LPS was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and was diluted in pyrogen-free 0.9% saline. Insulin was also purchased from Sigma-Aldrich (Merck KGaA), T0070907 was purchased from Selleck Chemicals (Houston, TX, USA). TRIzol reagent was obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Transcriptor First Strand cDNA Synthesis kit was purchased from Roche Diagnostics (Indianapolis, IN, USA). Anti-APN (ab22554), anti-PPARγ (ab45036), anti-Pref-1 (ab21682) and anti-CCAAT/enhancer binding protein α (C/EBPα) (ab40764) antibodies were purchased from Abcam (Cambridge, MA, USA); anti-β-actin antibody (KL002) was obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China); and horseradish peroxidase-labeled immunoglobulin G secondary antibody (goat anti-mouse lgG; SA00001-1 and goat anti-rabbit lgG; SA00001-2) was purchased from ProteinTech Group, Inc. (Chicago, IL, USA).

Patients provided written informed consent prior to tissue collection, and the present study was approved by the Ethics Committee of Human Investigation of Union Hospital, Huazhong University of Science and Technology (Wuhan, China), and conformed to the Helsinki Declaration. Donation procedures complied with the laws of China, and the specimens obtained were registered with the relevant governmental authorities of Hubei (China). Between March 2013 and August 2013, a total of 34 patients (40–75 years old, 25 male and 9 female) who had been planning cardiac surgery initially underwent selective coronary angiography (CAG). The patients were then divided into a CAD group (n=23) and a non-CAD control group (n=11), according to the results of CAG. EAT biopsy samples (average weight, 0.5–1.0 g) were collected from aortic root near the right coronary artery and were shock-frozen and immediately stored in liquid nitrogen for total RNA extraction. The mRNA expression levels of PPARγ, Pref-1 and C/EBPα were detected by qPCR.

The 3T3-L1 murine preadipocyte cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 3T3-L1 preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM; (Tianjin Hao Yang Biological Products Technology Co., Ltd., Tianjin, China) supplemented with 10% fetal bovine serum (FBS) (TBD) and penicillin (100 IU/ml), and streptomycin (100 mg/ml) (Wuhan Boster Biological Technology, Ltd., Wuhan, China) under an atmosphere of 5% CO₂ in air (v/v) at 37°C. The medium was replaced every 2–3 days. Differentiation was induced by replacing the medium with DMEM supplemented with 10% FBS, and 0.5 mM 3-isobutyl-1-methylaxanthine, 0.5 mM dexamethasone and 5 mg/ml insulin (MDI; Sigma-Aldrich; Merck KGaA). After 48 h of MDI induction, differentiation medium was replaced with DMEM supplemented with 10% FBS and 5 mg/ml insulin. The medium was then replaced every other day with DMEM containing 10% FBS until day 8. On day 8, the preadipocytes became mature adipocytes (14).

Cloning of human adiponectin gene from human adipose tissue and an adenovirus expressing apM1 (Ad-apM1) was assembled using pAxCAwt [Cyagen Biosciences (Guangzhou) Inc., Guangzhou, China]. The sequence was co-transfected into 293 cells (the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China) alongside Ad5DNA-TPC [Cyagen Biosciences (Guangzhou) Inc.] using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. The recombinant adenovirus was produced through homologous recombination. After 48 h, the sample was centrifuged for 20 min at 3,960 × g at 4°C and the viral supernatant was collected. High speed centrifugation was used to concentrate virus particles by ultra-speed centrifuge (Beckman, Fullerton, CA, USA) at 5,000 × g for 2.5 h. Virus was packed and stored at −80°C. Tissue culture infective dose 50 was used to determine virus titer. Western blotting was performed to verify the effects of APN overexpression on protein expression levels following infection of 3T3-L1 preadipocytes with the adenovirus (Fig. 1).

In a 6 wells Transwell plate (Corning, Corning, NY, USA), 3T3-L1 preadipocytes (1×10⁵ cells) in the inner chamber, were co-cultured with mature adipocytes (2×10⁵ cells) in outer chamber, which were differentiated from 3T3-L1 preadipocytes. The cells were divided into the following experimental groups: i) The control group, in which 3T3-L1 preadipocytes were induced to differentiate into adipocytes in the inner chamber, as aforementioned. Mature adipocytes were cultured in the outer chamber, with the addition of PBS, equal in volume to LPS in group ii. ii) The LPS stimulation group, in which mature adipocytes were stimulated with LPS (1 µg/ml, 18 h) to induce inflammation, as described in previous studies (15,16). The mature adipocytes in the outer chamber were co-cultured with 3T3-L1 preadipocytes in the inner chamber, which were induced to differentiate into mature adipocytes using MDI, as aforementioned. iii) The human APN recombinant adenovirus group (LPS + Ad-apM1), in which preadipocytes were infected with Ad-apM1 [multiplicity of infection (MOI), 100], as previously described (17). After a 48-h infection with the adenovirus preadipocytes were induced into mature adipocytes and were co-cultured with LPS-stimulated mature adipocytes. iv) The negative control (NC) group, in which preadipocytes were infected with an adenovirus containing empty plasmids (MOI, 100) as an NC; after 48 h, the cells were induced to differentiate into mature adipocytes and were co-cultured with mature adipocytes stimulated with LPS. v) The PPARγ-inhibited group (LPS + Ad-apM1 + T0070907), in which preadipocytes were infected with Ad-apM1 as aforementioned and were treated with 10 µM T0070907 24 h prior to being induced to differentiate into mature adipocytes via co-culturing with LPS-treated mature adipocytes. The time point and titer of adenoviruses and the dose of T0070907 used in the present study were determined during preliminary experiments. At day 8, subsequent experiments on the 3T3-L1 cells in the inner chamber were performed.

Cell viability of preadipocytes was determined following differentiation in the inner chamber using an MTT assay (Promega Corporation, Madison, WI, USA). MTT was directly dissolved in cell culture medium and was then incubated with live cells. Briefly, cells in the exponential growth phase were plated at 1×10⁴ cells/well in a 96-plate, and were incubated at 37°C with 20 µl MTT (5 mg/ml) in 100 µl cell culture medium for 4 h. After 4 h, the supernatant was discarded and 150 µl of DMSO was added to each well. The vortex was rotated for about 10 min to allow the crystals to dissolve. The absorbance of each well was measured at a wavelength of 490 nm using a Spectra Max Paradigm microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

Oil Red O (Sigma-Aldrich; Merck KGaA) staining was performed according to the manufacturer's protocol. A total of 8 days after the induction of adipocyte differentiation, adipocytes in the inner chamber were washed three times with PBS and were fixed with 10% formalin for 1 h at room temperature. The dishes were washed once with 60% isopropanol and were then left to dry completely. Subsequently, the cells were stained with 2 ml Oil Red O for 2 h at 37°C, rinsed with 60% isopropanol, and were thoroughly washed four times with PBS. Finally, images were captured using an inverted microscope (magnification, ×400). After extracting Oil Red O with 100% isopropanol, the absorbance of the extracted dye was determined spectrophotometrically at 490 nm wavelength using a Spectra Max Paradigm microplate reader (Molecular Devices, LLC) (18).

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from EAT and adipocytes in the inner chamber after 8 days of differentiation according to the manufacturer's protocol. RT was conducted using the Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics) according to the manufacturer's protocol. The oligonucleotide primer sequences, which were designed using Premier Primer 5.0 software (Premier Biosoft International, Palo Alto, CA, USA), are presented in Table I. β-actin was used as an internal control. The synthesized first-strand cDNA samples were subjected to qPCR using SYBR-Green PCR Master mix (Toyobo Life Science, Osaka, Japan) and PCR was performed using an ABI Prism 7700 Sequence Detector (Applied Biosystems; Thermo Fisher Scientific, Inc.). Thermocycling conditions were as follows: 2 min at 95°C followed by 40 cycles of 15 sec at 95°C, 15 sec at 60°C, 20 sec at 72°C, with a final extension step of 60°C for 30 min. Integrity of the PCR products was confirmed by dissociation curve analysis using 7500 Software version 2.0.4 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The quantification cycle (Cq) values were determined and the 2^−ΔΔCq method (19) was used to calculate relative gene expression.

Following 8 days of differentiation, cells harvested from the inner chamber were lysed with radioimmunoprecipitation assay lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA. The extracts were then centrifuged at 10,140 × g for 15 min at 4°C to remove insoluble material. Total protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. An equivalent amount of total protein (40 µg) per well was diluted with sample buffer containing 100 mM dithiothreitol and was heated at 98°C for 5 min. Subsequently, proteins were separated by 10–15% SDS-PAGE using gel apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and were transferred to polyvinylidene fluoride membranes. The membranes were then soaked in 5% non-fat dry milk for 2 h at room temperature and were incubated overnight at 4°C with primary antibodies (1:500) against β-actin, APN, PPARγ, Pref-1and C/EBPα. Subsequently, the membranes were washed with Tris-buffered saline containing 0.05% Tween (TBS-T) and were incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000) with agitation at room temperature for 1 h. The membranes were washed three times with TBS-T (20 min/wash) and immune complexes were visualized by enhanced chemiluminescence (Tiangen Biotech Co., Ltd., Beijing, China); band intensity was finally measured and semi-quantified (20). The resulting images were analyzed with Quantity One version 4.62 (Bio-Rad Laboratories, Inc.).

Data are presented as the mean ± standard error of the mean. The variance homogeneity test and one-way analysis of variance were performed by SPSS version 20.0 (IBM Corp., Armonk, NY, USA). The least significant difference method was used to compare between groups. P<0.05 was considered to indicate a statistically significant difference.

The mRNA expression levels of PPARγ, Pref-1 and C/EBPα in EAT were detected by qPCR. As presented in Fig. 2, compared with in the non-CAD control group, the mRNA expression levels of PPARγ and C/EBPα were significantly downregulated in the EAT from patients with CAD, whereas Pref-1 was upregulated. These results suggested that PPARγ, Pref-1 and C/EBPα may be involved in the occurrence and development of atherosclerosis.

In order to determine the effects of APN on the viability of 3T3-L1 cells co-cultured with LPS-stimulated adipocytes, an MTT assay was conducted. As presented in Fig. 3, APN overexpression significantly increased 3T3-L1 cell viability, whereas it was decreased in the LPS group. Conversely, the effects of APN on cell viability were reversed by T0070907, which is a selective inhibitor of PPARγ.

The differentiation of preadipocytes was examined by Oil Red O staining. As shown in Fig. 4A, cell volume and the number of lipid droplets was reduced in 3T3-L1 cells on day 8 following MDI-induced differentiation and co-culture with LPS-stimulated adipocytes compared with in the control group. However, this situation was markedly reversed in the LPS + Ad-apM1 group. As expected, the effects of APN were markedly attenuated in the NC and LPS + Ad-apM1 + T0070907 groups.

Following extraction of Oil Red O with 100% isopropanol, absorbance of the extracted dye was determined spectrophotometrically. As shown in Fig. 4B, the optical density (OD) value was markedly decreased in the LPS group compared with in the control group (P<0.05), whereas it was significantly increased in the LPS + Ad-apM1 group compared with in the LPS group (P<0.05). Conversely, the OD values were significantly decreased in the NC and LPS + Ad-apM1 + T0070907 groups.

The mRNA expression levels of MCP-1, IL-6, IL-8 and TNF-α were detected in 3T3-L1 preadipocytes by qPCR. As shown in Fig. 5, the mRNA expression levels of MCP-1, IL-6, IL-8 and TNF-α were significantly increased in the LPS group compared with in the control group (P<0.05). Conversely, the mRNA expression levels of MCP-1, IL-6, IL-8 and TNF-α were decreased in the APN overexpression group compared with in the LPS group (P<0.05). However, the effects of APN overexpression were markedly attenuated in the NC and LPS + Ad-apM1 + T0070907 groups.

To further investigate whether PPARγ expression was altered in response to APN overexpression, the protein expression levels of PPARγ were detected by western blotting. As shown in Fig. 6, the protein expression levels of PPARγ were significantly increased in the LPS + Ad-apM1 group compared with in the LPS group (P<0.05). However, there was a marked decrease in the expression of PPARγ in the NC and LPS + Ad-apM1 + T0070907 groups. These results suggested that APN may promote the differentiation of preadipocytes co-cultured with LPS-stimulated mature adipocytes via the upregulation of PPARγ.

As shown in Fig. 7, the inflammatory environment markedly increased Pref-1 expression compared with in the control group (P<0.05), whereas APN overexpression decreased the expression levels of Pref-1 compared with in the LPS group. Conversely, the expression levels of Pref-1 were significantly increased in the NC and LPS + Ad-apM1+T0070907 groups (P<0.05). Furthermore, the expression levels of C/EBPα were significantly downregulated in the LPS group compared with in the control groups (P<0.05), whereas its expression was increased in the LPS + Ad-apM1 group compared with in the LPS group (P<0.05). However, the expression levels of C/EBP α were markedly decreased in the NC and LPS + Ad-apM1 + T0070907 groups.