Trapa Japonica Flerov Extract Attenuates Lipid Accumulation through Downregulation of Adipogenic Transcription Factors in 3t3-l1 Cells

Obesity is a major human health problem associated with various diseases, including cardiac injury and type 2 diabetes. Trapa japonica Flerov (TJF) has been used in traditional oriental medicine to treat diabetes. In this study, we evaluated the inhibitory effect of and the mechanism underlying the effect of TJF extract on adipogenesis in 3T3-L1 cells. The effects of TJF extract on cell viability were analyzed using a


Introduction
Obesity is the result of imbalance between energy intake and energy expenditure, and greatly increases the risk of numerous associated diseases, including heart disease, hypertension, stroke, cancer, diabetes, and osteoarthritis [1] [2].The World Health Organization (WHO) has declared obesity to be one of the most serious social and health problems today [3].According to the Korea Health Statistics 2012, 17.3% of Koreans over the age of nineteen are obese [4].Thus, determining the molecular mechanisms regulating adipocytes has crucial implications for the development of therapeutics targeting obesity and obesity-associated diseases.
Reducing lipid accumulation through the inhibition of adipocyte differentiation may play a crucial role in preventing obesity.Many researchers have investigated novel anti-adipogenic agents as potential therapeutics to decrease or prevent obesity.Obesity is known to be associated with excessive growth of adipose tissue mass, through increases in both the number and the size of fat cells [5].Adipose tissue mass can be regulated by the suppression of adipogenesis, which is the development of mature fat cells from fibroblastic preadipocytes [6].Adipokines, such as peroxisome proliferator activated receptor (PPAR)γ and CCAAT/enhancer-binding (C/EBP)α, directly regulate the development of fat cells [7].In particular, PPARγ is highly expressed in adipose tissue, where it plays essential roles in adipogenesis [8].
The cellular and molecular mechanisms of adipocyte differentiation have been extensively investigated using preadipocyte culture systems.The 3T3-L1 cell line is one of the best characterized and reliable models for studying the conversion of preadipocytes to adipocytes.The formation and appearance of fat droplets in these cells also mimic live adipose tissue [9].Confluent 3T3-L1 preadipocytes can be differentiated synchronously by a defined adipogenic cocktail.Maximal differentiation is achieved upon treatment with the combination of insulin, a glucocorticoid, an agent that elevates intracellular cAMP levels, and fetal bovine serum [10].Approximately 24 h after induction by adipogenic cocktail, differentiating preadipocytes undergo a postconfluent mitosis and subsequent growth arrest [11].After the growth arrest, cells are committed to becoming adipocytes.The growth arrest is required for subsequent differentiation.Growth-arrested cells begin to express late markers of differentiation at d 3.These late markers consist of lipogenic and lipolytic enzymes, as well as other proteins responsible for modulating the mature adipocyte phenotype.The cells then round up, accumulate fat droplets and become terminally differentiated adipocytes by d 5 -7 [12].
In the preliminary stages of differentiation, transcription factors, such as C/EBPβ, are activated, followed by the activation of PPARγ and C/EBPα in the intermediate stage.In the later stages, fatty acid binding protein (FABP)4 and adiponectin induce differentiation into mature adipocytes [12]- [14].Adenosine monophosphateactivated protein kinase (AMPK), a sensor of cellular energy status, is also a potential molecular candidate regulating adipocyte differentiation [15].AMPK activation requires phosphorylation at Thr172 of the AMPKα subunit, thus allowing AMPK to regulate metabolic enzymes and transcription factors [16].Acetyl-CoA carboxylase (ACC), which is a critical enzyme for lipid biosynthesis, can also be phosphorylated and inactivated by AMPK [17].
Trapa japonica Flerov (water chestnut, TJF) has been used as food and herbal medicine.TJF is an annual aquatic plant found in lakes and ponds in various parts of the world, including Korea, Japan, China, India, and North America [18].The fruit contains approximately 80% starch, 5% protein, and a significant amount of vitamins [19].The recent investigation revealed polyphenols, eugeniin, 1,2,3,6-tetra-O-galloyl-β-d-glucopyranose, and trapain from the husk of the TJF [20].Most studies evaluating TJF have focused on its taxonomy and ecology [18] [21].TJF has been known to have anti-oxidative, anti-cancer and anti-diabetic effects, reduction of blood glucose levels, and inhibitory activity against α-amylase and α-glucosidase [20] [22]- [24]; however, the effects of TJF on anti-adipogenesis have not been investigated yet.In the present study, we examined whether TJF extract affects adipocyte differentiation, and the expression of adipogenic transcription factors and related proteins in 3T3-L1 cells.

Preparation of TJF Extract
Dried ripened TJF (containing the shell and fruit) was purchased from a Korean herbal medicine dealer in Gumediherb, Korea.Dried TJF extract was triturated, and a crude extract from the triturate was prepared by 75% aqueous ethanol extraction.Briefly, powdered TJF (30 g) was extracted twice with 75% aqueous ethanol (300 g) under reflux at 60˚C -90˚C for 4 h, and was filtered and evaporated under reduced pressure.The powdered extract was solubilized in DMSO and diluted for this experiment.

Determination of Total Polyphenol Contents
Total phenolic contents of TJF extract was determined using Folin-Ciocalteu assay [25].Briefly, 1 mg of TJF extract was dissolved in 1 mL of DW.Then, 100 μL of this solution was mixed with 100 μL of 50% Folin-Ciocalteu reagent, and 2 mL of 4% sodium carbonate (Na 2 CO 3 ).After incubation at RT for 30 min, the absorbance of the reaction mixtures were measured at 750 nm by a spectrophotometer (SHIMADZU, Japan).The standard calibration curve was prepared by using the gallic acid.
Polyphenols were analyzed by HPLC under the following conditions.HPLC was performed on the Agilent Technologies 1200 series coupled with UV detector, and auto-sampler with a 10 μL loop.HPLC analysis was carried out using an ZORBAX Eclipse XDB-C 18 (5 μm, 4.6 I.D × 250 mm, YMC Inc., USA).The separation was conducted using a linear gradient 0.1% v/v, trifluoroacetic acid in H 2 O to 0.1% v/v, trifluoroacetic acid in acetonitrile for 60 min at a flow rate of 0.8 mL/min with detector at UV 280 nm .

Measurement of Antioxidant Activity
The antioxidant properties of TJF extract were examined using DPPH and xanthine/xanthine oxidase.
The free radical scavenging activity, based on the scavenging of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma, USA) free radical, was determined using the method described by Fujita et al. [26].First, 1 mL of TJF extract was added to 2 mL DPPH solution (0.2 mM) and incubated at RT for 10 min.The absorbance was then measured at 517 nm using a microplate reader (Synergy, BioTek, USA).L-ascorbic acid (Sigma, USA) was used as a positive control.
Superoxide radicals were generated by xanthine/xanthine oxidase and measured using the method described by Noro et al. [27].In brief, TJF extract was mixed with 0.05 M Na 2 CO 3 , 3 mM xanthine, 3 mM EDTA, BSA and 0.75 mM NBT.After 10 min at 25˚C, xanthine oxidase was added.The absorbance of reaction mixture was read at 560 nm after 10 min incubation at 25˚C.Tocopherol (Sigma, USA) was used as a positive control.

Cytotoxicity (MTT) Assay
Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay.The 3T3-L1 cells were seeded at a density of 5 × 10 3 cells/well in a 96-well plate, and were cultured at 37˚C with 5% CO 2 .The cells were then treated with TJF extract at various concentrations for 24 h.Following incubation, the cells were treated with the MTT solution (Sigma) for 4 h at 37˚C.The supernatants were aspirated, and DMSO (Sigma) was added to each well.After incubation for 20 min, the absorbance was measured at 540 nm using a microplate reader (Synergy, BioTek, USA).Cytotoxicity is presented as a percentage of the optical density of the control group.

Determination of Lipid Accumulation by Oil Red O Staining
Oil red O staining was performed on day 8. Cells were washed twice with phosphate-buffered saline (PBS, Gibco) and fixed with 10% formalin for 1 h.Oil red O was then added to stain the cells.Thereafter, the cells were washed three times with water.After oil red O staining, the lipid droplets were dissolved with isopropanol, and the optical density was measured at 540 nm with a microplate reader.Fat content was presented as a percentage of the optical density of the control group.

Western Blot Analysis
Cells were rinsed twice with PBS and scraped into lysis buffer.Lysates were prepared with lysis buffer according to the manufacturer's instructions (Thermo, USA; iNtRON, Korea).The protein concentration was determined using a BCA Assay Reagent Kit (Thermo Fisher Scientific Inc., USA).Proteins present in the cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% gel, transferred onto polyvinylidene fluoride membranes (GE Healthcare, UK), blocked with 5% skim milk, and treated with primary antibodies for 2 h at room temperature (1:1000 dilution, Cell Signaling, USA).After incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, USA) at room temperature, immunoreactive proteins were detected with a chemiluminescent ECL assay kit (GE Healthcare, UK) according to the manufacturer's instructions.Bands were visualized using a ChemiDoc image analyzer (BIO-RAD, USA).

Statistical Analysis
Data were analyzed using Sigma Plot.Results were expressed as the mean ± S.D. of three independent experiments.Comparisons were performed using a one-way ANOVA followed by Duncan's multiple range test.A p-value < 0.05 was considered statistically significant.

Extraction Yield, Total Polyphenols Contents of TJF Extract
The yield obtained was 6.77%.The contents of total polyphenols in extract, were determined from regression equations of calibration curves and was expressed in gallic acid equivalents for TJF extract.TJF extract contains polyphenols of 46.58% ± 0.58% (including eugeniin and gallic acid as constituents accounting for 0.85% ± 0.01% and 0.21% ± 0.02%, respectively).

Effect of TJF Extract on the Viability of 3T3-L1 Cells
3T3-L1 cells were exposed to various concentrations of TJF extract, and cell viability was measured by MTT assay.TJF extract did not affect cell viability at concentrations up to 50 μg/mL (Figure 1), and there is no The data are presented as mean ± standard deviation (n = 3).a The antioxidative activities of the samples are expressed by the concentration that showed inhibition of 50% radical.cytotoxicity during the differentiation process (data not shown).Thus, TJF extract was used in a range of noncytotoxic concentrations (10 -50 μg/mL) in subsequent experiments.

Effect of TJF Extract on 3T3-L1 Differentiation
We evaluated the effects of TJF extract on adipocyte differentiation.Cultured 3T3-L1 cells were exposed to TJF extract at varying doses on day 0, and cell differentiation was induced with a hormonal mixture-containing medium.On day 8, differentiation was terminated, and lipid droplets were detected by oil red O staining.TJF extract significantly reduced lipid accumulation, as indicated by decreased oil red O staining (Figure 2(a)).The triglyceride content in 3T3-L1 adipocytes was decreased in a dose-dependent manner, corresponding to 94%, 83%, and 57% following treatment with 10, 20, and 50 μg/mL TJF extract, respectively, compared to vehicle treated control cells (100%, p < 0.05, Figure 2(b)).These results indicated that TJF extract may have been efficiently blocking adipocyte differentiation and have potential of anti-obesity effects in 3T3-L1 cells.

Effects of TJF Extract on the Expression Levels of Adipogenic Transcription Factors and Markers in 3T3-L1 Cells
3T3-L1 adipocyte differentiation requires the synergistic action of multiple transcription factors and adipogenic markers, including PPARγ, C/EBPα, and adiponectin.PPARγ and C/EBPα alone or in cooperation with each other induce the transcription of many adipocyte genes encoding proteins and enzymes involved in creating and maintaining the adipocyte phenotype [12].After induction of adipocyte differentiation, these transcription factors were transcriptionally induced and reached to maximum level by 3 -4 d [12] [28].We performed western blot analysis to determine whether TJF extract inhibited adipocyte differentiation by negatively regulating the expression of adipogenic related factors.As shown in Figure 3, TJF extract markedly inhibited the protein expression of PPARγ, C/EBPα, and adiponectin.

Effect of TJF Extract on the Expression Level of Terminal Markers of Adipogenesis in 3T3-L1 Cells
Since the adipogenic transcription factors were down-regulated by TJF extract, we further determined the expression of their downstream protein targets such as FABP4.Fatty acid binding protein (FABP)4, which binds fatty acids with high affinity and transports them to various compartments within the cell, is a key mediator of intracellular transport and metabolism of fatty acids in adipose tissues.FABP4 is massively expressed during adipogenesis and comprises up to 6% of total cytosolic proteins in a mature fat cell [29].Our western blot analysis revealed that FABP4, an important biomarker in adipogenesis, was down-regulated (Figure 4).

Effect of TJF Extract on the Activation of AMPK in 3T3-L1 Cells
Several reports suggest that naturally occurring compounds exert their anti-obesity effects via AMPK activation   Adiponectin [30].Therefore, we investigated the involvement of AMPK in the process of TJF extract induced inhibition of adipocyte differentiation.AMPK activation and its substrate ACC phosphorylation were examined.AMPK activation was directly observed by examining increases in phosphorylated AMPK, and indirectly by observing the phosphorylation level of ACC-Ser79, the best-characterized phosphorylation site on AMPK.As shown in Figure 5, AMPK phosphorylation increased at 20, 50 μg/mL, and phosphorylation of its substrate, ACC-Ser79 was also enhanced in a dose dependent manner.These results indicated that AMPK is involved in the inhibition process of adipocyte differentiation.

Discussion
Adipogenesis is the process by which preadipocytes differentiate to mature adipocytes and is accompanied by coordinated changes in cell morphology, gene expression, and hormone sensitivity.The 3T3-L1 cell line is one of the best characterized and reliable models for studying the conversion of preadipocytes into adipocytes [12], and is useful to rapidly screen and assess the adipogenic potential of various agents.
In this study, we observed that TJF extract significantly inhibited 3T3-L1 adipogenesis and fat accumulation (Figure 2).Several transcription factors have been identified that act in a sequential fashion to promote adipocyte differentiation.PPARγ and the C/EBP family serve as early markers of adipocyte differentiation.The C/EBP families include C/EBPα, C/EBPβ, and C/EBPδ.Transient expression of C/EBPβ and C/EBPδ occurs during the early stages of differentiation, followed by induced expression of PPARγ and C/EBPα.These proteins induce the expression of multiple adipocyte specific genes and are important for terminal adipocyte differentiation [31].TJF extract inhibits 3T3-L1 adipogenesis at a relatively early stage by regulating the expression of PPARγ and C/EBPα (Figure 3(a) & Figure 3(b)).Moreover, TJF extract significantly suppressed FABP4, a late adipogenesis marker downstream of PPARγ (Figure 4).Our results also show that TJF extract markedly inhibits the expression level of adiponectin (Figure 3(c)).Thus, the current study demonstrates that overexpression of adiponectin can enhance 3T3-L1 preadipocyte proliferation, accelerate adipocyte differentiation, and, in fully differentiated adipocytes, augment both lipid accumulation and insulin-responsive glucose transport [32].
One possible mechanism of TJF extract action could involve the activation of AMPK.AMPK, an energy sensor, plays a key role in the regulation of fatty acids.Several groups have demonstrated that AMPK activation reduced body weight gain and decreased liver and plasma triglyceride levels in vivo [33].Furthermore it was reported that AMPK affects adipocyte differentiation in vitro.Zhou et al. reported that A-769662, which is an AMPK activator, inhibited 3T3-L1 adipogenic differentiation by downregulating the expression of C/EBPβ and C/EBPδ, thereby inhibiting clonal expansion.Moreover, AMPK activation blocked the expression of PPARγ and C/EBPα, and their downstream adipogenic genes.TJF extract also activated AMPK via phosphorylation and phosphorylated and inactivated ACC, which is essential for lipid biosynthesis (Figure 5) [34].The AMPK cascade is an important therapeutic target for obesity because it promotes the expression or phosphorylation of downstream proteins involved in energy metabolism.

Conclusion
TJF extract inhibits adipocyte differentiation by downregulating the expression of PPARγ and C/EBPα and their downstream adipogenic target genes that are essential for adipocyte differentiation, and by activating AMPK.Other approaches will be necessary to identify the transcription factors and target genes during adipocyte differentiation.

Figure 1 .
Figure 1.Effects of TJF extract on the viability of 3T3-L1 adipocytes as measured by MTT assay.3T3-L1 cells were treated with various concentrations of TJF extract.After incubation for 24 h, the MTT assay was performed.The results represent the mean ± S.D. of three independent experiments.

Figure 2 .
Figure 2. Effect of TJF extract on adipocyte differentiation in 3T3-L1 cells.3T3-L1 cells were exposed to a hormonal mixture for 8 d in the presence or absence of TJF extract.(a) Fat drops were stained with oil red O; (b) Stained triglyceride content was quantified by measuring absorbance.The results represent the mean ± SD of three independent experiments.* p < 0.05 compared with control (DMSO vehicle) group.

Figure 3 .
Figure 3.Effect of TJF extract on PPARγ (a), C/EBPα (b), and adiponectin (c) expression in 3T3-L1 adipocytes.3T3-L1 cells were exposed to a hormonal mixture for 5 d in the presence or absence of TJF extract.Cell lysates were prepared and subjected to western blot analysis to detect PPARγ, C/EBPα, and adiponectin.

Figure 4 .Figure 5 .
Figure 4. Effect of TJF extract on FABP4 expression in 3T3-L1 adipocytes.3T3-L1 cells were fully exposed to a hormonal mixture for 8 d in the presence or absence of TJF extract.Cell lysates were prepared and subjected to western blot analysis to detect FABP4.