The Inhibitory Effect of Betanin on Adipogenesis in 3T3-L1 Adipocytes

Betanin, a natural pigment that presents ubiquitously in plants, has been reported to show biological effects. However, not much is known on the effectiveness of betanin in regulating fat accumulation. Therefore, the aim of this study is to explore the inhibitory effect of betanin on adipogenesis in 3T3-L1 adipocytes and its mechanism action. The results show betanin significantly inhibited oil red O-stained material (OROSM) and triglyceride levels in 3T3-L1 adipocytes, indicating betanin inhibited lipid accumulation in 3T3-L1 adipocytes. In addition, the peroxisome proliferator–activated receptor γ (PPARγ) expression was significantly inhibited in the betanin-treated adipocytes, implying that betanin suppressed the cellular PPARγexpression in 3T3-L1 adipocytes. Moreover, the suppression of lipid accumulation by betanin occurred by decreasing the gene expression of PPARγ, CCAAT-enhancer-binding protein α (C/EBPα) and sterol regulatory element binding protein 1c (SREBP-1c). Taken together, these findings suggest betanin may be a mediator of adipocyte accumulation, leading to the inhibition of lipogenesis in 3T3-L1 adipocytes and betanin is therefore potentially useful for designing new antiadipogenic


Introduction
Overweight and obesity are defined by the World Health Organization (WHO) as abnormal or excessive fat accumulation that presents a risk to health. Obesity is a global medical issue that can cause metabolic syndrome, hypertension, atherosclerosis, diabetes, cancers and sleep disorders [1]. Further, a WHO survey in 2016 indicated that over 340 million children and adolescents aged 5-19 were overweight or obese. Moreover, most of the world's population lives in countries where overweight and obesity kills more people than underweight [2]. Consequently, emergence of metabolic syndrome incidence has led to an increased need for therapeutic and preventive strategies. Lifestyle changes such as improving eating habits and increasing physical activity, along with potential treatments that target specific molecules for regulation of metabolic pathways are recommended approaches to preventing obesity [3].
Many studies point to the health benefits of phytochemicals. Epidemiological evidence has shown inverse associations between intakes of the dietary phytochemicals present in natural sources and risk of developing chronic diseases. In recent years, therefore, many studies have focused on the biological effects of natural sources in preventing and treating overweight and obesity.
Betanin (betanin-5-O-β-glucoside) is a water soluble pigment belonging to the betalain family that presents ubiquitously in plants. Betanin is widely used a colorant in cosmetics, food products and pharmaceuticals [4,5]. Recently, the biological activities of betanin have been reported. For example, betanin showed antioxidant [6,7], anti-inflammatory [8], anti-carcinogenic [9], antihypertensive [10], and heptoprotective [11] properties. Apart from these, in the previous studies by the authors reported that the presence of bioactive compounds such as betanin, rutin, and kaempferol in Djulis may partly account for the antiadipogenesis of Djulis [7]. Given that betanin has shown biological effects, it is also possible that fat accumulation may be regulated by betanin. However, not much is known about the antiadipogenic effect of betanin. Therefore, the aim of this study is to evaluate the antiadipogenesis of betanin.

Chemicals
Betanin and Oil red O solution were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Anti-PPARγ and anti-β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were reagent grade.

Cell Viability Assay and Adipocytic Differentiation
3T3-L1 cells (BCRC Number: 60159) were purchased from Bioresources collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan.). Cell viability was determined by the MTT assay [12]. 3T3-L1 preadipocytes were planted into 6 well plates and maintained in DMEM supplemented with 10 % bovine calf serum at 37°C in a humidified 5% CO 2 incubator. Adipocytic differentiation was done as described by the method of Chyau et al. [7].

Measurement of Triglyceride Content
3T3-L1 adipocytes were harvested 8 days after the initiation of differentiation. The cells were incubated with 10-50 μM of betanin for 72 h at 37°C in a humidified 5% CO 2 incubator. Cells were collected and lysed in lysis buffer (1% Triton X-100 in PBS) for 30 min on ice box. The intracellular triglyceride content was measured using a commercial triglyceride assay kit (Cayman Chemical, Ann Arbor, MI, USA). The protein concentration was determined with the Pierce TM BCA protein assay kit (Thermo Suientific, Rockford, IL, USA) using bovine serum albumin as a standard [7].

Western Blot Assay
To determine peroxisome proliferator-activated receptor γ (PPARγ) expression, western blot assay was done. 3T3-L1 adipocytes were harvested 8 days after the initiation with 10-50 μM of betanin for 24 h at 37°C in a humidified 5% CO 2 incubator. The cells were collected and lysed in ice-cold lysis buffer (20 mM Tris-HCl (pH7.2), 2 mM EDTA, 500 μM sodium orthovanadate, 1% Triton X-100, 0.1% SDS, 10 mM NaF, 10 μg/ml leupeptin and 1 mM PMSF). The protein concentration was estimated with the Pierce TM BCA protein assay kit (Thermo Suientific) using bovine serum albumin as a standard. Total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% polyacrylamide, and blotted for 1.5 h at 100 V. The proteins in the gel were transferred to a PVDF membrane. Then, the membrane was blocked with 5% BSA in PBST (0.1% v/v Tween-20 in PBS, pH7.2) for 1 h. β-actin and PPARγ of primary antibodies were used in this study. Goat anti-rabbit IgG-HRP and rabbit anti-rabbit IgG-HRP (Jackson, West Grove, Chester, PA, USA) were used as secondary antibodies, and ECL prime (Advansta Inc., Menlo Park, CA, USA) reagent was used for developing. Membranes were incubated with primary antibody at 4°C overnight and then with secondary antibody for 1 h. Membranes were washed in PBST for 10 min three times between each step. Blots were developed using the Western Bright ECL kit (Advansta Inc.), exposed to Gel Electrophoresis Documentation-Multi-Function Gel Image system (Topbio Co., Taipei, Taiwan). The relative expression of proteins was quantified densitometrically using the Image J software (Wayne Rasband, Madison, WI, USA) and calculated according to the reference band of β-actin [7].

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assay
To determine the mRNA expression level of PPARγ, CCAAT-enhancer-binding proteinsα(C/EBPα) and sterol regulatory element-binding protein 1c (SREBP-1c) in the 3T3L1 adipocytes, total RNA was extracted using Total RNA Isolation kit (NA017-0100, GeneDirex, Lais Vegas, NV, USA) according to the manufacturer's instructions. RNA quantity and purity were checked by spectrophotometric analysis at 260 and 280 nm. One microgram of total RNA was reverse transcribed into cDNA using GScript First-Strand Synthesis Kit

Statistical Analysis
All data were expressed as means ± SD, and ANOVA was conducted by using the SPSS software (SPSS Inc., Chicago, IL, USA). Analysis of variance was performed by ANOVA procedures. Significant differences between means were determined by Duncan's multiple range tests or a Dennett's test at a level of p<0.05.

Effect of Betanin on Cell Growth
To understand whether betanin affects the cytotoxicity of 3T3-L1 adipocytes under the test concentrations, the cell viability of 3T3-L1 was measured using MTT assay. None of the betanin levels in the range from 10-25 μM shows any cytotoxic effect on the survival of 3T3-L1 adipocytes. (Figure 1), indicating that betanin did not affect 3T3-L1 cell growth under the tested concentrations.  Next, to understand the effect of betanin on lipid accumulation in 3T3-L1 adipocytes, the cells were allowed to differentiate into adipocytes for 8 days, and then the effect of betanin on lipid accumulation was measured. Figure 2 shows the effect of betanin on adipogenesis. The microscopic images of oil red O-stained adipocytes are shown in Figure 2A, indicating that the number of lipid droplets in the differentiated cells treated with betanin decreased compared with the control. Quantitatively, the OROSM data showed that betanin levels at 10, 25 and 50 μM reduced lipid accumulation by 19.6, 26.1 and 32.6%, respectively ( Figure 2B), revealing the number of lipid droplets in the differentiated cells was significantly reduced in a concentration-dependent manner when the cells were treated with betanin. Figure 3 shows the effect of betanin on the intracellular triglyceride levels in 3T3-L1 adipocytes. Betanin levels at 10, 25 and 50 μM reduced intracellular triglyceride levels in 3T3-L1 adipocytes by 10.5, 16.6 and 18.1%, respectively, indicating the intracellular triglyceride levels in 3T3-L1 adipocytes treated with betanin were decreased compared with the control. PPARγ, a transcription factor, plays an important role in the induction and maintenance of differentiated adipocyte phenotype [15]. Therefore, to understand whether betanin reduces lipid accumulation through regulating PPARγ, the PPARγ protein expression was determined using western blot analysis. As shown in Figure 4, in the betanin-treated adipocytes, the PPARγ protein expression was decreased in a concentration-dependent manner. In addition, the effect of betanin on the expression of adipogenic genes was determined by quantitation PCR analysis ( Figure 5). As expected, in the betanin-treated adipocytes at different concentrations, PPARγ ( Figure 5A), C/EBPα ( Figure 5B) and SREBP-1c ( Figure 5C) mRNA levels were significantly down-regulated. In addition, a decline in protein levels of PPARγ was observed due to the direct relationship between mRNA and protein levels ( Figure 4). PPARγ is a potent inducer of adipogenesis, which can promote the transdifferentiation of myoblasts to adipocytes, particularly when coexpressed with C/EBPα [7,16,17]. In addition, C/EBPα is a key transcriptional factor in adipogenesis and its expression is activated by C/EBPβ, C/EBPδ and PPARγ in adipocytes [18]. This means PPARγ and C/EBPα emerge as master regulators of adipogenesis [19]. Furthermore, SREBP-1c, a key lipogenic transcriptional factor, directly activates the expression of more than 30 genes; SREBP-1c is dedicated to fatty acid biosynthesis and uptake of fatty acids, cholesterol and triglycerides [7,20,21]. Clearly, betanin suppressed the expression of adipogenic genes in the betanin-treated adipocytes; therefore, betanin was able to regulate the expression of adipogenic genes, such as PPARγ, C/EBPα and SREBP-1c, thereby inhibiting lipid accumulation in 3T3-L1 cells.

Conclusion
According to the results presented in this study, betanin inhibited lipid accumulation. The mechanism action may be attributed to down regulation of the gene expression of PPARγ, C/EBPα and SREBP-1c, leading to inhibition of adipose tissue formation. These findings offer new insight into prevention of adipogenesis. Supplementation with betanin could be a beneficial treatment to reduce adipogenesis. In addition, despite the mentioned benefits, betanin suffers from limited oral bioavailability (below 1%) due to incomplete oral absorption and low shelf life [4]. Therefore, scientific trials in vivo are needed to confirm the results.

Conflict of Interest
The authors declare no conflict of interest.