay Kit (Enzo Life Sciences, Farmingdale, NY, USA), according to the manufacturer’s instructions. Briefly, differentiated 3T3-L1 cells were treated with different concentrations of fisetin. Cells were extracted as described above, and then the protein concentration in the cell lysate was quantified with deacetylase buffer. Reactions with cell lysate were performed in the presence of 200 μM Fluor-de-Lys substrate. Then Fluor-de-Lys developer was treated for 30 min. Finally, the fluorescence was read using a fluorometric reader (Victor 3 Multilabel Plate Readers, PerkinElmer, Waltham, MA, USA) with excitation of 360 nm and emission at 460 nm. Negative controls were also included for normalization. RT-PCR Analysis. Cells were harvested, and total RNA was isolated using QIAzol-Regent (Qiagen, Germany). An equal quantity of total RNA was reverse transcribed using Omniscript Reverse Transcriptase (Qiagen) and Oligo-dT primer. The cDNA was amplified using Taq DNA polymerase (Cosmo Genetech, Korea) and primer sets. The primer sequences were as follows: adiponectin primers, 5′-TGGAGAGAAGGGAGAGAAAGG-3′ and 5′-TGGTCGTAGGTGA AGAGAACG-3′; GAPDH primers, 5′-CATGGCCTTCCGTGTTCCTAT-3′ and 5′-CCTGCTTCACCACCTTCTTGA-3′. PCR products were separated by electrophoresis in agarose gel containing ethidium bromide. PPAR Activity Assay. A PPAR Transcription Factor Assay Kit (Cayman) was used for the determination of PPARα and PPARγ DNA binding and transcription activity. Briefly, cells were harvested and placed in hypotonic buffer on ice for 15 min. Nonidet P-40 (10%) was added and gently mixed. The turbid lysate were clarified by centrifugation at 4 °C. The supernatant was transferred to a new tube as cytosolic fraction. The pellet was also resuspended with ice-
kinase 1 (S6K1) and the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in adipose tissue, suggesting that this compound regulates diet-induced obesity.19 However, there has been no study on the effect of fisetin on the regulation of adipokines, particularly adiponectin, in terms of obesity and adiposity. Therefore, this study aimed to investigate the effect of fisetin on the regulation of adiponectin expression in association with other related regulators such as SIRT1 and PPARs using 3T3-L1 preadipocyte cells, which finally differentiate to mature adipocytes.
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MATERIALS AND METHODS
Materials. Fisetin, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), insulin, oil red O, and sirtinol were obtained from SigmaAldrich (St. Louis, MO, USA). PPARα antagonist GW6471 was purchased from Tocris (Ellisville, MO, USA). PPARγ antagonist T0070907 was purchased from Cayman (Ann Arbor, MI, USA). Antiadiponectin, anti-PPARα, anti-PPARγ, anti-PGC-1, anti-acetyl-lysine, anti-AMPK, anti-phospho-AMPK (T172), and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SIRT1 siRNA and scrambled control siRNA-A were also purchased from Santa Cruz Biotechnology. Anti-rabbit IgG light chain was purchased from Aviva Systems Biology (San Diego, CA, USA). A mouse adiponectin Quantikine ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). Cell Culture and Differentiation. 3T3-L1 mouse preadipocytes were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). Preadipocytes were maintained under Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum (BCS) in a 37 °C humidified atmosphere containing 5% CO2. Passage numbers of cells used were between 10 and 20. Two days after full confluence, cells were induced to differentiate by exposure to 1 μM dexamethasone, 0.5 mM IBMX, and 10 μg/mL insulin in DMEM with 10% FBS for 72 h. Cells were replaced with DMEM supplemented with 10% FBS and 10 μg/mL insulin. Another 2 days later, the medium was changed to DMEM with 10% FBS. Thereafter, cells were maintained by exchanging media every 2 days with DMEM with 10% FBS until used in the experiments 10−14 days after initiation of the differentiation protocol. The cells were used for experiments whenever 80−90% of the cells exhibited the adipocyte phenotype by accumulation of lipid droplets. Oil Red O Staining. Cell medium was discarded from fully differentiated 3T3-L1 adipocytes. Cells were prefixed with 10% formalin for 5 min at room temperature. After prefix, formalin was discarded and the same volume of fresh formalin was added. Cells were incubated at room temperature for at least 1 h or more. The parafilm was wrapped over the plate to prevent drying. After fixing of the cells, formalin was discarded, and the cells were washed with 60% isopropanol. After cells were completely dried, oil red O staining working solution (Sigma-Aldrich) filtered at 0.2 μm pore size was added, and the cells were stained for 10−20 min. After staining, oil red O solution was removed and dH2O was added immediately. Cells were washed with distilled water four times. Cells were observed and 10469
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cold complete nuclear extraction buffer and rocked vigorously for 45 min. The lysate was clarified by centrifugation at 4 °C, and the supernatant was collected as nuclear fraction. The nuclear fractions were added to consensus dsDNA-coated 96-wells as a quantitatively equal amount. After primary antibody (anti-PPARα antibody or antiPPARγ antibody) incubation, horseradish peroxidase-conjugated secondary antibody was added, and the optical density of each well was measured using a Multiskan plate reader (Thermo Scientific, Waltham, MA, USA) with 450 nm wavelength. Small Interfering RNA (siRNA). For SIRT1 siRNA transfections, 3T3-L1 adipocytes, 2−3 days post differentiation, were transfected with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. Following 24 h of transfection with 40 nM siRNA, the protein level of SIRT1 was analyzed with anti-SIRT1 antibody. Statistical Analysis. Data are expressed as means ± SE. Statistical comparisons were performed by using one-way analysis of variance and Student’s t test. Differences were considered significant at p < 0.05.
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RESULTS Fisetin Up-regulates Adiponectin Expression in Differentiated 3T3-L1 Cells. Mouse 3T3-L1 preadipocytes
Figure 3. Effect of PPARs on fisetin-induced adiponectin upregulation. (A) Differentiated 3T3-L1 cells were pretreated with PPAR-γ antagonist (20 μM T0070907) or PPAR-α antagonist (20 μM GW6471) for 1 h and then treated with 1 μM fisetin for 24 h. Adiponectin levels released from cells were determined using adiponectin ELISA kit. (B) Differentiated 3T3-L1 cells were treated with the indicated concentration of fisetin for 24 h. PPARs activity was determined in nuclear extracts using a PPAR transcription factor assay kit. (∗) p < 0.05 by Student’s t test.
concentration from 1 nM to 10 μM for 24 h. The culture media were collected and subjected to ELISA. Adiponectin was secreted from 3T3-L1 adipocyte cells in response to fisetin treatment in a concentration-dependent manner (Figure 2A). The adiponectin expression levels in fisetin-treated 3T3-L1 cells were also determined by immunoblot analysis. As shown in Figure 2B, protein expression levels of adiponectin were upregulated upon the addition of fisetin in a similar pattern to adiponectin secretion. The mRNA levels of adiponectin were also up-regulated upon treatment with fisetin (Figure 2C). Fisetin Activates PPARs, Which Up-regulates Adiponectin Expression. To assess the effects of PPARs on the adiponectin production induced by fisetin treatment in 3T3-L1 cells, we used two selective antagonists, T0070907, a PPAR-γ antagonist, and GW6471, a PPAR-α antagonist. Differentiated 3T3-L1 cells were pretreated with T0070907 or GW6471 for 1 h and then treated with 1 μM fisetin for 24 h. As shown in Figure 3A, fisetin-induced adiponectin production was significantly inhibited by both antagonists. In addition, fisetin induced the activation of the PPARs in a concentrationdependent manner, reaching a maximum at 1 μM (Figure 3B). Fisetin Induces SIRT1 Activation. To determine whether SIRT1 is involved in fisetin-induced adiponectin production,
Figure 2. Effect of fisetin on adiponectin expression and secretion in 3T3-L1 adipocytes. Differentiated 3T3-L1 cells were treated with the indicated concentration of fisetin for 24 h. (A) Adiponectin levels released from 3T3-L1 cells were determined using adiponectin ELISA kit. (B) Protein levels of adiponectin in 3T3-L1 cell lysate were analyzed by immunoblot assay using anti-adiponectin antibody. (C) Total RNA was purified from 3T3-L1 cells and subjected to RT-PCR analyses using primers specific for adiponectin. (∗) p < 0.05 by Student’s t test.
were differentiated to mature adipocytes as described under Materials and Methods. Cell morphology showed that differentiated 3T3-L1 cells had many lipid droplets indicating lipid accumulation, whereas these were not observed in undifferentiated preadipocytes (Figure 1A,B). The oil red O staining was executed to confirm differentiation and lipid droplet formation with red pigments (Figure 1C). Differentiated 3T3-L1 cells were exposed to fisetin with range of 10470
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Figure 4. Effect of SIRT1 on fisetin-induced adiponectin up-regulation. Differentiated 3T3-L1 cells were pretreated with SIRT1 inhibitor (60 μM sirtinol) for 1 h and then treated with 1 μM fisetin for 24 h. (A) Adiponectin levels released from 3T3-L1 cells were determined using adiponectin ELISA kit. (B) Total RNA was purified from 3T3-L1 cells and subjected to RT-PCR analyses using primers specific for adiponectin. (C) PPAR-γ and PPAR-α activities were determined in nuclear extracts using PPAR transcription factor assay kit. (D) Differentiated 3T3-L1 cells were treated with the indicated concentration of fisetin for 24 h. SIRT1 activity was determined with a deacetylase fluorometric activity assay kit. (∗) p < 0.05 by Student’s t test.
we examined the effect of treatment with a SIRT1 inhibitor, sirtinol, on fisetin-induced adiponectin expression and secretion in 3T3-L1 cells. Fisetin-induced adiponectin release was significantly suppressed by treatment with sirtinol (Figure 4A). RT-PCR data also showed that inhibition of SIRT1 leads to a significant decrease in expression level of adiponectin induced by fisetin (Figure 4B). Moreover, the SIRT1 inhibitor suppressed fisetin-induced PPAR activation (Figure 4C). To check whether fisetin affected SIRT1 activity, we measured the intracellular SIRT1 activity in fisetin-treated adipocytes. It was found that fisetin increased the SIRT1 activity in a concentration-dependent manner (Figure 4D). Fisetin Promotes PPARs−PGC-1 Interaction. We examined whether fisetin treatment alters the acetylation level of PGC-1 and the interaction between PPARs and PGC-1 in 3T3-L1 cells. Fisetin-treated cell lysates were immunoprecipitated with anti-acetyl lysine antibody and immunoblotted with anti-PGC-1 antibody. As shown in Figure 5A, fisetin reduced the acetylation level of PGC-1. Also, an immunoprecipitation study using anti-PPAR antibodies showed that the interaction of PPARs and PGC-1 was significantly elevated in fisetintreated cells (Figure 5B,C). Effects of SIRT1 Knock-Down on PPARs and PGC-1 in Fisetin-Treated Adipocytes. To confirm the molecular mechanisms of SIRT1-mediated adiponectin expression in fisetin-treated adipocytes, we used siRNA to silence SIRT1 gene expression. The SIRT1 protein level was significantly decreased in the cells transfected with SIRT siRNA compared
Figure 5. Interaction between PPARs and PGC-1. Differentiated 3T3L1 cells were treated with 1 μM fisetin for 24 h. Cell lysate was immunoprecipitated with antiacetyl-lysine antibody (A), anti-PPAR-α antibody (B), or anti-PPAR-γ antibody (C). Then immunoprecipitant samples were analyzed by immunoblot assay with anti-PGC-1 antibody.
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fisetin promoted the deacetylation of PGC-1 and its interaction with PPARs. We also confirmed an important role of SIRT1mediated deacetylation in regulating the PPARs−PGC-1 interaction and adiponectin production induced by fisetin. This is the first study showing the beneficial effect of fisetin on the adipokine regulation in adipocytes, thereby being considered to contribute to the modulation of the pathogenesis of obesity-linked metabolic disorders and vascular diseases. Previous studies demonstrated that several polyphenols and bioactive natural products promoted the expression and secretion of adiponectin in adipocytes.12−14 According to Yen et al., polyphenolic compounds (i.e., resveratrol, quercetin, and p-coumaric acid) increased the levels of secreted adiponectin and antioxidant enzymes and inhibited the production of cytokines and intracellular reactive oxygen species in the tumor necrosis factor receptor-stimulated 3T3-L1 adipocytes.12 In this study, we also found that fisetin, one of flavonoids, increased adiponectin gene expression and its secretion in 3T3-L1 cells. Fisetin is a naturally occurring flavonoid, commonly found in strawberries and other fruits, that belongs to the flavonoid groups of plant polyphenols.17−20 Fisetin was reported to exert a wide range of biological activities such as anti-inflammatory,21 antioxidant,22 and anticancer activities.23 Also, Lee and Bae reported an antiadiposity effect of fisetin, which suppressed preadipocyte proliferation at early stages of differentiation, partly mediated by cell cycle arrest during adipogenesis.24 We also tested several fisetin analogues for adiponectin production using ELISA in differentiated 3T3-L1 cells. However, the other compounds including apigenin did not exhibit considerable adiponectin production (data not shown). Adiponectin is the most abundantly secreted adipokine, particularly derived from the adipocytes, which is involved in a variety of pathophysiological processes such as obesity, diabetes, and CVDs and exerts various beneficial effects as an antidiabetic, antiatherogenic, and anti-inflammatory.6−9 Various studies reported that PPARα and PPARγ are important transcription factors involved in adipogenesis and adipokine expression.25−27 The initiation of adiponectin gene promotion was known to require the formation of PPARγ and retinoid X receptor (RXR) or PPARα−RXR complex,28 thereby the complex binding with adiponectin promoter region for the activation. As an essential initiation condition for its activity, PPAR−RXR complexes were reported to require PGC-1 to bind with the complex itself to invoke transcriptional activation, which up-regulates adiponectin gene expression. 28,29 In addition, the deacetylation of SIRT1, a family of sirtuins that are characterized as its deacetylase activity against nonhistone protein, was reported to participate in the regulation of various transcription factors, for example, PGC-1, forkhead box-type O transcription factors, liver X receptor, RXR p53, mammalian target of rapamycin complex 1, and insulin receptor substrate 2.30−34 According to recent studies, SIRT1 binds with PGC-1 and deacetylases PGC-1 to restore its activity to work as a PPAR coactivator, thereby binding with the PPARγ−RXR or PPARα−RXR complex.35−37 In our study, PPARs and SIRT1 were significantly activated by fisetin treatment in 3T3-L1 cells, and fisetin-induced adiponectin secretions were inhibited by PPAR antagonists and SIRT1 inhibitor. Fisetin-induced PPAR activations were also prevented by SIRT1 inhibition and knockdown, and the deacetylation of PGC-1 and its interaction with PPARs were promoted by fisetin. Therefore, our data suggested that fisetin can up-regulate adiponectin expression by increasing SIRT1-
Figure 6. Effects of SIRT1 knock-down on PPARs−PGC-1 interaction and adiponectin production. (A) Differentiated 3T3-L1 cells were transfected with SIRT1 siRNA or control siRNA. After 24 h, SIRT1 levels were analyzed by immunoblot assay using anti-SIRT1 antibody. (B) After 24 h of transfection with SIRT1 siRNA, 3T3-L1 cells were incubated with 1 μM fisetin for 24 h. Cell lysate was immunoprecipitated with antiacetyl-lysine antibody, anti-PPAR-α antibody, or anti-PPAR-γ antibody. Then immunoprecipitant samples were analyzed by immunoblot assay with anti-PGC-1 antibody. (C) Differentiated 3T3-L1 cells transfected with SIRT1 siRNA were treated with 1 μM fisetin for 24 h. Adiponectin levels released from cells were determined using an adiponectin ELISA kit. (D) Expression of adiponectin was determined by RT-PCR analysis. (E) Protein levels of adiponectin in cell lysate were analyzed by immunoblot assay using anti-adiponectin antibody. (∗) p < 0.05 by Student’s t test.
to control siRNA (Figure 6A). We found that SIRT1 knockdown led to a decrease in deacetylation of PGC-1 in fisetin-treated cells (Figure 6B). Knockdown of SIRT1 also largely decreased fisetin-induced interaction of PPARs and PGC-1 (Figure 6B). To check the changes of adiponectin production by SIRT1 knockdown, we measured the secreted adiponectin in the culture medium. Fisetin-stimulated adiponectin production was significantly blocked by SIRT1 knockdown (Figure 6C), and there was a high correlation with mRNA expression and protein level in the cells (Figure 6D,E).
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DISCUSSION This study shows that fisetin, a dietary flavonoid, significantly promoted the expression of adiponectin through the activation of SIRT1-deacetylase and PPARs in adipocytes. In this study, we found increased expression and secretion of adiponectin in 3T3-L1 cells upon treatment with fisetin in a concentrationdependent manner. The activities of PPARs and SIRT1 were also increased by the stimulation of fisetin. In addition, fisetininduced adiponectin secretion was inhibited by PPARs antagonists. The up-regulation of adiponectin and the activation of PPARs by fisetin were also prevented by SIRT1 inhibitor and knockdown with SIRT1 siRNA. Furthermore, 10472
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(6) Yamauchi, T.; Kamon, J.; Waki, H.; Terauchi, Y.; Kubota, N.; Hara, K.; Mori, Y.; Ide, T.; Murakami, K.; Tsuboyama-Kasaoka, N.; Ezaki, O.; Akanuma, Y.; Gavrilova, O.; Vinson, C.; Reitman, M. L.; Kagechika, H.; Shudo, K.; Yoda, M.; Nakano, Y.; Tobe, K.; Nagai, R.; Kimura, S.; Tomita, M.; Froguel, P.; Kadowaki, T. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat. Med. 2001, 7, 941−946. (7) Weyer, C.; Funahashi, T.; Tanaka, S.; Hotta, K.; Matsuzawa, Y.; Pratley, R. E.; Tataranni, P. A. Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J. Clin. Endocrinol. Metab. 2001, 86, 1930−1935. (8) Hotta, K.; Funahashi, T.; Arita, Y.; Takahashi, M.; Matsuda, M.; Okamoto, Y.; Iwahashi, H.; Kuriyama, H.; Ouchi, N.; Maeda, K.; Nishida, M.; Kihara, S.; Sakai, N.; Nakajima, T.; Hasegawa, K.; Muraguchi, M.; Ohmoto, Y.; Nakamura, T.; Yamashita, S.; Hanafusa, T.; Matsuzawa, Y. Plasma concentrations of a novel, adiposespecific protein, adiponectin, in type 2 diabetic patients. Arterioscler. Thromb. Vasc. Biol. 2000, 20, 1595−1599. (9) Iwashima, Y.; Katsuya, T.; Ishikawa, K.; Ouchi, N.; Ohishi, M.; Sugimoto, K.; Fu, Y.; Motone, M.; Yamamoto, K.; Matsuo, A.; Ohashi, K.; Kihara, S.; Funahashi, T.; Rakugi, H.; Matsuzawa, Y.; Ogihara, T. Hypoadiponectinemia is an independent risk factor for hypertension. Hypertension 2004, 43, 1318−1323. (10) Yim, M. J.; Hosokawa, M.; Mizushina, Y.; Yoshida, H.; Saito, Y.; Miyashita, K. Suppressive effects of amarouciaxanthin A on 3T3-L1 adipocyte differentiation through down-regulation of PPARγ and C/ EBPα mRNA Expression. J. Agric. Food Chem. 2011, 59, 1646−1652. (11) Huang, B.; Yuan, H. D.; Kim, D. Y.; Quan, H. Y.; Chung, S. H. Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor-γ (PPARγ) and AMP-activated protein kinase (AMPK) pathways. J. Agric. Food Chem. 2011, 59, 3666−3673. (12) Yen, G. C.; Chen, Y. C.; Chang, W. T.; Hsu, C. L. Effects of polyphenolic compounds on tumor necrosis factor-α (TNF-α)induced changes of adipokines and oxidative stress in 3T3-L1 adipocytes. J. Agric. Food Chem. 2011, 59, 546−551. (13) Kim, O. Y.; Lee, S. M.; Do, H.; Moon, J.; Lee, K. H.; Cha, Y. J.; Shin, M. J. Influence of quercetin-rich onion peel extracts on adipokine expression in the visceral adipose tissue of rats. Phytother. Res. 2012, 26, 432−437. (14) Wang, A.; Liu, M.; Liu, X.; Dong, L. Q.; Glickman, R. D.; Slaga, T. J.; Zhou, Z.; Liu, F. Up-regulation of adiponectin by resveratrol: the essential roles of the Akt/FOXO1 and AMP-activated protein kinase signaling pathways and DsbA-L. J. Biol. Chem. 2011, 286, 60−66. (15) Hertog, M. G. L.; Feskens, E. J. M.; Hollman, P. C. H.; Katan, M. B.; Kromhout, D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 1993, 342, 1007−1011. (16) Moon, J.; Do, H. J.; Kim, O. Y.; Shin, M. J. Antiobesity effects of quercetin-rich onion peel extract on the differentiation of 3T3-L1 preadipocytes and the adipogenesis in high fat-fed rats. Food Chem. Toxicol. 2013, 58, 347−354. (17) Khan, N.; Syed, D. N.; Ahmad, N.; Mukhtar, H. Fisetin: a dietary antioxidant for health promotion. Antioxid. Redox Signal. 2013, 19, 151−162. (18) Wood, J. G.; Rogina, B.; Lavu, S.; Howitz, K.; Helfand, S. L.; Tatar, M.; Sinclair, D. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature 2004, 430, 686−689. (19) Jung, C. H.; Kim, H.; Ahn, J.; Jeon, T. I.; Lee, D. H.; Ha, T. Y. Fisetin regulates obesity by targeting mTORC1 signaling. J. Nutr. Biochem. 2013, 24, 1547−1554. (20) Adhami, V. M.; Syed, D. N.; Khan, N.; Mukhtar, H. Dietary flavonoid fisetin: a novel dual inhibitor of PI3K/Akt and mTOR for prostate cancer management. Biochem. Pharmacol. 2012, 84, 1277− 1281. (21) Wu, M. Y.; Hung, S. K.; Fu, S. L. Immunosuppressive effects of fisetin in ovalbumin-induced asthma through inhibition of NF-κB activity. J. Agric. Food Chem. 2011, 59, 10496−10504.
deacetylase activity, which catalyzes PGC-1 deacetylation, leading to an increase in the interaction with PPAR complex and ultimately to transcriptional activation. Our findings may have clinical importance given that adiponectin has multiple beneficial effects on obesity-related medical complications.38 Moreover, fisetin is thought to prevent adipocyte differentiation, thereby maintaining preadipocytes in an undifferentiated state,24 whereas in certain conditions such as differentiated state, cells can encounter the different responses that are activated to promote adiponectin production with another physiological signal under fisetin treatment. Hence, fisetin could have potential applications in the prevention and treatment of diseases, alternative medicine, and other therapeutic approaches by modulating the concentration or improving the bioavailability. In summary, this study showed that fisetin, a dietary flavonoid, increased adiponectin gene transcription by inducing SIRT1-deacetylase activity. The activation of SIRT1 by fisetin may be connected to PGC-1 deacetylation, which increases its interaction with PPAR complex, which triggers up-regulation of adiponectin promoters. This is the first report of the beneficial effect of fisetin on the regulation of adipokine derived from adipocytes. The discovery of fisetin as an up-regulator of adiponectin expression that functions to enhance insulin sensitivity, suppress inflammation, and increase energy expenditure may exert its beneficial biofunction, thereby fisetin being considered as a contributor for modulating the pathogenesis of obesity-linked metabolic disorders.
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AUTHOR INFORMATION
Corresponding Author
*(J.H.C.) Fax: +82-31-881-7077. E-mail:
[email protected]. Funding
Basic Science Research Program (Grant 2006-2005303) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED PPARs, peroxisome proliferator-activated receptors; SIRT1, silent mating type information regulation 2 homologue 1; CVDs, cardiovascular diseases; C/EBPs, CCAAT/enhancerbinding proteins; AMPK, AMP-activated protein kinase; S6K1, p70 ribosomal S6 kinase 1; mTORC1, mammalian target of rapamycin complex 1; RXR, retinoid X receptor; IBMX, 3isobutyl-1-methylxanthine
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REFERENCES
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