Life Sciences 101 (2014) 64–72

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Apigenin isolated from Daphne genkwa Siebold et Zucc. inhibits 3T3-L1 preadipocyte differentiation through a modulation of mitotic clonal expansion Mi-Ae Kim, Kyungsu Kang, Hee-Ju Lee, Myungsuk Kim, Chul Young Kim, Chu Won Nho ⁎ Functional Food Center, Korea Institute of Science and Technology, Gangneung, Gangwon-do 210-340, Republic of Korea

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Article history: Received 27 November 2013 Accepted 14 February 2014 Available online 26 February 2014 Keywords: Preadipocyte differentiation Mitotic clonal expansion CCAAT/enhancer binding protein Daphne genkwa Sieboldet et Zuccarini Apigenin 3T3-L1

a b s t r a c t Aims: Obesity develops when energy intake chronically exceeds total energy expenditure. We sought to assess whether the flavonoid-rich fraction of crude extracts from Daphne genkwa Siebold et Zuccarini (GFF) might inhibit adipogenesis of 3T3-L1 cells. Main methods: Cell viability of 3T3-L1 preadipocytes was assessed by MTT assays, and lipid accumulation was measured by Oil Red O. Adipogenesis related factors were checked by Western blot analysis. Flow cytometry was used to analyze the mitotic cell cycle during the mitotic clonal expansion phase. Key findings: Among five flavonoids isolated from GFF, only apigenin potently inhibited the differentiation of 3T3-L1 cells. Apigenin reduced CCAAT/enhancer binding protein (C/EBP) α and peroxisome proliferator-activated receptor γ levels. Apigenin-treated 3T3-L1 cells failed to undergo clonal expansion during the early phase of adipocyte differentiation. Apigenin arrested cell cycle progression at the G0/G1 phase. This effect was associated with a marked decrease in cyclin D1 and cyclin-dependent kinase 4 expression, with the concomitant and sustained expression of p27Kip1. In addition, apigenin inhibited the DNA-binding activity of C/EBPβ in differentiating 3T3-L1 cells by downregulating the 35 kDa isoform of C/EBPβ (liver-enriched activating protein) and up-regulating the expression of two different sets of C/EBP inhibitors: C/EBP homologous protein and the phospho-liver-enriched inhibitory protein isoform of C/EBPβ. Significance: These findings suggest that apigenin can prevent 3T3-L1 preadipocyte differentiation by the inhibition of the mitotic clonal expansion and the adipogenesis related factors and upregulation of the expression of multiple C/EBPβ inhibitors. © 2014 Elsevier Inc. All rights reserved.

Introduction Obesity is a public health threat worldwide because it is a major risk factor for type 2 diabetes mellitus, hypertension, atherosclerosis, cancer, and cardiovascular disease (Calle et al., 2003; Kannel et al., 1991). There is considerable research interest in the discovery of compounds with anti-obesity activity, particularly those that act through appetite suppression, inhibition of nutrient absorption, increased energy expenditure, or modulation of fat storage (Bray and Tartaglia, 2000; Wang et al., 2004). Currently approved drugs for the long-term treatment of obesity include sibutramine, which inhibits food intake, and orlistat, which blocks fat digestion, but both have undesirable side effects. Accordingly, dietary bioactives derived from natural products are attractive alternatives to synthetic anti-obesity drugs. The main criteria

⁎ Corresponding author. Tel.: +82 33 650 3651; fax: +82 33 650 3657. E-mail address: [email protected] (C.W. Nho).

http://dx.doi.org/10.1016/j.lfs.2014.02.012 0024-3205/© 2014 Elsevier Inc. All rights reserved.

for studying underlying mechanisms of anti-obesity phytochemicals are inhibition of preadipocyte proliferation and differentiation, inhibition of lipogenesis, activation of lipolysis, and increased fat oxidation (Rayalam et al., 2008). Therefore, the identification of natural products, together with an understanding of their underlying mechanisms of action, may help to prevent the initiation and progression of obesity and its associated diseases. Adipogenesis involves two major events: preadipocyte proliferation, and adipocyte differentiation. The transition between both processes is a tightly regulated process in which cell cycle regulators and differentiating factors interact, creating a cascade of events leading to preadipocyte commitment to the adipocyte phenotype (Fajas, 2003). Since the development of the murine adipose 3T3 cell culture system, 3T3-L1 and 3T3F442A cells have become a popular in vitro model for studying adipocyte differentiation (Green and Kehinde, 1975). In the presence of a standard adipogenic cocktail comprising 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin, growth-arrested 3T3-L1 preadipocytes synchronously re-enter the cell cycle, undergo two rounds of mitosis (known as the mitotic clonal expansion (MCE)), and then exit the cell cycle and

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commence terminal differentiation (characterized by morphological changes, lipid accumulation, and the expression of almost all genes characteristic of fat cells) (Tang et al., 2003a,b; Tang and Lane, 1999). Several members of the CCAAT/enhancer binding proteins (C/EBP family), as well as peroxisome proliferator-activated receptor γ (PPARγ), participate in a transcriptional cascade during adipogenesis (Rosen et al., 2002; Rosen and Macdougald, 2006). Although C/EBPβ is expressed immediately on induction, it requires a long lag period (~14 h) for localization to centromeres and DNA-binding activity, which are critical for the expression of two principal adipogenic factors, C/EBPα and PPARγ (Tang et al., 2003a, b; Macdougald and Lane, 1995; Christy et al., 1991). Recently, the clinical importance of herbal drugs has received considerable attention, and flavonoids, which are often found in herbal drugs and foods, are known to possess many useful biological properties. The dried flowers of Daphne genkwa Siebold et Zuccarini (Thymelaeaceae) (“Genkwa Flos”), a Chinese herbal medicine distributed primarily in mainland China and Korea, are traditionally used for their abortifacient, anticancer, antitussive, diuretic, and antiinflammatory effects (Zhou, 1991; Hong et al., 2011; Bae et al., 2012). The present study examined several compounds isolated from the flavonoid-rich fraction of D. genkwa Siebold et Zuccarini crude extracts (GFFs) for their ability to inhibit the differentiation of 3T3-L1 cells into adipocytes. Only apigenin potently inhibited 3T3-L1 differentiation. Apigenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4one) is a naturally occurring plant flavone that is abundant in common fruits and vegetables; it is also a bioactive flavonoid that possesses antiinflammatory, antioxidant, and anticancer properties (Nielsen et al., 1999; Yang et al., 2001; Shukla and Gupta, 2010; Patel et al., 2007). Although obesity is associated with increased oxidative stress and inflammation in adipose tissue, the mechanisms underlying the beneficial role of apigenin during adipose tissue development are still under investigation. Previous studies show that apigenin impairs 3T3-L1 preadipocyte differentiation (Bandyopadhyay et al., 2006; Phrakonkham et al., 2008). Furthermore, apigenin inhibits food intake in C57BL/6J mice (Myoung et al., 2010). Recently, Ono and Fujimori demonstrated that apigenin mediates anti-adipogenic effects by activating AMPK in 3T3-L1 cells (Ono and Fujimori, 2011). However, the molecular mechanisms by which apigenin acts during adipogenesis are not fully understood. Here, we examined the role of apigenin and attempted to identify the mechanisms underlying its effects on adipogenic differentiation using 3T3-L1 cells. We found that GFF inhibited the proliferation and differentiation of 3T3-L1 cells. Apigenin, the active ingredient in GFF, suppressed preadipocyte growth and differentiation by inhibiting MCE; specifically, it induced cell cycle arrest at the G0/G1 phase during the early stages of adipocyte differentiation. The mechanisms by which apigenin inhibited MCE include down- and up-regulations of positive (such as cyclin D1 and cyclin-dependent kinase (CDK) 4) and negative cell cycle regulators (p27Kip1), respectively. Apigenin also down-regulated the expression of the adipogenic transcriptional factors, C/EBPα, C/EBPβ, and PPARγ. In addition, apigenin inhibited C/EBPβ activity in differentiating 3T3-L1 cells by up-regulating the expression of two different sets of C/EBP inhibitors: the phospholiver-enriched inhibitory protein (LIP) isoform of C/EBPβ and C/EBP homologous protein (CHOP)-10. Materials and methods Materials Dulbecco's-modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Hyclone (South Logan, UT, USA) and newborn calf serum (NBCS) was purchased from Gibco (Grand Island, NY, USA). Insulin, dexamethasone, IBMX, troglitazone, propidium iodide (PI), and Oil Red O were obtained from Sigma-Aldrich (St. Louis, MO, USA).

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Plant material Dried flower buds of D. genkwa were purchased from the Kyungdong oriental herbal market, Seoul, Korea, and identified by one of the authors (C.Y. Kim). The voucher specimen (KG-24) was deposited at the Functional Food Center, KIST Gangneung Institute, Korea. Extraction and isolation of five compounds Dried flower buds of D. genkwa (600 g) were obtained by ultrasonic extraction in ethanol at room temperature for 3 h. The ethanol extract (1 kg) of D. genkwa was suspended in distilled water and partitioned sequentially with n-hexane, ethyl acetate, and n-butanol. The ethyl acetate fraction (10 g) was subjected to chromatography on an ODS column using an H2O-methanol gradient system (60:40 → 0:100) to yield seven fractions (fractions 1–7). Compounds 1 (15 mg) and 2 (23 mg) were isolated from fraction 2 (1 g) by Sephadex LH-20 column chromatography (methanol). Compound 3 (20 mg) was purified from fraction 3 (500 mg) using Sephadex LH-20 column chromatography (methanol). Compounds 4 (30 mg) and 5 (25 mg) were isolated from fraction 6 (2 g) using a Sephadex LH-20 (methanol) column and then purified by preparative RP-HPLC (YMC J'sphere-H80; 4 μm, 20 × 250 mm, ACN-H2O = 35:65). Purity of all isolated compounds is above 97%. Cell culture and adipocyte differentiation 3T3-L1 preadipocytes were maintained in DMEM supplemented with 10% NBCS, 100 units/mL penicillin, and 100 μg/mL streptomycin. For differentiation, 3T3-L1 cells were seeded into 6-well plates at a density of 5 × 104 cells per well. When the cells reached confluence, they were cultured in a differentiation medium (DMI: DMEM supplemented with 10% FBS, dexamethasone (1 μM), IBMX (0.5 mM), insulin (5 μg/mL), and the PPARγ agonist troglitazone (1 μM), either with or without various compounds) for 2 days. Fresh medium containing insulin, troglitazone, and various compounds was added to the differentiating cells every 2 days. At 6–8 days post-differentiation, cells were fixed with 4% paraformaldehyde at room temperature for 1 h, washed twice with distilled water, and stained for at least 1 h at room temperature in freshly diluted Oil Red O stock solution (six parts Oil Red O stock solution and four parts H2O; Oil Red O stock solution is 0.5% Oil Red O in isopropanol). To quantify intracellular triglyceride content, stained parts were dissolved with propanol, followed by measurement with a spectrophotometer at 510 nm. Cell viability and proliferation 3T3-L1 cells were seeded at 1 × 104 cells/well in 96-well plates and allowed to attach overnight at 37 °C. They were then incubated in a differentiation medium containing various concentrations of apigenin (0–70 μM). After 2 days, the medium containing DMSO or apigenin was replaced with a medium containing 10% EZ-Cytox solution (Daeil Lab Service, Yongin, Korea). After 1 h of incubation at 37 °C, cell viability was determined by measuring the absorbance at 450 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT). All assays were performed in triplicate. The cytotoxic effect of each treatment was expressed as a percentage of cell viability relative to 0.1% DMSO-treated cells and was calculated as follows: (A450 mm-treated cells) / (A450 nm-untreated cells) × 100. For the cell proliferation assays, 3T3-L1 cells were seeded into 6-well plates at a density of 5 × 104 cells/well and cultured in DMEM containing 10% NBCS, 100 units/mL penicillin, and 100 μg/mL streptomycin. When the cells reached confluence, they were incubated in a differentiation medium supplemented with (or without) 70 μM apigenin. At indicated time points, cells were harvested and counted using a hemocytometer. Under microscope examination, cells were counted

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from four different fields of the hemocytometer. Dead cells were excluded by Trypan Blue staining. Western blot analysis After adipogenic stimulation for the designated times, cell monolayers were lysed in a cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA) containing a protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride. The proteins (35–50 μg) were separated in 10% (for PPARγ) or 12% (for C/EBPs, cyclin D1, CDK4, and p27Kip1) SDS-PAGE gels and then transferred to PVDF membranes (Amersham Pharmacia Biotech., Amersham, UK). The membranes were then incubated with PPARγ, C/EBPα, C/EBPβ, phospho-C/EBPβ (Thr235), CHOP, CDK4, cyclin D1, p27 and α-tubulin (1:1000, Cell Signaling, Danvers, MA, USA) primary antibodies, followed by anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and visualized using the ECL advanced system (GE Healthcare, Hatfield, UK). Flow cytometric analysis Confluent 3T3-L1 cells were stimulated with differentiation media in the absence or presence of 70 μM apigenin for the indicated times. Both detached and adherent cells were collected by trypsinization and washed with phosphate buffered saline (PBS). The cells were then stained with PBS containing 20 μg/mL of RNase A and 40 μg/mL of PI for 20 min at room temperature. The fluorescence intensity of the PIstained cells was measured using CellQuest Pro™ and a ModFit LT V3.0 software (Becton Dickinson, San Jose, CA, USA). Immunohistochemistry 3T3-L1 preadipocytes were plated on glass coverslips in a 24-well culture plate and allowed to reach confluence. The cells were then induced to differentiate as described above. At 4 h and 24 h postinduction, the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature followed by 100% methanol for 15 min at − 20 °C, and blocked with PBS containing 5% (vol/vol) goat serum and 0.3% Triton X-100 for 1 h. Cells were then incubated overnight at 4 °C with an anti-C/EBPβ antibody (1:50 dilution) in PBS Tween 20 containing 5% (vol/vol) goat serum, and washed with PBS. The cells were incubated with an Alexa Fluor 488-conjugated antirabbit secondary antibody (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature before mounting on coverslips in a VECTASHIELD mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Images were obtained using a Leica TCS SP5 confocal microscope (Leica, Welzlar, Germany). Statistical analysis Experiments were repeated three times, and the results were expressed as the mean ± standard error (SE). Student's t-test was used for statistical analysis and p b 0.05 was considered significant. Statistical differences were reported as *p b 0.05, ** p b 0.01 or ***p b 0.001. Results GFF and flavonoids isolated from GFF inhibit adipogenic cell differentiation To test the anti-adipogenic potential of GFF, 3T3-L1 cells were stimulated with a standard adipogenic cocktail (dexamethasone, IBMX, and insulin) together with a PPARγ agonist and GFF (25, 50, 75 or 100 μg/mL). The effects on adipogenesis in 3T3-L1 cells were measured using Oil Red O staining, and cellular lipid accumulation was determined after differentiation. As shown in Fig. 1A, GFF decreased lipid accumulation in a dosedependent manner at concentrations of 25, 50 and 75 μg/mL, with no

cytotoxic effects. A phytochemical study was performed on GFF, and five compounds were isolated to identify its active ingredient. Spectroscopic methods and a comparison with the literature identified compounds 1–5 as apigenin 7-O-glucuronide (1), genkwanin-3′-O-beta-Dprimveroside (2), tiliroside (3), apigenin (4), and genkwanin (5). The chemical structures of these compounds are shown in Fig. 1B. To select the optimal concentrations of these identified compounds, a cell viability assay was performed using the differentiation of 3T3-L1 cells. The five isolated compounds had no effect on cell viability at a concentration of 30 μM and showed little effect at 70 μM (a maximum reduction in cell viability of 30%; Fig. 1C, right panel). Of the five flavonoids tested, apigenin showed the greatest inhibitory effect on lipid droplet accumulation (29% at 70 μM; Fig. 1C. left panel). Apigenin inhibits the differentiation of 3T3-L1 preadipocytes We next investigated the effects of apigenin on cell differentiation induced by different adipogenic stimuli. Apigenin caused a dosedependent inhibition of differentiation induced by different adipogenic stimuli, namely, DMI in the presence or absence of troglitazone (a PPARγ agonist), and DI (DI: DMEM supplemented with 10% FBS, dexamethasone (1 μM), and insulin (5 μg/mL)) containing troglitazone (Fig. 2A). Furthermore, expression of C/EBPα and PPARγ was significantly lower in differentiating adipocytes treated with apigenin than in control cells (Fig. 2B). 3T3-L1 adipocyte differentiation, initiated by exposure to a standard adipogenic cocktail, occurs via early (days 0–2; D0–D2), middle (D2–D4), and late (after D4) stages. To determine the critical stage of adipocyte differentiation specifically affected by apigenin treatment, 3T3-L1 cells exposed to DMI containing a PPARγ agonist were treated with 70 μM apigenin for different times. Long-term (D0–D6) and early- to mid-term (D0–D4) treatments prevented adipogenesis the most. When cells were exposed to apigenin during the early differentiation stage (D0–D2), the formation of lipid droplets was markedly reduced by 60%. However, application of apigenin during midstage (D2–D4), mid-late stage (D2–D6), and late-stage (D4–D6) differentiation did not suppress lipid accumulation. These results suggest that the complete inhibition of adipocyte differentiation by apigenin is largely limited to D0–D2 and, to a lesser extent, the post-mitotic stages of adipocyte differentiation. Apigenin inhibits MCE during the early stages of adipocyte differentiation After exposure to adipogenic inducers, post-confluent, growtharrested 3T3-L1 preadipocytes undergo two sequential rounds of mitosis over 2 days. These mitoses, referred to as MCE, result in an approximate 4-fold increase in cell number (Tang et al., 2003a,b). To determine whether apigenin affects MCE, we examined the effects of apigenin on the proliferation of differentiating 3T3-L1 cells using a cell number counting assay. The number of DMI-treated preadipocytes increased 4-fold from D0 to D3 (Fig. 3A). By contrast, preadipocytes treated with DMI plus apigenin failed to proliferate to a significant extent. These findings show that apigenin treatment of preadipocytes blocks the MCE phase of differentiation. To assess whether apigenin-induced growth inhibition is mediated by changes in the cell cycle, we next examined the effect of apigenin on cell cycle distribution. When compared with vehicle-treated control cells (54% cells in the G0/G1 phase), apigenin treatment (for 16 and 24 h post-induction of adipocyte differentiation) resulted in clear cell cycle arrest at the G0/G1 phase, with of 87% of cells in G0/G1 after 16 h of treatment, and 84% in G0/G1 after 24 h (Fig. 3B, C). This increase in the G0/G1 cell population was accompanied by a concomitant decrease in the number of cells in the S and G2/M phases of the cell cycle. Entry into the cell cycle is regulated by the sequential activation of cyclin-dependent kinases (CDKs) and their associated corresponding regulatory cyclins. For example, the G1–S transition is regulated by complexes formed between cyclin D and CDK4 or CDK6, and between cyclin E and CDK2 (Sherr and Roberts, 1995). As shown

M.-A. Kim et al. / Life Sciences 101 (2014) 64–72

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Fig. 1. Effects of GFF and its isolated compounds on lipid accumulation in 3T3-L1 cells (A, C, left panel). Post-confluent 3T3-L1 preadipocytes (day 0) were cultured in the differentiation medium containing various concentrations of GFF (0, 25, 50, 75 or 100 μg/mL) and compounds isolated from GFF (70 μM) from day 0 to 6. Cell differentiation was examined at day 7 by Oil Red O staining. Stained intracellular oil droplets were eluted with isopropanol and quantified by spectrophotometrical analysis at 510 nm (A and C, right panel). Post-confluent 3T3-L1 cells were incubated with DMI (differentiation medium) and various concentrations (0–100 μg/mL) of GFF and 30 and 70 μM of compounds isolated from GFF for 2 days. Cell viability was determined by the WST assay. Data are represented as the mean ± SEM of triplicate experiments *p b 0.05, **p b 0.01, and ***p b 0.001, as compared to differentiated control. DMI: differentiation medium. (B) Chemical structures of the isolated compounds from GFF.

in Fig. 3D, cyclin D1 expression was up-regulated under control conditions, reaching a peak at 12 h post-induction and then decreasing markedly thereafter. By contrast, apigenin treatment down-regulated cyclin D1 as early as 4 h after treatment. This decrease in cyclin D1 expression positively correlated with a decrease in CDK4 expression. Cyclin–CDK

complexes are further modulated by cyclin-dependent kinase inhibitors (CDKIs), including p15INK4b, p16INK4a, p18INK4c, and p19INK4d (members of the INK4 family), and p21Cip1, p27Kip1, and p57Kip2 (members of the CIP/KIP family). Cell-to-cell contact in confluent 3T3-L1 cells abrogates cell cycle progression by inducing p27Kip1, which suppresses the activity

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Apigenin exposure time (days) Fig. 2. Inhibition of adipocyte differentiation by apigenin. (A) Effects of apigenin from GFF on adipocyte differentiation in 3T3-L1 cells in a dose dependent manner. Two-day postconfluent 3T3-L1 preadipocytes were subjected to different adipogenic hormone mixtures in the presence of dimethyl sulfoxide (DMSO) or 30 and 70 μM of apigenin. After 6 or 7 days of treatment of 3T3-L1 cells with apigenin or not, the cells were subjected to Oil Red O staining and microscopic view (C, second panel). DMI: differentiation medium, DI: DMEM supplemented with 10% FBS, dexamethasone (1 μM), and insulin (5 μg/mL). (B) Effects of apigenin on protein expression of adipogenic transcription factor PPARγ and C/EBPα. The levels of protein were determined by Western blotting. (C) Effects of apigenin of lipid accumulation in 3T3-L1 cells treated for different periods. Cells were subjected to adipocyte differentiation for 6 days with 70 μM apigenin at the indicated time points. After these treatments, cells were subjected to Oil Red O staining and quantitative analysis of Oil Red O-stained intracellular lipid.

of CDK4–cyclin D. As shown in Fig. 3D, p27Kip1 protein was abundantly expressed during the early stage of G1 under control conditions, corresponding to 1–4 h post-induction. Expression diminished by 12–24 h post-induction, leading to entry into the S-phase of the cell cycle. By contrast, apigenin treatment maintained p27Kip1 expression at high levels in 3T3-L1 cells, which was still clearly evident at the 16 and 24 h time points. FACS and Western blot analyses revealed that the apigenin-induced delay of entry into the S-phase was associated with a decrease in the expression of positive cell cycle regulators such as cyclin D1 and CDK4, along with increased expression levels of negative cell cycle regulators such as p27Kip1.

The above results show that the inhibition of adipocyte differentiation by apigenin is largely limited to the D0–D2 stage of differentiation and is related to cell cycle arrest in the G0/G1 phase. Since C/EBPβ is required for both MCE of preadipocytes and for transactivation of C/EBPα and PPARγ, we examined the expression of C/EBPβ isoforms and C/EBPβ binding to the C/EBPβ consensus sequence by immunofluorescence microscopy (Zhang et al., 2004; Tang et al., 2003a,b; Li et al., 2007). C/EBPβ is expressed early in the differentiation program and exists as three isoforms: 38 kDa (liver-enriched activating protein (LAP*)), 35 kDa (LAP), and 20 kDa (LIP). LAP contains both the activation and the bZIP domains, whereas only the latter is present in LIP. Therefore, LIP can act as a dominant-negative inhibitor of C/EBP function by forming non-functional heterodimers with other members (Fig. 4A) (Ramji and Foka, 2002). Apigenin treatment reduced the expression of the full-length 35 kDa form of C/EBPβ (LAP) (Fig. 4B, top panel). C/EBPβ isoforms are expressed early (within 2–4 h) after hormone induction, but are not immediately active. The transcriptional activity of C/EBPβ is regulated by several mechanisms, including post-translational modification and association with other proteins. The sequential phosphorylation of C/EBPβ is necessary for its acquisition of DNA-binding activity. C/EBPβ immediately undergoes a priming phosphorylation (on Thr188) by extracellular signal-regulated kinase (ERK1/2) and maintains its phosphorylated form through CDK2/cyclin A. However, when C/EBPβ is phosphorylated (on Ser184 or Thr179) by GSK3β, which occurs concomitantly with re-entry into the cell cycle at the G1/S boundary (Li et al., 2007; Tang and Lane, 2012; Prusty et al., 2002), the acquisition of its DNA binding and transactivation capacities is delayed until ~ 16 h after induction. To investigate the effect of apigenin on the transactivation of C/EBPβ, we looked for changes in the levels of the phospho-C/EBPβ (Thr188 in LAP, and Thr37P in LIP) isoforms at time points up to 48 h. As shown in the middle panel of Fig. 4B, apigenin treatment increased expression of the 20 kD isoform of phospho-C/EBPβ (LIP, Thr37P), but had no significant effect on the full-length 35 kD isoform (LAP), at both 20 and 30 h post-induction. As a result, the phospho-C/EBPβ LIP/LAP ratio was markedly increased with respect to that in control cells. C/EBP-homologous protein (CHOP-10) has a short non-functional DNA-binding domain (Ron and Habener, 1992; Batchvarova et al., 1995) and bZIP type C-terminal leucine zipper dimerization domain (Fig. 4A), and can heterodimerize with other isoforms to produce dominant-negative C/EBP dimers, which are unable to bind to their cognate DNA enhancer. As preadipocytes reach the S-phase, CHOP-10 is down-regulated, apparently releasing C/EBPβ from inhibitory constraint and enabling C/EBPβ DNA-binding activity (Tang and Lane, 2000). However, as shown in the bottom panel of Fig. 4B, in contrast to control cells, apigenin treatment markedly increased CHOP-10 protein expression at 30 h post-induction. The effect of apigenin on C/EBPβ localization to centromeres was examined by immunofluorescence microscopy. A previous study showed that bright fluorescent spots in mouse nuclei stained with DAPI are due to heterochromatic satellite DNA, and that major species of mouse satellite DNA contain eight repeats of a consensus C/EBP binding site (Horz and Altenburger, 1981). As C/EBPβ acquires its DNA-binding activity, it becomes localized to centromeres, resulting in a characteristic punctate pattern on immunofluorescence analyses. This pattern is observed from 12 to 16 h post-induction and coincides with preadipocyte entry into the S-phase and the onset of MCE. As shown in Fig. 4C, at 20 h post-induction, C/EBPβ exhibited punctate staining in DMI-treated preadipocytes, indicative of C/EBPβ acquiring DNA-binding ability. By contrast, when differentiating cells were treated with apigenin, C/EBPβ immunofluorescence remained diffused at the 20 h time point. These results demonstrate that apigenin inhibits the DNA-binding activity of C/EBPβ in differentiating 3T3-L1 cells by down-regulating

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Time (h) 0 4 9 12 16 20 24 4 9 12 16 20 24 control 70uM apigenin

Fig. 3. Inhibition of the MCE process of adipocyte differentiation by apigenin. (A) 3T3-L1 preadipocytes were induced to differentiate in the presence or absence of apigenin (70 μM). Cell number was determined 0, 1, 2, 3, 4 and 5 days after induction. (B) 3T3-L1 preadipocytes were cultured in the differentiation medium treated with DMSO (control) or 70 μM apigenin for 16 or 24 h, stained with propidium iodide, and analyzed by flow cytometry. (C) Percentage of cells in the G0–G1, S and G2-M phases of the cell cycle was determined using the ModFit LT software and values plotted are averaged from two different experiments. (D) Time course of the protein expression of cyclin D1, CDK4 and p27Kip1 during adipocyte differentiation. Whole-cell lysates were prepared at indicated time points and subjected to Western blotting. Numbers on top of the bands indicate changes in protein levels compared with control as determined by densitometric scanning of the immunoreactive bands and corrected for α-tubulin loading control.

the 35 kDa isoform of C/EBPβ (LAP) and up-regulating the expression of two different sets of C/EBP inhibitors: the CHOP and the phospho-LIP isoform of C/EBPβ. Discussion Obesity develops when energy intake chronically exceeds total energy expenditure. Excessive caloric intake relative to expenditure produces a metabolic state that promotes hyperplasia (increase in number) and hypertrophy (increase in size) of adipocytes (Shepherd et al.,

1993). The rise in adipocyte number involves the conversion of mesenchymal stem cells to preadipocytes, which then differentiate into adipocytes. All anti-obesity medications currently approved by the FDA act to repress energy intake, either by suppressing appetite or by inhibiting intestinal fat absorption (e.g., orlistat). However, side effects include depression, oily bowel movements, and steatorrhea; therefore, there is an urgent need for alternative approaches. Natural products are attractive alternatives that have potentially fewer side effects than chemically synthesized anti-obesity drugs. Phytochemicals such as epigallocatechin-3-gallate, genistein, and resveratrol are potential medicinal agents that inhibit the

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A

22

93

220

296

38kDa

LAP* AD

RD1 RD2

bZip

22

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LAP

35kDa 296

152

LIP

20kDa 296

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CHOP-10

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α-tubulin Time (h) 0 4 24 30 48 control PhosphoC/EBPβ

35kDa, LAP(Thr188P)

CHOP-10

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20kDa, LIP (Thr37P)

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0h

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20 h

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C/EBPβ 20 µM

20 µ M

20 µM

20 µM

20 µ M

20 µM

20 µ M

20 µM

20 µM

20 µ M

DAPI Preadipocyte

Control

Apigenin

Fig. 4. Inhibition of DNA-binding activity of C/EBPβ by apigenin treatment in the early stage of 3T3-L1 adipocyte differentiation. (A) Schematic presentation of the three isoforms of the C/EBPβ and CHOP-10. The schema shows the positions of N-terminal activation domains of LAP*/LAP and the bZIP domain common to the three isoforms of C/EBPβ and CHOP-10 that mediates homo/heterodimerization and DNA binding. (B) Day 0 post-confluent 3T3-L1 preadipocytes were induced to differentiation in the presence or absence of apigenin, whole-cell extracts were prepared at times indicated, and an equal amount of protein was separated by SDS-PAGE, and immunoblotted with anti-C/EBPβ, anti-phosphorylated C/EBPβ (Thr188P) and anti-CHOP-10. (C) Preadipocytes were pated on coverslips, and induced to differentiation with/out apigenin (70 μm). Cells were fixed and stained with C/EBPβ antibody and FITC-conjugated secondary antibody to detect localization of C/EBPβ at 4 or 20 h after induction. Cells were also stained with DAPI and photographed with a fluorescent microscope. The scale bar corresponds to 20 μm.

differentiation of preadipocytes, stimulate lipolysis, and induce apoptosis of existing adipocytes, thereby reducing the amount of adipose tissue (Gonzalez-Castejon and Rodriguez-Casado, 2011). The present study showed that GFF inhibits adipogenesis in a dose-dependent manner, without significant cytotoxicity. Among the five flavonoids isolated from GFF, only apigenin potently inhibited 3T3-L1 differentiation (Fig. 1C). Furthermore, apigenin blocked adipogenic differentiation induced by various adipogenic stimuli in a dose-dependent manner (Fig. 2A). The inhibitory role of apigenin was highlighted by apigenin-induced decreases in C/EBPα and PPARγ protein expression (Fig. 2B). Recently, the preventive role of apigenin in obesity was tested in an animal model. Myoung et al. showed that apigenin inhibits food intake of C57BL/6J mice. Bandyopadhyay et al. and Ono and Fujimori used the 3T3-L1 cell line to demonstrate a potential preventive function for apigenin, in which apigenin suppressed adipocyte differentiation and reduced PPARγ and

C/EBPα mRNA levels. Furthermore, Phrakonkham et al. demonstrate that apigenin among xenoestrogens differentially impair 3T3-L1 preadipocyte differentiation. However, the detailed molecular mechanisms underlying apigenin-induced adipocyte differentiation were not fully explored. Adipogenesis involves both preadipocyte proliferation and adipogenic differentiation. When growth-arrested 3T3-L1 preadipocytes are stimulated with a standard adipogenic cocktail comprising IBMX, dexamethasone, and insulin, they re-enter the cell cycle and undergo several rounds of mitosis, referred to as MCE. The cells then exit the cell cycle, lose their fibroblastic morphology, accumulate cytoplasmic triglycerides, and acquire the appearance and metabolic features of adipocytes (Green and Kehinde, 1975; Tang and Lane, 2012). MCE is an essential step in the adipocyte differentiation program. Thus, blocking DNA replication by various means (e.g., using inhibitors of DNA polymerase or by

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blocking cell cycle progression) prevents differentiation. Curcumin, rehmannia, and piceatannol inhibit adipocyte differentiation during the early stages (Kim et al., 2011; Jiang et al., 2008; Kwon et al., 2012). Similarly, apigenin inhibited the formation of lipid droplets at the early stage of adipocyte differentiation (D0–D2) (Fig. 2C). We further demonstrated that apigenin blocks MCE at the early stage of adipocyte differentiation, as evidenced by a significant suppression of cell proliferation from D0 to D3 post-induction of differentiation, delayed cell cycle progression, and suppressed of cyclin D and CDK4 levels with concomitant and sustained expression of p27Kip1 (Fig. 3). A growing body of evidence suggests that apigenin may also play a regulatory role in cell cycle progression in various malignant cell types (Shukla and Gupta, 2007, 2010; Takagaki et al., 2005). Of note, recent studies demonstrate that C/EBPβ, one of the critical transcription factors during the early stage of adipogenesis, is required for both MCE of preadipocytes (by turning on histone H4) and for terminal differentiation of 3T3-L1 adipocytes (by activating two principal adipogenic factors, C/EBPα and PPARγ) (Zhang et al., 2011; Lane et al., 1999). To examine the effects of apigenin on C/EBPβ activity, we first examined the expression of C/EBPβ in response to apigenin treatment. Apigenin suppressed C/EBPβ expression during the early differentiation stage (D0–D2) (Fig. 4B, top panel). Second, we examined the expression levels of phosphorylated C/EBPβ isoforms. Translation of C/EBPβ mRNA from different initiation codons leads to the synthesis of two transcriptional activators (LAP* and LAP) and a transcriptional repressor (LIP) (Fig. 4A). The LIP/LAP ratio is a critical factor in C/EBPβ-mediated gene transcription (Raught et al., 1995; Li et al., 2008; Abdou et al., 2011). Apigenin-treated 3T3-L1 cells showed similar levels of phosphorylated C/EBPβ-LAP (Thr188) to control cells, with a concomitant increase in the levels of phosphorylated C/EBPβ-LIP (Thr37); this resulted in a marked increase in the phospho-C/EBPβ-LIP/LAP ratio (Fig. 4B; middle panel). Third, the results showed that apigenin increased the expression of CHOP-10 (Fig. 4B, bottom), a dominant-negative C/EBP isoform that readily forms heterodimers with C/EBPβ and prevents C/EBPβ from binding to DNA (Tang and Lane, 2000). Dormant under normal growth conditions, the CHOP gene is induced to a high level during cellular stress caused by toxins, metabolic inhibitors, and nutrient deprivation (Wang et al., 1996). Notably (and as we showed for apigenin), genistein, berberine and the protease inhibitor ALLN all inhibit 3T3-L1 adipogenesis by up-regulating CHOP-10 (Tang and Lane, 2000; Pham et al., 2011; Harmon et al., 2002). The apigenin-induced changes in C/EBP isoform expression imply that C/EBPβ DNA binding may be impaired in apigenin-treated cells. Two lines of evidence support this view: (1) the downstream effects of decreased C/EBPβ binding, i.e., the expression of C/EBPα and PPARγ, were prevented (Fig. 2B), and cells did not differentiate, as indicated by the failure to accumulate cytoplasmic triglycerides (Fig. 2A); and (2) C/EBPβ did not become centromere associated when differentiating cells were treated with apigenin (Fig. 4C). The latter point is of particular importance, as centromeric satellite DNA contains multiple C/EBP consensus-binding sites (Tang and Lane, 1999). C/EBPβ possesses DNA-binding activity and exhibits centromeric localization as preadipocytes synchronously enter the S-phase at the onset of MCE (Tang and Lane, 2000). C/EBPβ is required for MCE during adipocyte differentiation, liver regeneration, inflammation, and cellular proliferation. Thus, it will be interesting to investigate how C/EBP family proteins interact in different combinations, and to identify how their altered interactions affect MCE under the influence of apigenin (Lane et al., 1999; Greenbaum et al., 1998; Mohan et al., 2008; Umek et al., 1991). Conclusions In summary, we showed that GFF inhibits the adipogenesis of 3T3-L1 cells, and that apigenin is the active ingredient responsible for these effects. Furthermore, apigenin inhibits adipogenesis by targeting the early

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biochemical and cellular events that occur during adipogenesis, including MCE. Apigenin down-regulates the expression of the early adipogenic transcriptional factors, C/EBPβ, PPARγ and C/EBPα, and up-regulates the expression of multiple C/EBPβ inhibitors, including the phosphorylated 20 kD isoform of C/EBPβ (LIP) and CHOP-10. Conflict of interest statement The authors declare that there are no conflicts of interest.

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