Adipogenesis is the cell differentiation process from preadipocytes into adipocytes and the critical action in the development of obesity. In the present study, we conducted
Obesity is a major contributor to the development of type 2 diabetes, hyperglycemia, and cancer. Along with diet, exercise, behavior modification, and medications are used in the treatment of overweight or obese people. Among these treatments, medications are an important part of the treatment process for morbid obesity, but weight-loss drugs can have serious side effects. Sibutramine and orlistat have been used in the treatment of obesity for the past two decades. However, sibutramine, which suppresses the appetite, increases the risk of heart attack and stroke in patients with a history of cardiovascular disease [
Samsoeum (SSE,
In the present study, we evaluated inhibitory effect of SSE on 3T3-L1 adipocytes treated with SSE. We also investigated its mechanisms of action by examining its effects on the expressions of genes and proteins involved in lipid metabolisms.
The 12 herbal medicines forming SSE were purchased from Omniherb (Yeongcheon, Korea) and HMAX (Jecheon, Korea). The origin of these herbal medicines was taxonomically confirmed by Professor Je Hyun Lee (Dongguk University, Gyeongju, Korea). A voucher specimen (2008–KE28–1~KE28–12) has been deposited at the K-herb Research Center, Korea Institute of Oriental Medicine.
SSE decoction comprising the 12 herbal medicines including Perillae Folium, Puerariae Radix, Pinelliae Tuber, Angelicae Decursive Radix, Ginseng Radix Alba, Poria Sclerotium, Aurantii Fructus Immaturus, Platycodonis Radix, Glycyrrhizae Radix et Rhizoma, Citri Unshius Pericarpium, Zingiberis Rhizoma Crudus, and Zizyphi Fructus was mixed (Table
Composition of Samsoeum (SSE).
Herbal medicine | Scientific name | Supplier | Source | Amount (g) |
---|---|---|---|---|
Perillae Folium |
|
Omniherb | Geochang, Korea | 3.75 |
Puerariae Radix |
|
Omniherb | Jecheon, Korea | 3.75 |
Pinelliae Tuber |
|
HMAX | China | 3.75 |
Angelicae Decursivae Radix |
|
HMAX | China | 3.75 |
Ginseng Radix Alba |
|
Omniherb | Geumsan, Korea | 3.75 |
Poria Sclerotium |
|
Omniherb | Yeongcheon, Korea | 3.75 |
Aurantii Fructus Immaturus |
|
HMAX | China | 2.8125 |
Platycodonis Radix |
|
Omniherb | Yeongcheon, Korea | 2.8125 |
Glycyrrhizae Radix et Rhizoma |
|
HMAX | China | 2.8125 |
Citri Unshius Pericarpium |
|
Omniherb | Jeju, Korea | 2.8125 |
Zingiberis Rhizoma Crudus |
|
Omniherb | Yeongcheon, Korea | 3.75 |
Zizyphi Fructus |
|
Omniherb | Yeongcheon, Korea | 3.75 |
|
||||
Total amount | 41.25 |
The mouse 3T3-L1 preadipocyte cell line was obtained from the American Type Culture Collection (CL-173, ATCC, Rockville, MD). The cells were cultured in DMEM (Gibco BRL, Carlsbad, CA) supplemented with 10% newborn calf serum (Gibco BRL, Carlsbad, CA) at 37°C. For adipocyte differentiation, the cells were stimulated with 3T3-L1 differentiation medium containing isobutylmethylxanthine, dexamethasone, and insulin (MDI) (Zen-Bio Inc., Research Triangle Park, NC) for 48 h after reaching a confluent state. The medium was switched to DMEM containing 10% FBS and 1
Undifferentiated 3T3-L1 cells were treated with various concentrations of SSE for 24 h. To produce differentiated adipocyte cells, 3T3-L1 preadipocytes were differentiated for 8 days by stimulating them by SSE. CCK-8 solution (Dojindo, Kumamoto, Japan) was added, and the cells were incubated for 4 h. After incubation, the absorbance was read at 450 nm on a microplate reader (Benchmark Plus, Bio-Rad. Hercules, CA).
The differentiated 3T3-L1 cells were fixed with 10% formalin for 15 min at room temperature and washed with 70% ethanol and PBS. The cells were stained with Oil Red O (Sigma-Aldrich, St. Louis, MO) for 5 min and then washed with PBS. Cell images were collected using an Olympus CKX41 inverted microscopy (Olympus, Tokyo, Japan). Stained oil droplets were dissolved in isopropyl alcohol and measured by reading the absorbance at 520 nm using microplate reader (Benchmark Plus, Bio-Rad. Hercules, CA).
The triglyceride concentration was measured enzymatically using a commercial kit (BioVision Inc., Milpitas, CA). Briefly, the 3T3-L1 adipocytes treated with SSE were homogenized in 5% NP-40 assay buffer and the sample to solubilize all triglycerides. The sample was mixed with lipase and triglyceride reaction mixture. After a 1 h incubation, the sample absorbance was measured at 570 nm using microplate reader (Benchmark Plus, Bio-Rad. Hercules, CA).
After the induction of adipocyte differentiation with treating with SSE, 3T3-L1 cells were washed twice with PBS. GPDH activity was measured using a commercial kit (TAKARA, Tokyo, Japan) and by monitoring the dihydroxyacetone phosphate-dependent oxidation of NADH at 340 nm. GPDH activity was expressed as unit/mg of protein.
Leptin concentration was measured using a mouse leptin immunoassay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. In brief, the culture supernatant was collected from the differentiated 3T3-L1 adipocytes that has been treated with or without SSE. Equal amounts of the supernatants (50
Total RNA was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA). Real-time RT-PCR analysis was performed using an Applied Biosystems 7300 Real-time PCR system and the SYBR green fluorescence quantification system (Applied Biosystems, Foster City, CA) to quantify the amplicons. cDNA was synthesized using 100 ng of RNA in a reverse transcription reaction. The PCR conditions were 50 cycles of 95°C (30 s), 55°C (30 s), and a standard denaturation curve. The primer sequences are listed in the 5′ to 3′ orientation in Table
List of primer sequences for real-time RT-PCR.
Gene | Primer sequences | |
---|---|---|
|
Forward | 5′-ACAATGAATACGGCTACAGCAACAG-3′ |
Reverse | 5′-GGTGGTCCAGGGTTTCTTACTCC-3′ | |
|
||
|
Forward | 5′-TATGGAGTGACATAGAGTGTGCT-3′ |
Reverse | 5′-CCACTTCAATCCACCCAGAAAG-3′ | |
|
||
|
Forward | 5′-CAAGAACAGCAACGAGTACCG-3′ |
Reverse | 5′-GTCACTGGTCAACTCCAGCAC-3′ | |
|
||
|
Forward | 5′-CAAGAACAGCAACGAGTACCG-3′ |
Reverse | 5′-GTCACTGGTCAACTCCAGCAC-3′ | |
|
||
|
Forward | 5′-CTGCTGGCGTAGCAGGAAGT-3′ |
Reverse | 5′-CTGGAAAGTGCCTCCATTG-3′ | |
|
||
|
Forward | 5′-CAAGAACAGCAACGAGTACCG-3′ |
Reverse | 5′-GTCACTGGTCAACTCCAGCAC-3′ |
The protein was extracted using Mammalian Cell Lysis Buffer (Sigma-Aldrich, St. Louis, MO) containing protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). The protein concentration was measured using a protein assay reagent (Bio-Rad Lab, Hercules, CA). The proteins were resolved by 8–12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Nonspecific biding sites were blocked with 5% (w/v) skim milk, and the membranes were incubated with the primary antibodies anti-phospho-p38, phospho-JNK, phospho-ERK1/2 (Cell Signaling Tech., Danvers, MA), and
Puerarin, daidzin, liquiritin, naringin, glycyrrhizin (all purity ≥98.0%), and hesperidin (purity ≥92.0%) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Neohesperidin (purity ≥99.0%) was obtained from ChromaDex (Irvine, CA). The HPLC-grade reagents methanol, acetonitrile, and water were obtained from J. T. Baker (Phillipsburg, NJ). Glacial acetic acid was obtained from Merck KGaA (Darmstadt, Germany).
A standard stock solution of the 7 compounds puerarin, daidzin, liquiritin, naringin, hesperidin, neohesperidin, and glycyrrhizin was dissolved in methanol at concentration of 1.0 mg/mL. For HPLC analysis, 200 mg of lyophilized SSE extract was dissolved in 20 mL of distilled water, and the solution was filtered through a SmartPor GHP 0.2
Analysis of the 7 compounds in SSE sample was performed using a Shimadzu LC-20A HPLC system (Shimadzu Co., Kyoto, Japan) comprising a solvent delivery unit, an online degasser, a column oven, an autosampler, and a PDA detector. The data processor was LCsolution software (version 1.24). The analytical column used was a Gemini C18 (250 × 4.6 mm; particle size 5
All data were presented as mean ± standard error of the mean (SEM). Group differences were assessed by one-way ANOVA and Tukey’s multiple comparison post hoc test using GraphPad InStat ver.3.10 (GraphPad software Inc., San Diego, CA). The significance of the differences between the sample and normal control at
To determine whether SSE had toxic effect, we used a CCK-8 assay to examine the cytotoxicity of SEE. Both preadipocytes and adipocytes were exposed to a concentration range of 31.5 to 1000
Cytotoxic effects of SSE extract in undifferentiated and differentiated 3T3-L1 cells. (a) 3T3-L1 preadipocytes were treated with various concentrations of SSE (0, 31.25, 62.5, 125, 250, or 500
During adipogenesis, triglycerides are stored in the form of lipid droplets in adipocytes [
Inhibitory effect of SSE extract on triglyceride production in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes by incubation with isobutylmethylxanthine, dexamethasone, and insulin (MDI) for 8 days. The cells were treated with or without SSE or GW9662 (20
GPDH is an enzyme that generates glycerol-3-phosphate from dihydroxyacetone phosphate in adipocytes for lipid biosynthesis [
Inhibitory effects of SSE on GPDH activity and leptin production in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes by incubation with isobutylmethylxanthine, dexamethasone, and insulin (MDI) for 8 days. The cells were exposed to various concentrations of SSE (0, 25, 50, 100, 200, or 400
Adipogenesis is accompanied by changes in the expression of adipogenesis-related transcriptional factors and specific molecular markers [
Effects of SSE on mRNA expression of lipid metabolism-related genes in 3T3-L1 adipocytes. (a) 3T3-L1 preadipocytes were differentiated into adipocytes by incubation with isobutylmethylxanthine, dexamethasone, and insulin (MDI) for 6 days. The cells were exposed to various concentrations of SSE (0, 25, 50, 100, 200, or 400
The MAPK pathways play a role in the regulation of each step in the process of adipogenesis [
Effects of SSE on phosphorylation of the MAPK family proteins in 3T3-L1 adipocytes. (a) 3T3-L1 preadipocytes were differentiated into adipocytes by incubation with isobutylmethylxanthine, dexamethasone, and insulin (MDI) for 4 days. The cells were exposed to various concentrations of SSE (0, 25, 50, 100, 200, or 400
We performed the simultaneous determination of seven components for quality control of SSE using HPLC coupled with photodiode array (PDA) detector. The regression equation of each compound was calculated by plotting the peak area (
Regression data, linear range, correlation coefficient, LOD, and LOQ for marker compounds (
Compound | Linear range ( |
Regression equation |
Correlation coefficient ( |
LOD |
LOQ |
---|---|---|---|---|---|
Puerarin | 1.56–200.00 |
|
0.9997 | 0.05 | 0.16 |
Daidzin | 0.78–100.00 |
|
0.9998 | 0.05 | 0.17 |
Liquiritin | 0.78–100.00 |
|
1.0000 | 0.06 | 0.19 |
Naringin | 1.95–250.00 |
|
0.9999 | 0.06 | 0.18 |
Hesperidin | 1.56–200.00 |
|
1.0000 | 0.06 | 0.18 |
Neohesperidin | 1.56–200.00 |
|
0.9999 | 0.04 | 0.14 |
Glycyrrhizin | 3.13–100.00 |
|
0.9999 | 0.52 | 1.74 |
Contents of seven compounds in the SSE by HPLC (
Compound | Mean (mg/g) | SD | RSD (%) | Source |
---|---|---|---|---|
Puerarin | 5.29 | 0.01 | 0.22 | PR |
Daidzin | 1.26 | 0.01 | 1.15 | PR |
Liquiritin | 2.27 | 0.03 | 1.20 | GRR |
Naringin | 10.38 | 0.05 | 0.49 | AFI, CUP |
Hesperidin | 5.64 | 0.05 | 0.87 | AFI, CUP |
Neohesperidin | 6.01 | 0.11 | 1.80 | AFI, CUP |
Glycyrrhizin | 3.96 | 0.04 | 0.97 | GRR |
Three-dimensional chromatogram of SSE by HPLC-PDA. HPLC conditions, column: Gemini C18 column (250 × 4.6 mm, 5
The field of herbal medicine includes the use and study of herbal plants for the purpose of preventing and treating various diseases. Herbs are attractive candidates for new drug development compared with synthetic chemical agents because they elicit fewer adverse effects of herbs. Recent studies have shown that many herbal plants have antiobesity activity by regulating adipogenesis. Lee et al. reported antiobesity effects of Aster glehni extract in both
In the current study, we found that the traditional Korean herbal formula SSE inhibited adipogenesis in 3T3-L1 cells. Adipogenesis was induced by adding the differentiation stimulators MDI to 3T3-L1 preadipocytes. The differentiated adipose cells exhibited increased triglyceride accumulation, GPDH activation, and leptin production. By contrast, SSE treatment exerted strong inhibitory effects on triglyceride accumulation, GPDH activity, and leptin production in adipocytes (Figures
Adipogenesis is a multistep process that is regulated by a cascade of various transcription factors. PPAR-
MAPK represents a family of proteins involved in various cellular processes such as cell survival, differentiation, and proliferation [
Antiobesity effects have been reported for 9 of the 12 SSE components except for
HPLC-PDA method is a convenient, widely used, and powerful approach for the rapid identification of constituents in botanical extracts and plants important in traditional Chinese medicine [
In conclusion, our data demonstrate that SSE has the inhibitory effects on adipogenesis in 3T3-L1 adipocytes as indicated by a significant reduction in triglyceride accumulation without cytotoxicity. The inhibitory effects of SSE may be mediated through the suppression of PPAR-
The authors have declared that no conflict of interests exists.
This work was supported by a grant from the Korean Institute of Oriental Medicine (no. K14030).