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 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and the anti-adipogenic effect was measured by oil red O staining. The expression of peroxisomal proliferator activated receptor (PPAR) γ, CCAAT/enhancer-binding protein- α (C/EBP) α, adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), adiponectin, and fatty acid binding protein (FABP)4 involved in adipogenesis was determined by western blot analysis. TJF extract effectively inhibited lipid accumulation and the expression of PPAR γ and C/EBP α in 3T3-L1 cells. TJF also increased the phosphorylation of AMPK and ACC, and decreased the expression of adiponectin and FABP4. These results indicate that TJF extract exerts its anti-obesity effect through the downregulation of adipogenic transcription factors and adipogenic marker genes.
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 [
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 [
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 [
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 [
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 [
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.
Total phenolic contents of TJF extract was determined using Folin-Ciocalteu assay [
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-C18 (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 H2O to 0.1% v/v, trifluoroacetic acid in acetonitrile for 60 min at a flow rate of 0.8 mL/min with detector at UV280 nm.
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. [
Superoxide radicals were generated by xanthine/xanthine oxidase and measured using the method described by Noro et al. [
3T3-L1 cells were obtained from the American Type Culture Collection (ATCC, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Gaithersburg, MD, USA) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% fetal calf serum (FCS, Gibco). Cells were incubated at 37˚C in a humidified atmosphere containing 5% CO2.
3T3-L1 cells were grown to confluence in DMEM containing 10% FCS at 37˚C in a humidified atmosphere of 5% CO2. One day after confluence (designated “day 0”), cell differentiation was induced with a hormonal mixture containing 0.5 mM isobutylmethylxanthine (IBMX, Sigma, USA), 0.25 μM dexamethasone (DEX, Sigma), 10 μg/mL insulin (Sigma), and 10% fetal bovine serum (FBS, Gibco, Gaithersburg). On day 2 and day 5, the medium was replaced with DMEM containing 10% FBS and 10 μg/mL insulin only. 3T3-L1 cells were treated with DMSO vehicle and varying concentrations of TJF extract during the differentiation process.
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 × 103 cells/well in a 96-well plate, and were cultured at 37˚C with 5% CO2. 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.
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.
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).
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.
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).
The antioxidant activity of T. japonica husk extract was studied [
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 (
Sample | DPPH Scavenginga (EC50 μg/mL) | Superoxide Scavenginga (EC50 μg/mL) |
---|---|---|
TJF extract | 7.17 ± 0.86 | 107.55 ± 2.41 |
L-ascorbic acid | 4.14 ± 0.11 | - |
Tocopherol | - | 225.61 ± 4.56 |
The data are presented as mean ± standard deviation (n = 3). aThe 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 non- cytotoxic concentrations (10 - 50 μg/mL) in subsequent experiments.
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 (
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 [
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 [
Several reports suggest that naturally occurring compounds exert their anti-obesity effects via AMPK activation
[
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 [
In this study, we observed that TJF extract significantly inhibited 3T3-L1 adipogenesis and fat accumulation (
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 [
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 (
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.
This work was supported by Chungcheong Institute for Regional Program Evaluation Promotion Project (R0002893) of the Ministry of Trade, Industry and Energy Republic of Korea.
1) Effect of the TJF extract on the viability of differentiated 3T3-L1 adipocytes at d 8.
2) Relative intensity of p-AMPK (