Traditional Korean Herbal Formula Samsoeum Attenuates Adipogenesis by Regulating the Phosphorylation of ERK1/2 in 3T3-L1 Cells

Abstract

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 in vitro analyses to investigate the inhibitory effects of Samsoeum (SSE), a traditional herbal decoction. SSE had no significant cytotoxic effect against either the undifferentiated or differentiated 3T3-L1 cells. Oil Red O staining results showed that SSE significantly inhibited fat accumulation in adipocytes. SSE treatment consistently reduced the intracellular triglyceride content in the cells. SSE significantly inactivated glycerol-3-phosphate dehydrogenase (GPDH), a major link between carbohydrate and lipid metabolisms in 3T3-L1 adipocytes, and markedly inhibited the production of leptin, an important adipokine, in differentiated cells. SSE markedly suppressed the mRNA expression of the adipogenesis-related genes peroxisome proliferator-activated receptor-gamma (PPAR-γ), CCAAT/enhancer binding protein-alpha (C/EBP-α), fatty acid synthase (FAS), lipoprotein lipase (LPL), and fatty acid binding protein 4 (FABP4). Importantly, SSE increased the phosphorylation of ERK1/2, but not p38 MAPK and JNK, in adipose cells. Overall, our results indicate that SSE exerts antiadipogenic activity and modulates expressions of adipogenesis-related genes and ERK1/2 activation in adipocytes.

Figure 1

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 μg/mL) for 24 h. (b) 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, 62.5, 125, 250, 500, or 1000 μg/mL) during the differentiation period. Cell viability was determined using a CCK-8 assay kit by measuring the absorbance at 450 nm. Data are presented as mean ± SEM.

Figure 2

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 μM) during the differentiation period. ((a) and (b)) Lipid accumulation in the cells was analyzed by Oil Red O staining. (a) The stained cells were visualized on an Olympus CKX41 inverted microscopy at ×200 of magnification. (b) Stained oil droplets were dissolved in isopropyl alcohol and quantified by reading the absorbance at 520 nm. (c) The triglyceride content was measured enzymatically using a commercial kit (BioVision Inc.) at 570 nm. Data are presented as mean ± SEM. ^∗∗ p < 0.01 and ^∗∗∗ p < 0.001 versus differentiated cells.

Figure 3

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 μg/mL) during the differentiation period. (a) GPDH activity of the cells was assessed by measuring the decrease in NADH at 340 nm using a TAKARA glycerol-3-phosphate dehydrogenase activity assay kit. (b) Culture supernatant was collected from the SSE-treated cells. Leptin production was determined by ELISA by subtracting the value measured at 450 nm using a mouse leptin immunoassay kit (R&D Systems). Data are presented as the mean ± SEM. ^∗∗∗ p < 0.01 compared with the differentiated control.

Figure 4

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 μg/mL) during the differentiation period. Total RNA was isolated and subjected to real-time RT-PCR for PPAR-γ (a) and C/EBP-α (b), FAS (c), LPL (d), and FABP4 (e). β-actin was used as a housekeeping gene.

Figure 5

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 μg/mL) during the differentiation period. Cell lysates were prepared and subjected to immunoblotting for phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK. (b) The graph represents the relative levels of phosphorylation of MAPKs. (c) 3T3-L1 cells were treated with SSE and/or ERK inhibitor PD98059. Cell lysates were prepared and subjected to immunoblotting for phospho-ERK1/2.

Figure 6

Three-dimensional chromatogram of SSE by HPLC-PDA. HPLC conditions, column: Gemini C₁₈ column (250 × 4.6 mm, 5 μm; mobile phase: 1.0% (v/v) acetic acid in water and 1.0% (v/v) acetic acid in acetonitrile; gradient elution: 5–70% B for 0–40 min, 70–100% B for 40–45 min, 100% B for 45–50 min, and 100–5% B for 55 min; flow rate: 1.0 mL/min; column oven temperature: 40°C; injection volume: 10 μL).