AdipoRon, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), dexamethasone (Dex), 3-isobutyl-1-methylxanthine (IBMX), indomethacin, insulin and Oil Red O were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Minimum essential Eagle's medium with Earle's Balanced Salts (MEM-EBSS) was purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Monoclonal antibodies, including rabbit anti-PPARγ (cat no. 2443), rabbit anti-FABP4 (cat no. 3544), rabbit anti-C/EBPβ (cat no. 3087), rabbit anti-C/EBPα (cat no. 2295), rabbit anti-AMPKα (cat no. 5831), rabbit anti-phosphorylated-acetyl-CoA carboxylase (p-ACC; cat no. 11818), rabbit anti-adiponectin (cat no. 2789) and rabbit anti-GAPDH (cat no. 5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal anti-p-AMPKα1/2 antibody (cat no. sc-33524) and goat polyclonal anti-AdipoR1 antibody (cat no. sc-46748) and anti-AdipoR2 antibody (cat no. sc-46751) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

The C3H10T1/2 mouse embryonic mesenchymal stem cell line was purchased from the Chinese Academy of Medical Sciences (Beijing, China). Cells were cultured in MEM-EBSS supplemented with 10% FBS at 37°C in a 5% CO₂ atmosphere until adipocyte differentiation. Two days after reaching confluence (day 0), the C3H10T1/2 cells were cultured in differentiation medium (DM; MEM/EBSS containing 10% FBS, 1 µM Dex, 0.5 mM IBMX, 50 mM indomethacin and 1 µg/ml insulin) for 2 days to induce differentiation. Following incubation, MEM/EBSS supplemented with 10% FBS and 1 µg/ml insulin was added for 2 days as a differentiation medium and was changed every 2 days.

Cell viability was detected by using a CCK-8 kit, according to the manufacturer's instructions. Briefly, C3H10T1/2 cells were seeded in 96-well plates at a density of 5×10⁴ cells/ml at 37°C in a humidified 5% CO₂ atmosphere and treated with increasing doses of AdipoRon (1, 5, 10, 20 and 40 µM) for 24, 48 or 72 h. Subsequently, 10 µl of kit reagent was added to each well and incubated at 37°C for 2 h. The plates were scanned with a microplate reader (Benchmark; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm.

Cells were cultured in 24-well plates with a seeding density of 5×10⁴ cells/ml and differentiated as described above. On day 8, the cells were stained with ORO as previously described (2). Briefly, the cells were fixed with 10% formalin (Solarbio, Beijing, China) at 4°C for 30 min and stained with ORO at room temperature for 30 min following washes with phosphate-buffered saline. Oil droplets in the cells were observed under an inverted microscope (Olympus-CKX41; Olympus Corporation, Tokyo, Japan). The ORO-stained plates were then washed and treated with 100% isopropanol (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and lipid accumulation was detected by measuring the absorbance at 490 nm using a Benchmark microplate reader (Bio-Rad Laboratories, Inc.).

Cells were cultured in 6-well plates with a seeding density of 5×10⁴ cells/ml and differentiated as described above. Cells were lysed in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) supplemented with a protease inhibitor cocktail and phosphatase inhibitors (Roche Molecular Diagnostics, Pleasanton, CA, USA) for 20 min and centrifuged at 12,000 × g for 20 min at 4°C. Protein concentration was determined using the Bicinchoninic Acid Protein Assay reagent kit (Beijing Solarbio Science & Technology Co., Ltd.). A total of 30 µg of protein lysates from each sample were separated on 10 or 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was incubated with 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween-20 (TBST) (both from Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, followed by hybridization with primary antibodies against PPARγ (1:1,000), C/EBPβ (1:1,000), C/EBPα (1:1,000), FABP4 (1:2,000), adiponectin (1:500), AMPKα (1:1,000), p-ACC (1:500), GAPDH (1:1,000), p-AMPKα1/2 (1:200), AdipoR1 (1:200) and AdipoR2 (1:200) overnight at 4°C. Following three washes with TBST, the membrane was incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (cat no. sc2004; 1:10,000; Santa Cruz Biotechnology, Inc.) or a horseradish peroxidase-conjugated rabbit anti-goat IgG secondary antibody (cat no. ZB-2306; 1:10,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) for 1 h at room temperature. Following three washes with TBST, the bands were visualized with an Enhanced Chemiluminescence Substrate (Thermo Fisher Scientific, Inc.). Densitometry of the western blot bands was performed using ImageJ software version 1.31 (National Institutes of Health, Bethesda, ML, USA).

Cells were cultured in 6-well plates with a seeding density of 5×10⁴ cells/ml and differentiated as described above. RNA was extracted from C3H10T1/2 cells on day 8 using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A High Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to synthesize the cDNA, according to the manufacturer's protocol. mRNA expression levels were determined using an ABI 7500 Fast real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and a SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers were obtained from Genotech (Shanghai, China) and the sequences are provided in Table I. GAPDH was used as the internal control. qPCR was performed using the following previous described cycling conditions (15): Initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Each experiment was performed in triplicate. Gene transcript levels were calculated using the 2^−ΔΔCq method (16).

All experiments were performed at least three times. The results are expressed as the mean ± standard deviations (SD). The data were analyzed using Student's t-test or one-way analysis of variance followed by least significance difference test. All analyses were performed with GraphPad Prism version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Following treatment for 24, 48 or 72 h, AdipoRon exhibited no significant effects on C3H10T1/2 cell viability at concentrations ≤40 µM (Fig. 1A). Therefore, 5–20 µM AdipoRon was used in all subsequent experiments. The antiadipogenic effects of AdipoRon treatment were not due to cytotoxicity. C3H10T1/2 cells were induced into adipocytes for 8 days in the presence or absence of various doses of AdipoRon (0–20 µM) to investigate the effects of AdipoRon on adipocyte differentiation. Based on the results of ORO staining, lipid accumulation in C3H10T1/2 cells was dose-dependently decreased by AdipoRon (Fig. 1B and C). Protein expression of the adiponectin receptors, AdipoR1 and AdipoR2, were detected in C3H10T1/2 cells (Fig. 1D).

Adipogenesis is controlled by numerous transcription factors; the present study examined the effects of various concentrations of AdipoRon (0–20 µM) on the mRNA expression levels C/EBPβ, C/EBPα and PPARγ, to investigate whether AdipoRon inhibits adipogenesis by downregulating the expression of these adipogenic transcription factors. AdipoRon treatment significantly reduced the expression levels of the C/EBPβ, C/EBPα and PPARγ mRNAs in a dose-dependent manner (Fig. 2A-C). In addition, treatment with 20 µM AdipoRon was used to examine the expression levels of the C/EBPβ, C/EBPα and PPARγ proteins. The expression levels of the C/EBPβ, C/EBPα and PPARγ proteins were decreased by 20 µM AdipoRon compared with those of the controls (Fig. 2D).

As AdipoRon treatment significantly downregulated the expression of adipogenic transcription factors, the effects of various concentrations of AdipoRon on the expression levels of adipogenesis-related genes were also examined. AdipoRon exposure dose-dependently suppressed the mRNA expression of FAS, leptin, stearoyl-CoA desaturase (SCD)-1, adiponectin and FABP4 (Fig. 3A-E). Consistent with the changes in mRNA expression levels, 20 µM AdipoRon decreased the expression levels of the adiponectin and FABP4 proteins (Fig. 3F).

AdipoRon treatment significantly increased the levels of p-AMPK and p-ACC, which is the downstream target of AMPK (Fig. 4A-C). The AMPK activator AICAR was used as a positive control to further explore the association between the activation of AMPK signaling and the antiadipogenic effects mediated by AdipoRon. As shown in Fig. 4D and E, both AICAR (1 mM) and AdipoRon (10 and 20 µM) were able to activate the AMPK signaling pathway. Both AICAR and AdipoRon treatments were also able to significantly reduce lipid accumulation and the expression of adipogenesis-related genes, PPARγ and adiponectin, in C3H10T1/2 cells (Fig. 4F-H).