Obesity is a state of excessive accumulation of adipocyte during adipogenesis1,2). The obese population is rapidly increasing worldwide due to the irregular diet and unhealthy lifestyle of individuals3,4). In addition, obesity is a major health problem and a risk factor for increasing the incidence of various human chronic diseases, such as hyperlipidemia, type 2 diabetes, heart disease, and cancer5-8). Several drugs have been developed to prevent and treat these obesity diseases. However, these drugs exert serious side effects, such as depression, gastrointestinal tract problems, and cardiovascular diseases9). Therefore, it is necessary to develop antiobesity products using natural extracts that possess good properties.
Excessive adipogenesis is a major cause of obesity. Adipogenesis is the process by which adipocytes develop and accumulate in the adipose tissue at various sites in the human body10). Adipocytes are formed by the proliferation and differentiation of preadipocytes into morphologically or biochemically mature adipocytes, which require the activation of important transcription factors. stimulation by hormones, including insulin, and regulation of adipocyte gene expression11). Therefore, the activity of transcription factors associated with the gene regulation of preadipocytes is also important for the regulation of obesity.
In this study, we investigated the effect of the
The 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Daegu, Korea) supplemented with 10% bovine calf serum (BCS; Gibco, Waltham, MA, USA) and 1% penicillin-streptomycin (P/S; Gibco) in a humidified incubator at 37℃ in 5%.
The 3T3-L1 pre-adipocytes were distributed in a 6-well plate at a density of 5× cells/well in DMEM supplemented with 10% BCS and 1% P/S at 37℃ in 5%. After incubating until confluency, the 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin (MDI) solution containing 1 μM DEX (Sigma Aldrich Co., Ltd.), 0.5 mM IBMX (Sigma Aldrich Co., Ltd.), and 10 μg/ml insulin (Sigma Aldrich Co., Ltd.) was used to induce the differentiation of cells in DMEM with 10% fetal bovine serum (FBS) for two days. After two days of differentiation induction, the medium was replaced with DMEM supplemented with 10% FBS (Gibco) and 10 μg/ml insulin every two days. This was followed by cell differentiation and incubation for 10 d. Every time the medium was changed, the cells were treated at 50, 100, and 200 μg/ml with
The 3T3-L1 pre-adipocytes were seeded at a density of 1 × cells/well in a 96-well plate and incubated at 37℃ in 5% for 24 h. Next, 3T3-L1 cells were treated with different concentrations (0, 50, 100, and 200 μg/ml) of
After 10 days of differentiation, the cells were washed with phosphate-buffered saline (PBS) and fixed with 10% formalin (Sigma Aldrich Co., Ltd.) for 1 h at room temperature. The cells were washed with 60% isopropyl alcohol (Fujifilm, Osaka, Japan) and stained for 10 minutes with 0.5% Oil-Red O solution (Sigma Aldrich Co., Ltd.). The Oil Red O solution was then removed and the cells were washed twice with distilled water. After Oil Red O staining, the cells were observed under an inverted microscope. Stained lipid droplets were dissolved in isopropanol and quantified at 500 nm using a microplate reader.
The differentiated 3T3-L1 cells were washed once with cold PBS and dissolved with radioimmunoprecipitation assay lysis buffer (cell signaling) and centrifuged at 12,000 rpm for 15 minutes at 4℃ to obtain the supernatant. The quantified protein was transferred to a polyvinylidene fluoride membrane after electrophoresis on 6-12% sodium dodecyl sulfate-polyacrylamide gel. The membrane was blocked with 5% nonfat milk in PBS-T buffer with 20% tween-20 for 1-2 h and incubated overnight with the following primary antibodies: anti-peroxisome proliferator-activated receptor-γ (PPAR-γ; Santa Cruz Biotechnology, Dallas, TX, USA), anti-CCAAT/ enhancer-binding protein-α (C/EBP-α; Santa Cruz Biotechnology), anti-sterol regulatory element-binding protein-1 (SREBP-1; Santa Cruz Biotechnology), anti-glyceraldehyde-3-phosphate dehydrogenase (Ab Frontier; GW Vitek, Seoul, Korea), anti-AMP-activated protein kinase (AMPK; Cell Signaling, Danvers, MA, USA), and anti-phosphorylated AMPK (p-AMPK; Cell Signaling) antibodies. After washing with PBS-T buffer, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or mouse anti-goat secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at room temperature. Proteins were visualized using a detection kit (Amersham BioSciences UK Ltd., Little Chalfont, UK) and quantified using a CS analyzer (ATTO, Tokyo, Japan).
All experimental data were presented as the mean±standard error of the mean of triplicate experiments. Data analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA) and Sigma Plot software (Systat Software Inc., Chicago, IL, USA). The significance of differences was determined using a one-way analysis of variance by post-hoc Tukey's test when relevant. P-value <0.05 was considered to be statistically significant.
The effect of
Fig. 2A shows the differentiation of the lipid droplet experimental protocol for 3T3-L1 preadipocytes.
The effects of
The differentiated control (+) increased compared to the undifferentiated control (-); however, AMPK levels were increased in the
Our study aimed to determine the ameliorative effect of the
PPAR-γ and C/EBP-α are transcription factors that play important roles in adipogenesis during the differentiation of preadipocytes to adipocytes17,18). C/EBP-α plays an important role in the late differentiation process of adipogenesis in adipocytes19). PPAR-γ is responsible for regulating the differentiation of adipocytes and its expression is associated with adipogenesis and fat storage20). PPAR-γ and C/EBP-α are master regulators of adipogenesis21-23). Our results showed that
Additionally, we assessed the AMPK signaling pathway, which regulates the transcription factors. Oxidation of fatty acids, lipid hydrolysis of triglycerides, and adipogenesis by adipocytes regulate the AMPK pathway24-26). AMPK is involved in adipocyte differentiation and adipogenesis regulation, and the activation of AMPK suppresses adipogenesis27,28). Activation of the regulatory pathway requires AMPK phosphorylation which inhibits lipid synthesis and upregulates lipid hydrolysis and fatty acid oxidation26,29,30). Several studies have shown th at AMPK inhibits adipogenesis by inactivating SREBP-1, a transcription factor that regulates lipid homeostasis and metabolism31-34). SREBP-1 induces gene expression associated with the regulation of PPAR-γ transcriptional activity and accumulation of lipids35). SREBP-1 is quickly induced in the early stages of preadipocyte differentiation and plays a role in promoting preadipocyte differentiation along with PPAR-γ. SREBP-1 promotes lipid metabolism by increasing the expression levels of several genes involved in lipid metabolism36,37). These results suggest that AMPK regulates various transcription factors, such as PPAR-γ, C/EBP-α, and SREBP-1, that are responsible for adipocyte differentiation and inhibition of adipogenesis34). Our results showed that
Obesity is an underlying condition for inflammatory and metabolic diseases and is often accompanied by a low-grade chronic inflammation38). Anemone, one of the main components in
In summary, we demonstrated that