Modulation of adipogenesis-related gene expression by ethanol extracts of Detam 1 soybean and Jati belanda leaf in 3T3-L1 cells
Abstract
In this study, we evaluated the effects of ethanol extracts of Detam 1 soybean, Jati belanda leaf, and the combination toward expression of peroxisome proliferator-activated receptor gamma (PPAR-gamma), CCAAT/enhancer-binding protein alpha (C/EBP-alpha), and stearoyl-CoA desaturase 1 (SCD1) genes in 3T3-L1 cells as anti-adipogenesis and anti-obesity. The differentiation of 3T3-L1 cells into adipocyte was conducted using induction medium consist of Dulbecco's Modified Eagle's Medium, 3-isobutyl-1-methylxanthine, insulin, dexamethasone, and fetal bovine serum. The expression of PPAR-gamma, C/EBP-alpha, and SCD1 gene was measured using real-time quantitative polymerase chain reaction (qPCR). Ethanol extract of Jati belanda at a concentration of 50 μg/mL was most effective to reduce PPAR-gamma, C/EBP-alpha, and SCD1 gene expression in 3T3-L1 cells. Ethanol extract of Detam 1 soybean failed to reduce PPAR-gamma gene expression, whilst in the concentration of 50 ug/mL it was able to significantly reduce the C/EBP-alpha and SCD1 gene expression. Both ethanol extracts of Detam 1 soybean and Jati belanda have potential as anti-adipogenesis and anti-obesity by suppressing adipogenesis-related gene expression, particularly C/EBP-alpha and SCD1.
Introduction
Obesity is one of the largest and fastest growing health problem both in the developed and developing countries (Sergent et al., 2012; Rodgers et al., 2012). In the cellular level, obesity is characterized by a rise in the number (hyperplasia) and/or size (hypertrophy) of adipocytes, which is fat storage cells that differentiate from fibroblast-like precursor cells in adipose tissue (Bunkrongcheap et al., 2014; Lim et al., 2014). Thus, the suppression of differentiation into adipocytes, known as adipogenesis and/or promoting the intracellular lipid breakdown are potential anti-obesity mechanism (Bunkrongcheap et al., 2014). The adipogenesis process involves a cascade of transcription factors, and several factors such as peroxisome proliferator-activated receptor gamma (PPAR-gamma) and CCAAT/enhancer-binding proteins (C/EBPs) are considered to play important role in the process (Lefterova and Lazar, 2009).
Black soybeans (Glycine max) have been widely used as a healthy food and medicinal herb in oriental medicine (Kim et al., 2012). Several soy isoflavonesin from soybean revealed to inhibit adipogenesis through down-regulation of adiponectin expression in 3T3-L1 adipocytes (Zhang et al., 2009; Yanagisawa et al., 2012). Guazuma ulmifolia Lam., known as "Jati belanda" in Indonesia has been popularly used as therapeutics for hypercholesterolemic, gastrointestinal disorder, diabetes mellitus, and to reduce weight in some parts of the world (Feltrin et al., 2012; Magos et al., 2008). Our previous study demonstrated that ethanol extract of Detam 1 soybean seeds and ethanol extract of Jati belanda leaves have potential as anti-obesity, it inhibited weight gain in male Wistar rat and possessed inhibitory activities toward glucose-6-phosphate dehydrogenase (G6PD), triglyceride (TG) and cholesterol (CHOL) in 3T3-L1 cell line (Hidayat et al., 2015a; Hidayat et al., 2015b).
In this research, we continue to study the anti-adipogenesis properties of ethanol extract of Detam 1 soybean (EEDS) and ethanol extract of Jati belanda leaves (EEJB) in 3T3-L1 cell line by analyzing the expression of PPAR-gamma and C/EBP-alpha genes which are important transcription factor in adipogenesis (Rosen et al., 2002) and SCD1 gene which plays a role in lipid storage in adipocytes (Ralston and Mutch, 2015).
Materials and Methods
Plant materials
Detam 1 black soybean variety, which is a high quality black soybean that has been approved by the Agricultural Ministry of Republic of Indonesia, was collected from the estate of Research Unit and Development of Legumes and Tuber in Malang, East Java, Indonesia. Jati belanda was collected from plantations of Bumi Herbal Dago, Bandung, West Java, Indonesia. The plant was identified by a herbarium staff from the Department of Biology, School of Life Sciences and Technology, Bandung Institute of Technology, Bandung, West Java, Indonesia.
Preparation of the extracts
Ethanol extractions of Detam 1 soybean and Jati belanda were carried out by simple maceration method (Hidayat et al., 2015b). Detam 1 soybean (3 kg) and Jati belanda (700 g) were extracted twice by ethanol for 7 days. The solvent was evaporated and thick liquid extract was collected (71 and 56 g for Detam 1 soybean and Jati belanda, respectively). The ethanol extracts of Detam 1 soybean and Jati belanda were stored at -20°C for further use.
Adipogenesis induction
Adipogenesis induction was conducted using adipogenesis assay kit (Abcam ab133102). After cells reached 80% confluence, the medium was replaced by induction medium (DMEM + 3-isobutyl-1-methylxanthine (IBMX) + insulin + dexamethasone + fetal bovine serum (FBS)) for positive control, growth medium for negative control, and treatment medium (DMEM + IBMX + insulin + dexamethasone + FBS + EEDS or EEJB) for treatment. Cells were incubated for 3 days at 37°C, humidified atmosphere, 5% CO2. Following that, the cells were washed with phosphate-buffered saline (PBS) 1x and the medium was replaced by insulin medium (DMEM + FBS + insulin) for positive control, culture medium for negative control, and insulin medium + EEDS or EEJB. The plate was incubated for 2 days at 37°C, humidified atmosphere and 5% CO2. The insulin medium was replaced again and more than 80% cells were differentiated after 7 days. The adipocytes were observed using microscope (Hidayat et al., 2015a; Huang et al., 2006).
Box 1: Cell culture (Mouse pre-adipocyte)
Requirements
Mouse pre-adipocytes cells, 3T3-L1 (ATCC®CL-173; CV Gamma Scientific Biolab, Indonesia); Dulbecco's Modified Eagle Medium (DMEM, Biowest) supplemented with 10% calf serum (Biowest) and 100 U/mL penicillin-streptomycin (Biowest); inverted microscope, Eppendorf micropipette; incubator with CO2 cylinder; phosphate buffer solution
Procedure
Step 1: Collect the fresh culture medium from the water bath
Step 2: Taking out the culture cells from the incubator
Step 3: Check the confluence of the cells using the inverted microscope
Step 4: Remove the culture medium
Step 5: Add phosphate buffer solution to remove the remaining debris
Step 6: Remove the phosphate buffer solution along the remaining debris
Step 7: Add fresh medium to the cell culture
Step 8: Incubate the cell culture 37°C, humidified atmosphere, 5% CO2
To see the Video Clip
RNA extraction and cDNA synthesis
RNA extraction was performed using aurum total RNA kit (Bio-Rad) according to the manufacturer's instructions. After the extraction, the quality of RNA was checked using electrophoresis. The RNA was then used for cDNA synthesis using mix iScript cDNA synthesis kit (Bio-Rad) at 25°C for 5 min, 42°C for 30 min, with a final step of 5 min at 85°C. The product was stored at -20°C.
Quantification of PPAR-gamma, C/EBP-alpha, and SCD1 expression by real-time qPCR
The peroxisome proliferator-activated receptor gamma (PPAR-gamma), CCAAT/enhancer-binding protein alpha (C/EBP-alpha), and stearoyl-CoA desaturase 1 (SCD1) genes expression along with the constitutively expressed β-actin gene was analyzed using real-time quantitative polymerase chain reaction (qPCR). The primers used in this study are shown in Table I. The real-time qPCR was conducted using real-time PikoReal (Thermo Scientific Inc.) with condition pre-incubation cycle at 95°C for 30 sec followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 59°C for 20 sec, and extension at 72°C for 10 sec.
Table I: Sequence of primers used in real-time quantitative PCR
| Primer | Forward | Reverse |
|---|---|---|
| PPAR-gamma | 5'-TTATCAAGGGTCCCAGTTTC-3' | 5'-TTATTCATCAGGGAGGCCAG-3' |
| C/EBP-alpha | 5'-GCCGAGATAAAGCCAACCAA-3' | 5'-CCTTGACCAAGGAGCTCTCA-3' |
| SCD1 | 5'-CTGTACGGGATCATACTGGTTC-3' | 5'-GCCGTGCCTTGTAAGTTCTG-3' |
| beta-Actin | 5'-TCTGGCACCACACCTTCTACAATG-3' | 5'-AGCACAGCCTGGATAGCAACG-3' |
Statistical analysis
Statistical analysis was performed with Statistical Package for the Social Sciences (SPSS) statistics version 17.0 software. One-way analysis of variance (ANOVA) was conducted, followed by Duncan post-hoc test and p<0.05 was considered to be significant. Data are presented as mean ± SD.
Results
PPARγ gene expression
The quantification of PPAR-gamma gene expression using qPCR revealed that only several treatments than positive control, which were EEJB 50 µg/mL and combination of EEJB and EEDS (20 µg/mL, 10 µg/mL) (Figure 1). The lowest expression of PPAR-gamma gene was found in 3T3-L1 cells treated with EEJB 50 µg/mL, while the highest expression of PPAR-gamma gene was found in 3T3-L1 cells treated with combination of EEJB and EEDS (10 µg/mL, 5 µg/mL).
Figure 1: Relative expression of adipogenesis-related gene, PPAR-gamma in 3T3-L1 cells. (a) Effects of single ethanol extract of Detam 1 soybean (EEDS) or ethanol extract of Jati belanda leaves (EEJB) treatments in concentration of 10 µg/mL and 50 µg/mL; (b) Effects of combination of ethanol extract ofJati belanda leaves and ethanol extract of Detam 1 soybean treatments in concentration of 10 µg/mL, 5 µg/mL and 20 µg/mL, 10 µg/mL (*p<0.05, significant compared to positive control, Duncan post-hoc test)
C/EBP-alpha gene expression
All 3T3-L1 cells treated with the EEJB and/or EEDS were found to express C/EBP-alpha mRNA lower than positive control, but the all combination of extracts treatments did not able to reduce the expression significantly (Figure 2). EEJB 50 µg/mL was the most active to reduce C/EBPα gene expression, whilst the combination of EEJB and EEDS (10 µg/mL, 5 µg/mL) was the least active.
Figure 2: Relative expression of adipogenesis-related gene, C/EBP-alpha in 3T3-L1 cells. (a) Effects of single ethanol extract of Detam 1 soybean (EEDS) or ethanol extract of Jati belanda leaves (EEJB) treatments in concentration of 10 µg/mL and 50 µg/mL; (b) Effects of combination of ethanol extract of Jati belanda leaves and ethanol extract of Detam 1 soybean treatments in concentration of 10 µg/mL, 5 µg/mL and 20 µg/mL, 10 µg/mL (*p<0.05, significant compared to positive control, Duncan post-hoc test)
SCD1 gene expression
In this research, EEJB and EEDS at the concentration of 50 µg/mL showed lower SCD1 gene expression by approximately twice than positive control (Figure 3). EEJB at the concentration of 50 µg/mL was able to reduce SCD1 gene expression the most, with relative expression value of 4.9. Meanwhile, the combination of EEJB and EEDS (10 µg/mL and 5 µg/mL) resulted in highest SCD1 gene expression, with relative expression value of 15.8.
Figure 3: Relative expression of adipogenesis-related gene, SCD1 in 3T3-L1 cells. (a) Effects of single ethanol extract of Detam 1 soybean (EEDS) or ethanol extract of Jati belanda leaves (EEJB) treatments in concentration of 10 µg/mL and 50 µg/mL; (b) Effects of combination of ethanol extract of Jati belanda leaves and ethanol extract of Detam 1 soybean treatments in concentration of 10 µg/mL, 5 µg/mL and 20 µg/mL, 10 µg/mL (*p<0.05, significant compared to positive control, Duncan post-hoc test)
Discussion
In this research, only EEJB and the combination of EEJB and EEDS treatments were able to reduce PPAR-gamma gene expression, but the reduction by combination of extracts was lowed compared to the reduction by EEJB alone. EEDS resulted in relative expression level similar to positive control, while other treatments seemed to induce the expression rather than suppress it. These results indicated that EEJB and EEDS can both reduce and increase PPAR-gamma gene expression depend on the concentration used, high concentration of extract resulted in low expression of PPAR-gamma mRNA while low concentration of extract given to the 3T3-L1 cells resulted in high expression of PPAR-gamma mRNA. PPAR-gamma belongs to the nuclear receptor super family of ligand-activated transcription factors, which is needed in sufficient quantity so adipocyte differentiation can take place (Lefterova and Lazar, 2009).
PPAR-gamma is abundant in adipose tissue, and serves as essential regulator of adipocyte differentiation and maintenance of mature adipocyte. It also controls several adipocyte genes involved in all pathways of lipid metabolism (Kershaw et al., 2007). Suppression of PPAR-gamma expression can block adipogenesis and lipogenesis (Huang et al., 2006), therefore the reduction of PPAR-gamma gene expression by EEJB in high concentration (50 µg/mL) in this study could be one mechanism in inhibiting adipogenesis in 3T3-L1 cells.
C/EBP-alpha is one of adipogenic transcription factors that plays a role in adipogenesis (Rosen, 2005). Based on the result, all 3T3-L1 cells treated with EEJB, EEDS, and combination of both resulted in C/EBP-alpha expression lower than cells without treatment (positive control), but the treatment of EEJB or EEDS alone showed more reduction than the combination of EEJB and EEDS. Single treatment of both EEDS and EEJB in high concentration was able to significantly down-regulate the C/EBP-alpha expression compared to the low concentration of extract (10 µg/mL), indicating that the properties of both EEDS and EEJB in down-regulating C/EBP-alpha expression were in concentration-dependent manner. C/EBP-alpha is major contributor of adipsin and leptin expression in fat cells (Rosen et al., 2002), and plays a role in maintaining PPAR-gamma levels and insulin sensitivity in differentiated adipocytes (Rosen, 2005). C/EBP-alpha and PPAR-gamma induce expression of each other and can act synergistically to promote adipogenesis, by activate promoters of some adipocyte-specific genes (Harmon et al., 2002). Therefore, suppression of C/EBP-alpha gene expression by EEJB and EEDS in this study also could inhibit adipogenesis in 3T3-L1 cells (Gwon et al., 2013).
Conclusion
EEDS and EEJB in high concentration had anti-adipogenesis and anti-obesity properties by down-regulated C/EBP-alpha and SCD1 gene expression in 3T3-L1 cells. EEJB in the concentration of 50 ug/mL was the most active in suppressing expression of C/EBPalpha and SCD1 gene. Moreover, it was also able to reduce the PPARγ gene expression.
Acknowledgement
We are thankful to I Dewa Gde Sathya Deva, Merry Afni, Ervi Afifah, Seila Arumwardana, Hayatun Nufus, Hanna Sari Widya Kusuma, Dwi Davidson Rihibiha from Biomolecular and Biomedical Research Center, Aretha Medika Utama, Bandung for their valuable assistance.
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