THP was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). 3T3-L1 mouse embryonic fibroblast cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS), and bovine calf serum (BCS), were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). All chemicals were purchased from Sigma-Aldrich; Merck KGaA. Rabbit anti-mouse polyclonal FAS (3180; 1:2,000), rabbit anti-mouse monoclonal SCD1 (2794; 1:2,000), rabbit anti-mouse polyclonal PPARγ (2435; 1:2,000), rabbit anti-mouse polyclonal C/EBPα (2295; 1:2,000), rabbit anti-mouse polyclonal phospho (p)-AMPKα (2531L; 1:2,000), rabbit anti-mouse polyclonal AMPKα (2532S; 1:2,000), rabbit anti-mouse polyclonal p-ACC (3661L; 1:2,000) and rabbit anti-mouse polyclonal ACC (3662; 1:2,000) antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, USA). SREBP1 (sc-366; 1:1,000), actin (sc-1616; 1:1,000) and horseradish peroxidase (HRP)-conjugated goat anti-mouse polyclonal IgG antibody (sc-1616; 1:1,000) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Reverse transcriptase was supplied by Promega Corporation (Madison, WI, USA). QuantiTect SYBR-Green PCR kit was purchased from Qiagen GmbH (Hilden, Germany).

Mouse 3T3-L1 pre-adipocytes were cultured at 37°C with 5% CO₂ atmosphere in DMEM medium, 10% BCS, 100 U/ml penicillin, 100 µg/ml streptomycin. For standard adipocyte differentiation, 3T3-L1 cells were plated at a density of 5×10⁵ cell/ml and incubated for 2 days until they reached confluence. Pre-adipocytes (designated day 0) were cultured in differentiation medium (DMEM, 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone and 10 µg/ml insulin) for 4 days, switched to post- differentiation medium containing 10% FBS and 10 µg/ml insulin, and then changed to 10% FBS medium for an additional 2 days. During adipocyte differentiation, 3T3-L1 cells were treated with THP at a concentration of 0, 10, 20 or 40 µM from day 0–4. The positive control was treated 10 µM of pioglitazone (PIO; Sigma-Aldrich; Merck KGaA) and the same concentration of differentiation standard adipogenic medium.

Cells were seeded at 5×10³ cells/well in 96-well plate and incubated in the presence or absence of THP (0, 10, 20 or 40 µM). Following 96 h of culturing, 20 µl of Cell Titer 96^® AQueous One Solution Cell Proliferation Assay kit reagent (Promega Corporation, Madison, WI, USA) was added to each well, incubated for 1 h, and absorbance at 490 nm was measured using a microplate reader.

For TG determination, cells were collected and lysed in lysis buffer (25 mM sucrose, 20 mM Tris-HCl, 1 mM EDTA and 1 mM EGTA). The cellular contents of TG were measured using Infinity™ triacyglycerol reagents (Asan Pharmaceuticals Co., Ltd., Seoul, Korea) according to the manufacturer's instructions. The protein concentration was determined by using a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's instructions.

For GPDH determination, cells were harvested on day 8, washed twice with PBS, and collected with a scraper into 25 mM Tris buffer containing 1 mM EDTA and 1 mM dithiothreitol (pH 7.5). The harvested cells were sonicated for 10 sec, and then centrifuged at 6,950 × g for 5 min at 4°C. The supernatants were analyzed using GPDH activity assay kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocols.

Following differentiation, culture medium was removed and cells were gently rinsed with PBS once. Cells were fixed with 10% formalin for 1 h at room temperature, and then stained with filtered Oil Red O solution (6:4 ratio of stock solution and water) for 2 h at room temperature. Finally, cells were washed with distilled water and dried. The images were captured using an Olympus IX71 inverted microscope (Olympus Corporation, Tokyo, Japan). The stained lipid droplets were dissolved in isopropanol and quantified by spectrophotometrical analysis (540 nm).

Cells were harvested, and total protein extracts were prepared using a PRO-PREP™ protein extraction solution (Intron Biotechnology, Inc., Seongnam, Korea) and insoluble protein was removed by centrifugation at 11,750 × g for 15 min at 4°C. The supernatant was collected from the lysates and protein concentrations were determined using a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. For western blotting, 40 µg protein per lane was separated on an 8% SDS-PAGE and then transferred to polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were incubated with blocking solution (tris-bufferd saline/Tween 20, TBST) containing 5% skim milk (w/v) for 1 h at room temperature, followed by incubation overnight at 4°C with the indicated primary antibodies and further incubated with the HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive proteins were visualized by enhanced chemiluminescence solution (GE Healthcare Life Sciences, Chalfont, UK) Image J software version 1.50 (http://rsb.info.nih.gov/ij/download.html; National Institutes of Health, Bethesda, MD, USA) was used for the quantification of the results of western blotting.

Total mRNA was isolated using an Easy-Blue kit (Intron Biotechnology, Inc., Seongnam, Korea) according to the manufacturer's instructions. RNA was quantified using a Nanodrop ND-1000 UV-Vis spectrophotometer (Nanodrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). SREBP1c, FAS, SCD1, GPAT, PPAR-γ, C/EBPα, aP2, CPT1 and β-actin mRNA levels were measured by a LightCycler Real-Time PCR system (Roche Applied Science, Penzberg, Germany) using the QuantiTect SYBR-Green PCR kit. mRNA levels were normalized to the housekeeping gene, β-actin. The primer sequences used were as follows; SREBP1c, sense 5′-GCGCTACCGGTCTTCTATCA-3′, anti-sense 5′-TGCTGCCAAAAGACAAGGG-3′; FAS, sense 5′-GATCCTGGAACGAGAACAC-3′, anti-sense 5′AGACTGTGGAACACGGTGGT-3′; SCD1, sense 5′-CGAGGGTTGTTGTTGATCTG-3′, anti-sense 5′-ATAGCACTGTTGGCCCTGGA-3′; GPAT, sense 5′-GGTAGTGGATACTCTGTCGTCCA-3′, anti-sense 5′-CAGCAACATCATTCGGT-3′; PPARγ, sense 5′-AGGCCGAGAAGGAGAAGCTGTTC-3′, anti-sense 5′-TGGCCACCTCTTTGCTCTTGCTC-3′; C/EBPα, sense 5′-GGGTGAGTTCATGGAGAATGG-3′, anti-sense 5′-CAGTTTGGCAAGAATCAGAGCA-3′; aP2, sense 5′-TCTCACCTGGCCTCTTCCTTTGGCTC-3′, anti-sense 5′-TTCCATCCAGGCCTCTTCCTTTGGCTC-3′; CPT1, sense 5′-TGTCCAAGTATCTGGCAGTCG-3′, anti-sense 5′-CATAGCCGTCAGCAACC-3′; β-actin sense 5′-GGACTCCTATGGTGGGTGACGAGG-3′, anti-sense 5′-GGGAGAGCATAGCCCTCGTAGAT-3′. RT-qPCR was performed in a 25 µl reaction mixture containing 1 µl cDNA and primers using the ABI PRISM 7000 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The double-stranded DNA-specific dye, SYBR-Green I, was incorporated into the PCR buffer provided by the QuantiTect SYBR-Green PCR kit (Qiagen GmbH) to quantitate the PCR products by using the ^∆∆Cq method (15). PCR was performed at 95°C for 30 sec, followed by 60°C for 30 sec, and 72°C for 1 min. The last cycle was followed by a final extension step of 72°C for 10 min.

Results were represented as mean ± standard error of the mean and differences between groups were analyzed by one-way analysis of variance followed by Student Newman Keuls. P<0.05 was considered to indicate a statistically significant difference. Calculations were performed using the SigmaStat software version 3.5 (Systat Software, Inc., Chicago, IL, USA).

To examine the cell viability following THP treatment, 3T3-L1 cells were treated with various concentrations of THP (0–40 µM) for 96 h. THP did not exhibit any cellular toxicity up to 40 µM (Fig. 1A), and thus concentrations from 0 to 40 µM of THP were employed in the subsequent studies. To explore the effect of THP on differentiation of pre-adipocytes into adipocytes, 3T3-L1 pre-adipocytes were treated with various concentrations of THP (0, 10, 20 or 40 µM) for 4 days.

On the day 8 of incubation, the change in the contents of TG in THP-treated adipocytes was examined. As demonstrated in Fig. 1B, THP significantly reduced TG accumulation in a concentration-dependent manner. Compared with the differentiation medium control level, TG contents were significantly reduced by 13.1% at 10 µM (P<0.01), 34.4% at 20 µM (P<0.001) and 44.7% at 40 µM (P<0.001) of THP. Additionally, lipid accumulation was quantified by Oil Red O staining. As presented in Fig. 2A, THP markedly inhibited lipid droplet accumulation in a concentration-dependent manner. Consistent with results of lipid droplet accumulation, absorbance value of the eluted dyes measured at 540 nm were concentration-dependently decreased (Fig. 2B). Additionally, the effect of THP on the activity of GPDH was examined, as cytosolic GPDH is important role for the synthesis of TG (16). As demonstrated in Fig. 2C, THP treatment of 3 T3-L1 adipocytes resulted in a marked decrease in GPDH activity in a dose-dependent manner. These results suggested that THP efficiently inhibited adipocyte differentiation in 3T3-L1 adipocytes and may have potential anti-obesity effects.

To investigate the THP on the expression of genes involved in lipid metabolism, the expressions SREBP1, FAS and SCD1 genes responsible for adipogenesis were examined using western blot analysis and RT-qPCR. THP significantly inhibited the expressions of proteins (Fig. 3A) and genes (Fig. 3B) SREBP1c, FAS and SCD1 in a concentration-dependent manner, which are all associated with adipogenesis. In addition, the gene expression of GPAT was inhibited in a concentration-dependent manner compared with the differentiation medium group (P<0.01; Fig. 3B). These results suggested that THP suppresses the adipogenesis gene expression of the SREBP1c target genes, such as FAS, SCD1 and GPAT, resulting in the inhibition of 3T3-L1 differentiation.

PPARγ and C/EBPα are the primary transcription factors involved in adipogenesis and lipogenesis (4). To further investigate the mechanism on 3T3-L1 differentiation, expression of PPARγ and C/EBP-α at the protein and mRNA level were monitored by western blot and RT-qPCR analyses, respectively. THP markedly suppressed expression of PPARγ and C/EBP-α protein (Fig. 4A) and mRNA (Fig. 4B) levels in a concentration-dependent manner. PPARγ and C/EBP-α promote terminal differentiation by trans-activating the expression of downstream adipocyte-specific genes, including aP2 (17). In the current results, aP2 gene expression was reduced in a concentration-dependent manner compared with the differentiation medium group (P<0.01; Fig. 4B). These results strongly indicated that THP suppresses the expression of PPARγ, C/EBP-α and aP2, resulting in the inhibition of 3T3-L1 differentiation.

To investigate whether the activation of AMPK is involved in the adipocyte TG lowering effect of THP, the phosphorylated forms of AMPKα (Thr¹⁷²), and its immediate substrate ACC (Ser⁷⁹) were detected using western blotting. As demonstrated in Fig. 5A and B, THP treatment markedly enhanced the level of p-AMPKα Thr¹⁷² (P<0.01) and p-ACC Ser⁷⁹ (P<0.01), indicating that AMPK/ACC signaling is involved in the suppression of adipogenesis caused by THP (Fig. 5A and B). AMPK leads to fatty acid β-oxidation through inactivation of ACC and increasing the expression of CPT-1 (8). The present results indicated that THP increased gene expression of CPT1 in a concentration-dependent manner (P<0.01; Fig. 5C) compared with the control group. These results indicated that THP may inhibit adipogenesis via AMPK activation.