Trapa japonica Flerov Extract Prevents Obesity by Regulating Adipogenesis and Lipolysis in Differentiated 3T3-L1 Cells

: Our study investigated that the anti-obesity effect of the Trapa japonica Flerov extract (TJ) in differentiated 3T3-L1 adipocytes. To this end, 3T3-L1 cells were treated with TJ during their differentiation period. On the last day of the cell culture, we tested intracellular cAMP, FA, glycerol release, TG, and performed Oil Red O staining and Western blot assays. On the part of adipogenesis, lipogenesis, and lipolysis mechanism, TJ increased the cAMP (maximum 125.4%) levels and glycerol release (maximum four times) and decreased FA (maximum 35.1%) and TG (maximum 35.7%) levels. Furthermore, the protein expression levels of each mechanism-related factor were regulated in a dose-dependent manner. These results indicate that TJ reduced lipid accumulation by max 53.6% and 47.9%, respectively, in adipogenesis and lipolysis mechanisms. We expect this effect of TJ to be due to its component, ellagic acid. In conclusion, we found that TJ inhibits TG synthesis during adipogenesis and lipogenesis, promotes lipolysis, and thus, indicating its potential as a functional food for obesity prevention.


Introduction
Obesity is a serious disease with an increasing prevalence of epidemics in the Western world, including the United States [1]. Obesity-related diseases have been known through numerous reports so far, and the resulting complications have a significant negative impact on the quality of life of modern people [2][3][4]. Moreover, adipose tissue is an important regulator of whole-body metabolism and energy homeostasis [5].
Adipogenesis is the process of generating mature adipocytes from preadipocytes and was based on studies using murine preadipocytes, 3T3-L1 cells, to accelerate lipid accumulation [6]. When differentiation of preadipocytes begins, specific transcription factors are combined to induce the expression of differentiation initiating factors [7,8]. Excessive action of carbohydrate absorption and insulin signaling in the body accelerates lipogenesis in adipocytes. Acetyl coenzyme A (acetyl-CoA), which plays an important role in the tricarboxylic acid (TCA) cycle during lipogenesis, moves from the intracellular mitochondrial membrane to the cytoplasm. Acetyl-CoA is also involved in the production of fatty acid (FA) and is converted to malonyl-CoA under the influence of the multifunctional enzyme acetyl-CoA carboxylase (ACC) [9]. In the body, not only the accumulation of fat but also the breakdown of fat is regulated by several hormones and various transcription factors [10,11]. In particular, in the case of the lipolysis process in which TG is degraded, several pathways exist, but the cAMP-PKA pathway leads to the activation of hormone-sensitive lipase (HSL), The 3T3-L1 cell line used in this study was purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were incubated, cultured, and differentiated according to our conventional protocols [19]. The cells were seeded at a density of 2 × 10 5 cells/well into a 6-well plate. When cells are in a 100% confluent state, the medium was changed to a differentiation medium containing 10% FBS with an adipogenic cocktail (1 µM dexamethasone, 10 µg/mL insulin, and 0.5 mM IBMX). A total of 3 days after that day, the existing medium was replaced with a new medium containing only insulin (10 µg/mL), and after another 3 days, it was replaced with a new medium containing only 10% FBS. The information of group in this study was as follows: normal control (NC, no treatment), differentiation control (DC), positive control 1 (catechin 100 µg/mL, PC1), positive control 2 (catechin 200 µg/mL, PC2), and TJ (10, 50, 100, and 200 µg/mL). The cells were treated with TJ daily during the differentiation period (9 days) in the adipogenesis experiment set and treated only the last 3 days in the lipolysis experiment set according to our previously established protocol [19,20].

Water Soluble Tetrazolium Salt (WST) Assay
The WST assay was performed for testing the cell viability using an EZ-Cytox kit (Daeil Lab Service, Seoul, Korea) according to our previous method [19]. The cells were seeded at a density of 1 × 10 4 cells/well into a 96-well plate. After the cells were attached to the bottom of the plate, TJ samples were added to each well at a concentration of 0-1000 µg/mL for 24 h. After 24 h, 20 µL/200 µL of EZ-Cytox reagent was added to each well and incubated for 3 h. After gently shaking the plate, the optical density (OD) was measured at 450 nm (ELISA reader; Bio-Rad Laboratories, Hercules, CA, USA).

Western Blotting
After completion of the 3T3-L1 differentiation experiments, the cells were harvested and homogenized with CelLytic TM MT Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO, USA) containing the Halt TM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Rockford, IL, USA). All processes, such as protein quantification, loading, and transfer, were performed according to our conventional methods [19]. The primary antibody information was as follows: phosphorylated mitogen-activated protein kinase . As the secondary antibody, anti-rabbit, anti-mouse, or anti-goat IgG HRP-conjugated secondary antibodies (1:2000; Bethyl) were used. The membrane exposure was performed using EzWestLumi plus and Ez-Capture II equipment (ATTO, Tokyo, Japan), and analysis was also performed using ATTO's CS Analyzer 3.0 software.

Enzyme-Linked Immunosorbent Assay (ELISA)
The cyclic-adenosine monophosphate (cAMP) level in differentiated 3T3-L1 cells was measured using a cAMP ELISA kit (Cell Biolabs Inc., San Diego, CA, USA). The TG and FA levels were measured using each Quantification Colorimetric Kit (Biovision Inc., Milpitas, CA, USA). All experiments were conducted according to the respective manufacturer's protocols and our conventional methods.

Oil Red O Staining
The staining protocols related to lipid accumulation followed our conventional methods [19]. After completion of the 3T3-L1 cell differentiation, the cells on the plate were washed with Dulbecco's phosphate-buffered saline and fixed with 10% formalin. After drying the cells with 60% isopropanol, the lipids of the cells were stained with Oil Red O working solution for 2 h, and then the stained cells were washed with distilled water and photographed. The level of lipid accumulation was determined by eluting the Oil Red O dye that had stained the lipids using 100% isopropanol and measuring the OD value at 520 nm.

Glycerol Release Assay
For the measurement of the free glycerol, a TG decomposition product, the glycerol phosphate oxidase (GPO)-TRINDER enzyme reaction protocol was used [19]. After completion of the 3T3-L1 cell differentiation, the free glycerol reagent (Sigma-Aldrich) was mixed with the cultured medium. After incubation, the OD value was measured at 540 nm. Glycerol standard solution (Sigma-Aldrich) was used to analyze the glycerol content by substituting the results of the OD value into the standard curve.

Statistical Analysis
All experiment data were expressed as mean ± standard deviation (SD). The significance of each experimental group was determined using one-way analysis of variance and Duncan's multiple range test with SPSS Statistics 22.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance was considered to be at the p < 0.05 level.

Effects of TJ on Cell Viability in 3T3-L1 Cells
As a result of the cell viability test for 3T3-L1 cells of TJ (0-1000 µg/mL), at least 80% cell viability was observed in concentrations up to 200 µg/mL. In the main experiment, TJ was applied within the range where cytotoxicity was not observed (Figure 1). nm. Glycerol standard solution (Sigma-Aldrich) was used to analyze the glycerol content by substituting the results of the OD value into the standard curve.

Statistical Analysis
All experiment data were expressed as mean ± standard deviation (SD). The significance of each experimental group was determined using one-way analysis of variance and Duncan's multiple range test with SPSS Statistics 22.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance was considered to be at the p < 0.05 level.

Effects of TJ on Cell Viability in 3T3-L1 Cells
As a result of the cell viability test for 3T3-L1 cells of TJ (0-1000 μg/mL), at least 80% cell viability was observed in concentrations up to 200 μg/mL. In the main experiment, TJ was applied within the range where cytotoxicity was not observed (Figure 1). Figure 1. Effects of the TJ on cell viability in 3T3-L1 cells. TJ (0-1000 μg/mL). The data are shown as mean ± standard deviation (SD; n = 3). * = means significant differences from the controls were evaluated using student's t-test: * p < 0.05. TJ, Trapa japonica Flerov extract.

The TJ Inhibited the Lipid Accumulation Related to the Adipogenesis Mechanism in Differentiated 3T3-L1 Cells
As a result of measuring the lipid accumulation on the process of inhibiting adipogenesis in differentiated 3T3-L1 cells by conducting Oil Red O staining, we confirmed that the OD value of the DC group (1.28 ± 0.03) significantly increased relative to that of the NC group (0.07 ± 0.02). Furthermore, the result of the PC1 (0.68 ± 0.04), PC2 (0.40 ± 0.09), and TJ groups (10: 1.02 ± 0.09, 50: 0.88 ± 0.03, 100: 0.67 ± 0.05, 200: 0.59 ± 0.03) significantly decreased in a dose-dependent manner relative to the DC group ( Figure 3A,B).

The TJ Inhibited the Lipid Accumulation Related to the Adipogenesis Mechanism in Differentiated 3T3-L1 Cells
As a result of measuring the lipid accumulation on the process of inhibiting adipogenesis in differentiated 3T3-L1 cells by conducting Oil Red O staining, we confirmed that the OD value of the DC group (1.28 ± 0.03) significantly increased relative to that of the NC group (0.07 ± 0.02). Furthermore, the result of the PC1 (0.68 ± 0.04), PC2 (0.40 ± 0.09), and TJ groups (10: 1.02 ± 0.09, 50: 0.88 ± 0.03, 100: 0.67 ± 0.05, 200: 0.59 ± 0.03) significantly decreased in a dose-dependent manner relative to the DC group ( Figure 3A,B).

The TJ Inhibited the Lipid Accumulation Related to the Lipolysis Mechanism in Differentiated 3T3-L1 Cells
As a result of measuring the lipid accumulation on the process of lipolysis in differentiated 3T3-L1 cells by conducting Oil Red O staining, we confirmed that the OD value of the DC group (1.31 ± 0.02) significantly increased relative to that of the NC group (0.07 ± 0.01). Furthermore, the result of the PC1 (0.87 ± 0.05), PC2 (0.56 ± 0.07), and TJ groups (10: 1.10 ± 0.10, 50: 0.95 ± 0.04, 100: 0.77 ± 0.05, 200: 0.68 ± 0.07) significantly decreased in a dose-dependent manner relative to the DC group ( Figure 7A,B).
As a result of measuring the lipid accumulation on the process of lipolysis in differentiated 3T3-L1 cells by conducting Oil Red O staining, we confirmed that the OD value of the DC group (1.31 ± 0.02) significantly increased relative to that of the NC group (0.07 ± 0.01). Furthermore, the result of the PC1 (0.87 ± 0.05), PC2 (0.56 ± 0.07), and TJ groups (10: 1.10 ± 0.10, 50: 0.95 ± 0.04, 100: 0.77 ± 0.05, 200: 0.68 ± 0.07) significantly decreased in a dose-dependent manner relative to the DC group ( Figure 7A,B). The data are shown as mean ± standard deviation (SD; n = 3), and statistical analyses were conducted by Duncan's multiple range tests after one-way ANOVA. Each lowercase alphabet indicates withinfigure differing significance (p < 0.05).

Discussion
Excessive fat accumulation in the body leads to a disease we call 'obesity', which is affected by changes in various economic, lifestyle, and nutritional conditions, and can be exposed to obesity and related diseases in all life cycles [21,22]. In addition, in the recent health functional food market, demand and interest in weight control foods for a healthy and ideal body is peaking. To satisfy these demands, research on the development of various natural materials that suppress fat accumulation or help in weight control is actively conducted [23][24][25]. TJ is a plant that is widely distributed in Asian countries and some Western countries and has been widely used for medicinal purposes [26]. In addition, according to a recent investigation, active substances such as polyphenols, trapain, 1,2,3,6tetra-O-galloyl-β-d-glucopyranose, and eugeniin were found in TJ [16]. In particular, the antidiabetic effect of TJ was prominent among several studies [27], and the result of controlling glucose metabolism is expected to be effective in the prevention or treatment of obesity. In our preliminary study, we confirmed the presence of ellagic acid in a component analysis test of TJ (data not shown). Ellagic acid is a dimeric derivative of gallic acid that is found in woody plants, nuts, grapes, and berries [28]. It is reported to exhibit key biological activities such as radical scavenging, chemoprevention, and antiviral [29]. Our study aimed to determine whether TJ exerts effects on the adipogenesis, lipogenesis, and lipolysis mechanisms of differentiated 3T3-L1 cells. Prior to this experiment, we conducted a cell viability test. 3T3-L1 cells treated with TJ were confirmed to be non-toxic up to the maximum concentration (200 μg/mL) used in the main experiment. Furthermore, we used green tea catechin as a positive control in this experiment. Green tea is one of the

Discussion
Excessive fat accumulation in the body leads to a disease we call 'obesity', which is affected by changes in various economic, lifestyle, and nutritional conditions, and can be exposed to obesity and related diseases in all life cycles [21,22]. In addition, in the recent health functional food market, demand and interest in weight control foods for a healthy and ideal body is peaking. To satisfy these demands, research on the development of various natural materials that suppress fat accumulation or help in weight control is actively conducted [23][24][25]. TJ is a plant that is widely distributed in Asian countries and some Western countries and has been widely used for medicinal purposes [26]. In addition, according to a recent investigation, active substances such as polyphenols, trapain, 1,2,3,6-tetra-O-galloyl-β-d-glucopyranose, and eugeniin were found in TJ [16]. In particular, the antidiabetic effect of TJ was prominent among several studies [27], and the result of controlling glucose metabolism is expected to be effective in the prevention or treatment of obesity. In our preliminary study, we confirmed the presence of ellagic acid in a component analysis test of TJ (data not shown). Ellagic acid is a dimeric derivative of gallic acid that is found in woody plants, nuts, grapes, and berries [28]. It is reported to exhibit key biological activities such as radical scavenging, chemoprevention, and antiviral [29]. Our study aimed to determine whether TJ exerts effects on the adipogenesis, lipogenesis, and lipolysis mechanisms of differentiated 3T3-L1 cells. Prior to this experiment, we conducted a cell viability test. 3T3-L1 cells treated with TJ were confirmed to be non-toxic up to the maximum concentration (200 µg/mL) used in the main experiment. Furthermore, we used green tea catechin as a positive control in this experiment. Green tea is one of the most popular teas in Asia and is known as a drink useful for weight control [30]. One of its active ingredients, catechin, has been reported as a substance that promotes fat oxidation, regulates body composition, and inhibits fat synthesis in the 3T3-L1 cell model [31].
To clarify the effect of the TJ on the adipogenesis and lipogenesis mechanisms during the differentiation period of adipocytes, we confirmed the expression and activity levels of adipogenesis-and lipogenesis-related proteins. Adipogenesis is the progress by which the differentiation of 3T3-L1 cells (preadipocytes) matures into adipocytes [32]. It begins with a reduction in cAMP levels and activation of CREB, which stimulates the expression of transcription factors such as PPARγ and C/EBPa [33]. Adipogenesis is regulated by these transcription factors that are also called the master regulators in the final step of differentiation [34]. PPARγ, called a ligand-activated transcription factor, can induce the expression of C/EBPa, which in turn mediates the expression levels of adipogenesisrelated genes, including adiponectin, FABP4, G6Pase, and leptin [35,36]. In our previous study, we confirmed that the activity of cAMP and MAPK decreased in the adipogenesisinduced model, and the expression levels of C/EBPβ, C/EBPa, and PPARγ increased when adipocytes were differentiated [20]. In the present study, TJ increased cAMP activity and decreased the protein levels of CREB, C/EBPβ, C/EBPa, and PPARγ compared to the differentiation induction group. Due to this, the protein levels of FABP4, leptin, and G6Pase were also regulated.
In the lipogenesis mechanism, SREBP1c with various functions plays a crucial role. It stimulates ligands for nuclear receptors and PPARγ and regulates FA synthesis by activating lipogenesis-related factors, including ACC and FAS [37][38][39]. ACC is the rate-limiting enzyme in de novo FA synthesis and has been reported to be the enzyme responsible for the production of malonyl-CoA [40,41]. It has been reported that AMPK acts as an energy sensor that regulates lipid metabolism in cells, and the active form suppresses adipogenesis by down-regulating the expression of SREBP1c [42,43]. In addition, LPL is also an important factor in lipogenesis metabolism because it is also involved in FA synthesis and lipoprotein metabolism [44]. In the present study, TJ decreased the expression of SREBP1c by activating AMPK compared to the control group. In addition, it induced the activity of ACC and decreased the protein expression of FAS and LPL, thereby reducing the intracellular FA level. These results also affected the reduction in lipid accumulation, leading to a decrease in TG.
In the lipolysis mechanism, cAMP levels are regulated by the expression of PDE3B, and the PDE3 gene forms encode the PDE3A and 3B forms that act in the process of decomposing cAMP to 5 AMP. PDE3B is expressed in insulin-sensitive cells, such as hepatocytes, pancreatic β-cells, while adipocytes play a key role in glycogenolysis and lipolysis mechanisms. Lipolysis is mainly stimulated by the activation of the cAMP-PKA pathway, and since a decrease in the expression of PDE3B is related to a decrease in the hydrolysis of cAMP, the cAMP-PKA pathway can be stimulated [12,45]. In the next step, HSL and perilipin are phosphorylated by activation of PKA. This step is related to the efficiency of lipolysis: HSL migrates from the cytoplasm to the lipid droplets and activates this step, which ultimately affects the hydrolysis of TG [11,13,46]. Perilipin exists in the form of surrounding lipid droplets and can protect TG from several lipolytic enzymes [13,47]. It has been reported that an absence or decrease in the level of perilipin promotes basal lipolysis [46,47]. As a result of confirming the effect of TJ on the lipolysis mechanism using our established experimental model, TJ treatment increased cAMP levels and decreased the expression of PDE3B compared to the differentiation control group. In addition, TJ reduced the expression of ATGL, increased the expression levels of PKA, activated AMPK and HSL phosphorylation, and decreased the perilipin in the adipocytes. Furthermore, TJ reduced the intracellular FA content compared to that of the differentiation control group. This led to the reduction in TG content, and glycerol release was finally induced to promote lipolysis.
In our study, we evaluated the anti-obesity effect of Chinese TJ (extracted with 20% ethyl alcohol), which had not been attempted before, by various mechanisms. Although there have been reports of previously known adipogenesis inhibitory effects of TJ [15], we expected that the efficacy would be different depending on the country of origin and extraction process, and in order to open the possibility of future development as a health functional food, we evaluated the maximum effective dose within the range of non-toxicity. Although there were limitations to the markers showing effects at high concentrations, we differentiated them by identifying various factors of the mechanism extending to the previously unreported lipogenesis inhibitory activity and lipolysis activity of TJ. In addition, as a result of the comparison with green tea catechin, some markers showed similar effects at the same concentration (200 µg/mL), and in the case of other markers, a limitation was found that did not show as much effect as catechin. However, it will continue to be tested through additional studies on TJ, and we intend to leave the possibility open even to clinical studies.

Conclusions
In conclusion, TJ regulated the expression of adipogenesis-related proteins by reducing the expression of transcription factors at the cellular level. In the lipogenesis models, related factors in the lipogenesis mechanism were regulated through SREBP1c and acetyl-CoA, while TG and FA production, as well as glycerol release, were also finally regulated. Notably, TJ not only regulated the expression levels of AMPK and ATGL in the lipolysis mechanism but also activated the cAMP-PKA pathway to regulate protein levels, leading to degradation of TG ( Figure A1). Given that the effect of TJ on adipogenesis, lipogenesis, and lipolysis mechanisms in vitro have been substantiated, we expect that it will be developed as a natural agent to prevent obesity.