A 293T cell line and 3T3-L1 preadipocyte cell line (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life Technologies, Grand Island, NY, USA), containing 10% fetal bovine serum (Gibco Life Technologies) and 1% penicillin-streptomycin, at 37°C in saturated humidity with 5% CO₂. The culture solution of the cells was exchanged on a daily basis and trypsinization was processed every two days. Cells at a logarithmic growth phase were seeded for the experiment.

Effects of Compound Q (J&K Chemical Co., Ltd., Shanghai, China) on the coactivator recruitment of PPARγ and the transcriptional activity of the RXRα-PPARγ heterodimer were analyzed. At a logarithmic phase of cell growth, the 293T cells were seeded in a 24-well cell culture plate. When the cells reached a density of 50–70%, the medium was replaced with serum-free DMEM. Plasmids were transfected into the cells using a Calcium Phosphate Transfection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). To assess the ability of the compound for PPARγ coactivator recruitment, PPARγ-LBD, UAS-TK-Luc reporter and an internal reference plasmid, pRL-SV40 (all from Promega Corporation, Madison, WI, USA), were transfected in the 293T cells. When assaying the effects of the compound on the transcriptional activity of the RXRα-PPARγ heterodimer, the full-length plasmids of RXRα and PPARγ, PPRE-Luc and the internal reference plasmid, pRL-SV40, were transfected into the 293T cells. After 6 h of transfection, the medium was replaced with complete medium containing 10% fetal bovine serum. In addition, Compound Q was added to the medium for 18 h of culturing. The medium from the culture dish was removed and the plates were washed with phosphate-buffered saline (PBS). To each well, 100 μl cell lysis buffer was added and incubated at 37°C for 20 min to enable cell lysis. Next, with reference to the instructions in the luciferase kit (Dual-Luciferase^® Reporter Assay system; Promega Corporation), the activity of the firefly luciferase and internal reference, fluorescein, was analyzed.

A 3T3-L1 preadipocyte differentiation assay was conducted according to the reported classic procedure (14). Briefly, 3T3-L1 preadipocytes at a logarithmic growth phase were seeded into six-well cell culture plates. The cell density was allowed to reach 100%, and this time point was set as day 0. On day 4, the complete medium containing 0.115 mg/l methylisobutylxanthine (MIX), 0.39 mg/l dexamethasone (DEX) and 1 mg/l insulin was cultured for three days. The solution was further stimulated through the removal of old medium and the addition of fresh medium with 1 mg/l insulin, which was cultured for an additional three days. Following these stimulations, the differentiated adipocytes were cultured in normal medium (DMEM containing 10% FBS) for 1–2 days prior to carrying out further experiments.

To observe the effects of Compound Q on adipocyte differentiation, a negative control [dimethyl sulfoxide (DMSO)], positive control (Ros) and Compound Q at various concentrations (1, 10 and 20 μM) were added to the differentiation medium. After adipocyte stimulation for 6 days, the differentiation medium was removed and the cells were washed three times with PBS. The solution was stained using a Oil Red O kit (Nanjing Jiancheng Bioengineering Institute) according to manufacturer’s protocol, to investigate the lipid accumulation in adipocytes. Images were photographed for observation using a BX 50 microscope (Olympus Corporation, Tokyo, Japan) (15).

Mature 3T3-L1 adipocytes that had undergone differentiation were cultured in six-well plates and processed with different compounds, including a negative control (DMSO), positive control (Ros) and Compound Q at various concentrations (1, 10 and 20 μM), that were added to the complete medium for 24 h. Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and reversed transcribed to cDNA using a PrimeScript^TM RT kit (Takara Biotechnology, Co., Ltd., Dalian, China). SYBR Green Real Time PCR Master Mix (Toyobo Co., Ltd., Tokyo, Japan) was used for PCR, which was performed in a DNA Engine Opticon 2 device (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primers used in the experiment were as follows: GAPDH forward, 5′-GTATGACTCCACTCACGGCAAA-3′ and reverse, 5′-GGTCTCGCTCCTGGAAGATG-3′; fatty acid synthase (FAS) forward, 5′-CTGAGATCCCAGCACTTCTTGA-3′ and reverse, 5′-GCCTCCGAAGCCAAATGAG-3′; CCAAT/enhancer-binding protein-α (C/EBPα) forward, 5′-CTGAGATCCCAGCACTTCTTGA-3′ and reverse, 5′-CACGGCTCAGCTGTTCCA-3′; adipocyte fatty acid binding protein 2 (aP2) forward, 5′-CATGGCCAAGCC CAACAT-3′ and reverse, 5′-CGCCCAGTTTGAAGG AAATC-3′; HMG-CoA forward, 5′-CATGCAGATTCT GGCAGTCAGT-3′ and reverse, 5′-CGGCTTCACAAACCA CAGTCT-3′. These genes were selected as they are PPARγ-regulated and the main genes involved in lipid metabolism. All the samples were assayed according to the manufacturer’s instructions.

A mammalian one-hybrid system was carried out to screen compounds which were able to directly bind and influence the recruitment of co-activators to PPARγ-LBD. In the mammalian one-hybrid system, the Gal4/UAS system was used to investigate the activity regulation of PPARγ, whether the co-transfection of Gal4-PPARγ-LBD and UAS-TK-Luc into 293T cells was able to express the Gal4-PPARγ-LBD protein, and whether the potential compound was able to activate or inhibit PPARγ-LBD and its ability to influence the expression of TK-Luc. A transcriptional activation system was also carried out to investigate the transcription activity of PPARγ. As the co-transfection of pGL3-PPRE-Luc and pcDNA3.1-PPARγ into 293T cells results in PPARγ expression, whether the potential compound was able to activate or inhibit PPARγ activity, and increase or decrease the transcription of PPRE-Luc was investigated. In the mammalian one-hybrid and transcriptional activation systems, SV40 was used as the control for transfection efficiency.

Data are shown as mean ± standard error of the mean. A student’s t-test was performed for the comparison of two groups and one-way analysis of variance was carried out for the comparison of >2 groups by GraphPad Prism 5 software (GraphPad, San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, a mammalian one-hybrid method using a luciferase-reporter system was used for the experiment. Compound Q (Fig. 1A) was shown to depress the coactivator recruitment of PPARγ in a concentration-dependent manner (Fig 1B). In addition, with regard to transcriptional activation, Compound Q was revealed to reverse the transcriptional activity of the Ros-activated RXRα-PPARγ heterodimer in a concentration-dependent manner (Fig. 1C). The observations indicated that as a PPARγ antagonist, Compound Q is able to reduced the transcriptional activity of the RXRα-PPARγ heterodimer.

As PPARγ is a key moderator of lipid metabolism and a vital gene involved in adipocyte differentiation, a previous study reported that PPARγ agonists, such as Ros, are able to significantly promote adipocyte differentiation (14). Thus, the present study further investigated the effects of Compound Q on adipocyte differentiation.

Compound Q at various concentrations, or the negative or positive control, were added to the differentiation medium. At the end of the differentiation assay, staining with Oil Red O demonstrated that the positive control, Ros, was able to significantly enhance adipocyte differentiation, while Compound Q decreased the formation of grease drops in adipocytes and the formation of adipocytes in a concentration-dependent manner (Fig. 2). This indicates that Compound Q has the potential to inhibit triglyceride accumulation in adipose tissue and ameliorate obesity in obese patients.

Adipocyte differentiation is a complex process involving the regulation of multiple genes. In order to study the mechanism underlying the Compound Q-induced depression of adipocyte differentiation, quantitative PCR was used to analyze the effects of Compound Q on the expression levels of key genes involved in lipid metabolism. As shown in Fig. 3, following the culture of adipocytes for 24 h with Compound Q at various concentrations, the positive control (Ros) or the negative control (DMSO), the total RNA of the different cells was extracted. Following quantitative PCR, Ros was shown to enhance the expression levels of FAS, C/EBPα, aP2 and HMG-CoA. By contrast, treatment with Compound Q decreased the expression levels of the genes associated with lipid metabolism in a concentration-dependent manner.