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Dulbecco’s modified Eagle’s medium (DMEM), Bovine Calf Serum (BCS), and penicillin-streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). Fetal Bovine Serum (FBS) was purchased from ATLAS Biologicals (Fort Collins, CO). The 3-isobutylmethylxanthine, insulin, and dexamethasone were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Oil-red O (ORO) solution was purchased from Sigma-Aldrich (MO, USA). Isopropanol was obtained from Daejung Chemical (Seoul, Korea). Primary antibodies against β-actin, CCAAT/enhancer-binding protein (C/EBP) α, Peroxisome proliferator-activated receptor (PPAR) γ, Fatty acid synthase (FAS), Mammalian target of rapamycin (mTOR), p-mTOR, AKT, p-AKT, Insulin receptor substrates 1 (IRS1), p-IRS1 and Phosphoinositide 3-kinase (PI3K) 110α subunit were purchased from Cell Signaling Technology (Danvers, MA, USA). Adipocyte fatty acid-binding protein 2 (ap2) was purchased from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-linked anti-rabbit IgG and HRP-linked anti-mouse IgG were purchased from GenDEPOT (Barker, TX, USA).

The 3T3-L1 mouse preadipocytes were purchased from the American Type Culture Collection (Rockville, MD, USA), and maintained in DMEM containing 10% BCS with 1% penicillin-streptomycin at 37 ℃ in an incubator containing a humidified atmosphere with 5% CO₂. To differentiate 3T3-L1 preadipocytes into mature adipocytes (defined as Day 0), fully confluent 3T3-L1 preadipocytes were treated with differentiation medium containing DMEM, 10% fetal bovine serum, 0.5 mM 3-isobutylmethylxanthine, 5 μg/mL insulin, and 1 μM dexamethasone (MDI differentiation medium). After two days incubation, the cell medium was replaced by DMEM supplemented with 10% FBS and 5 μg/mL of insulin (Day 2). After another two days, the medium was changed with DMEM containing 10% FBS (Day 4). These cells were completely differentiated into mature adipocytes on Day 6. To determine the effect of ATE on adipogenesis, differentiated preadipocytes were treated with presence or absence of ATE for 4 d. Roots of Adenophora triphylla var. japonica extract was prepared by same method as previously reported¹⁴⁾. Dried A. triphylla var. japonica was extracted with 70% aqueous ethyl alcohol for 24 hours at 70 ℃ and was then filtered. The filtered extract was concentrated with a vacuum evaporation and dried.

After differentiation (Day 8), 3T3-L1 cells were washed with phosphate-buffered saline (PBS), fixed with 10% (v/v) formalin solution (Sigma-Aldrich, MO, USA) for 1 h, and dried. Fresh Oil-red O working solutions were prepared by mixing stock solution with distilled water (6:4), followed by incubation for 1 h at room temperature (RT), and further filtration. Cells were stained with Oil-red O working solution for 4 h, washed with distilled water, and photographed with ECLIPSE Ts2 microscope (Nikon Corporation, Tokyo, Japan). Stained lipid droplets were extracted with isopropanol, and the absorbance was measured at 520 nm by microplate reader (Tecan, Mannedorf, Switzerland).

Cell viability was determined by MTT assay. The cells were treated with various concentrations of ATE (0, 100, 300, and 500 μg/mL) for 24 h. MTT solution (5 mg/mL) was added to each of the wells, and the cells were incubated for 3 h at 37 ℃. The supernatants were removed, and DMSO was added to dissolve the formazan crystals. Absorbance at 570 nm was measured by microplate plate reader (Tecan, Mannedorf, Switzerland).

3T3-L1 cells were lysed in CelLytic buffer (Sigma-Aldrich, MO, USA). The lysates were centrifuged at 12,000 rpm for 15 min at 4 ℃, and the protein concentrations were measured by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were separated by 10% SDS-PAGE, and transferred to Immobilon-P membrane (Millipore, Bedford, Mass). The membranes were initially blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h, and then incubated with specific primary antibodies against β-actin, C/EBPα, PPARγ, ap2, FAS, mTOR, p-mTOR, AKT, p-AKT, IRS1, p-IRS1 and PI3K110α for overnight at 4 ℃. The membranes were then incubated in the corresponding horseradish peroxidase-conjugated anti-rabbit, anti-mouse immunoglobulin G (GenDEPOT, Barker, TX, USA) for 1 h at 23 ℃. Detection was performed with ECL solution (GenDEPOT, Barker, TX, USA), and the intensity of bands was detected by LuminoGraph (Atto, Tokyo, Japan).

All data were presented as mean ± standard deviation. The values were compared using Student’s t-test. One-way analysis of variance (ANOVA) was conducted using the software Origin 7 (Microcal Software, USA). Values of P < 0.05 were considered to indicate statistical significance.

The potential effects of ATE on the 3T3-Ll preadipocytes and cell viability were evaluated by MTT assay. ATE had no cytotoxic activity at various concentrations (0, 100, 300, and 500 μg/mL) (Fig. 1A).

Fig. 1. 
Effects of ATE on cell viability and adipocyte differentiation in 3T3-L1 preadipocytes. (A) 3T3-L1 cells were treated with various concentrations of ATE (0, 100, 300, and 500 μg/mL) for 24 h. The cell viability was evaluated by MTT assay, as described in the Materials and methods. (B) Lipid droplets were photographed after Oil-Red O staining, and lipid content was then quantified by measuring absorbance. Each percentage value in treated cells was calculated with respect to control cells. #P < 0.05, compared with the vehicle control. *P < 0.05 and **P < 0.01, when compared to only MDI differentiation medium treated-control group.

Previous study¹⁴⁾ has found that ATE treatment inhibited lipid accumulation, and affected the expression levels of adipogenesis-related proteins in 3T3-L1 adipocyte. That study also explained that further studies were needed on the expression levels of other genes related to adipogenesis. Therefore, to investigate the effects of ATE on adipogenic differentiation, 3T3-L1 preadipocytes were treated with various concentrations of ATE (0, 100, 300, and 500 μg/mL). Adipogenesis was tested with Oil-red O staining of lipid droplets and contents. The results show that ATE decreased the lipid accumulation in 3T3-L1 adipocytes without cell cytotoxicity (Fig. 1B). A reduction was observed in the lipid accumulation in the percentage of 3.15-44.05%.

Expression levels of adipogenic key transcriptional factors, such as C/EBPα and PPARγ, and other adipocyte-specific gene, such as ap2 and FAS, were also examined by western blotting. The results of western blotting analysis showed that C/EBPα, PPARγ, ap2 and FAS protein levels in MDI-induced 3T3-L1 adipocytes were significantly increased over the control, whereas ATE inhibited the expression levels of C/EBPα, PPARγ, ap2 and FAS by 89.94%, 85.78%, 69.10%, and 75.43% respectively (Fig. 2).

Fig. 2. 
Effects of ATE on the expression of adipogenesis related proteins. Western blot analysis was applied to investigate the expression of adipogenesis related proteins. Proteins were extracted at day 8 from the cells treated with or without ATE during differentiation. Adipogenesis related protein expression levels of C/EBPα, PPARγ, ap2 and FAS. Western blot bands were analyzed using Image J software. β-actin was used as a loading control. #P < 0.05, compared with the vehicle control. *P < 0.05 and **P < 0.01, when compared to only MDI differentiation medium treated-control group.

To further investigate the mechanism of ATE in adipogenesis, we examined the expression of mTOR signaling pathway proteins, which have important roles in adipogenesis. mTOR is one of the upstream factors of C/EBPα and PPARγ, which stimulates adipogenesis. Fig. 3A shows that ATE downregulates the phosphorylation of mTOR with an inhibition percentage of 52.09%, without affecting total mTOR expression. To further investigate the upstream mechanism of ATE in adipogenesis, we confirmed the IRS/PI3K/AKT signaling pathway proteins, which constitute the upper mechanism of mTOR signaling. Proteins expression levels were determined via western blot analysis. The stimulation with MDI caused the phosphorylation of IRS1, induction of PI3K110α, and phosphorylation of AKT. However, the phosphorylation of AKT, (Fig. 3B), IRS1, and the protein expression levels of PI3K110α (Fig. 3C) were significantly decreased in ATE treated cells by 67.24%, 63.31%, and 71.66%, respectively. Whereas, ATE did not show any effect on the expression of total IRS1 and AKT.

Fig. 3. 
Effects of ATE on the IRS/PI3K/AKT and mTOR signaling pathway. Western blot analysis was applied to investigate the expression of IRS/PI3K/AKT and mTOR signaling factors. (A) The protein expression levels of mTOR and phosphorylation of mTOR. (B) The protein expression levels of AKT and phosphorylation of AKT. (C) The protein expression levels of AKT signaling upstream factors, such as IRS1, p-IRS1, and PI3K110α. Western blot bands were analyzed using Image J software. β-actin was used as a loading control. #P < 0.05, compared with the vehicle control. *P < 0.05 and **P < 0.01, when compared to only MDI differentiation medium treated-control group.