Previously isolated and characterized human T-MSCs (8,24) were cultured in Dulbecco's modified Eagle's medium (DMEM) high-glucose medium (Welgene, Inc., Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS; Welgene, Inc.), 100 IU/ml penicillin and 100 µg/ml streptomycin (Welgene, Inc.). Cells were maintained at 37°C in a humidified 5% CO₂ atmosphere. When cells reached 80% confluency, fresh culture medium was added and the cells were incubated for an additional 48 h. T-MSC CM was harvested and concentrated 10-fold using 3-kDa cut-off Amicon Ultra centrifugal filter units (Merck KGaA, Darmstadt, Germany) at a speed of 3,800 × g for 30 min at room temperature.

A total of 12 male SAMP6 mice (age, 4 months; weight, 47.04±2.87 g) were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and maintained at 21–23°C with 51–54% humidity and a 12-h light/dark cycle under conventional conditions, with food and water supplied ad libitum. At 7 months of age (weight, 52.64±4.66 g), mice were divided into two groups (n=6 mice/group) and injected via tail vein with either culture medium alone (control) or culture supernatant that was collected and concentrated after a 48 h incubation of 0.8×10⁶ T-MSCs (T-MSC CM). Treatments were performed twice a week for 2 weeks using freshly prepared CM. Mice were sacrificed at 9 months of age by cervical dislocation, and whole blood and organs were harvested for further analysis. Experiments and procedures were approved by the Animal Ethics Committee at Ewha Womans University School of Medicine (Seoul, Korea; no. ESM 14-0278), and all experiments were performed in accordance with relevant guidelines and regulations.

Mouse epididymal adipose tissue (eAT) was isolated and fixed with a 4% paraformaldehyde solution overnight at 4°C. Paraffin-embedded eAT was sectioned at 4 µm and subjected to H&E staining. Briefly, cells were stained with 0.7% hematoxylin for 2 min and then with 5% eosin for 1.5 min at room temperature. Stained sections were observed using an Olympus BX50 light microscope (Olympus Corporation, Tokyo, Japan) and images were captured under ×100 magnification.

Blood samples (~200 µl) were collected in a tube containing heparin sodium (JW Pharmaceutical, Seoul, Korea) from the facial vein of 7- and 9-month-old mice following 6 h of fasting. Plasma proteins were separated by centrifugation at 1,000 × g for 10 min at room temperature. The adiponectin concentration was measured using the Adiponectin Total, HMW ELISA kit (cat. no. 47-ADPMS-E01; Alpco Diagnostics, Salem, NH, USA), according to the manufacturer's protocol.

The mouse preadipocyte cell line 3T3-L1 was purchased from the Korean Cell Line Bank (Seoul, Korea) and maintained in DMEM high-glucose medium supplemented with 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. After reaching 100% confluency, the medium was changed and cells were maintained for an additional 3 days. Cells were subsequently induced to differentiate to adipocytes (day 0). To induce adipocyte differentiation, high-glucose DMEM containing 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin supplemented with 2 mg/ml insulin, 0.25 µM dexamethasone and 0.5 mM 3-isobutyl-1-methylxanthine (all from Sigma-Aldrich; Merck KGaA) was used. On day 3, the medium was replaced with medium containing insulin only and cells were cultured for another 4 days at 37°C in a humidified 5% CO₂ atmosphere. Concentrated culture medium or T-MSC CM was added between days 7 and 10. On day 10, the cells were rinsed with PBS and incubated in serum-free medium overnight. Culture supernatants and cells were harvested the next day for further analyses. To induce oxidative stress in mature adipocytes, cells were pretreated with 50 mU/ml glucose oxidase (Sigma-Aldrich; Merck KGaA) for 2 h, followed by overnight treatment with T-MSC CM at 37°C.

To harvest adipocytes, the eAT was weighed and finely minced. Minced tissue was transferred to a 4-times greater volume of Hank's Balanced Salt Solution buffer (Welgene, Inc.) containing 1 mg/ml collagenase I (Sigma-Aldrich; Merck KGaA) and 0.28 M fatty acid-free bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA). Tissue digestion was performed by incubating the tissue sample for 2 h at 37°C in a shaking incubator. Digested tissue was filtered through a cell strainer and centrifuged at 100 × g for 15 min at room temperature. The floating adipocyte fraction was transferred to a new tube for RNA extraction.

To extract RNA from mouse eAT or 3T3-L1 adipocytes (9.5×10⁵), TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used. Tissue homogenization was performed using a TissueRuptor (Qiagen GmbH, Hilden, Germany), and following phase separation by centrifugation at 12,000 × g for 15 min at 4°C, RNA was isolated using NucleoSpin^® RNA Clean-up kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), according to the manufacturer's protocol. After determination of the concentration and purity of the RNA samples using BioPhotometer D30 (Eppendorf, Hamburg, Germany), 1 µg RNA was used for cDNA synthesis using a ReverTra Ace-α-kit (Toyobo Co., Ltd., Osaka, Japan). Reverse transcription was performed by incubation at 30°C for 10 min, at 42°C for 20 min and at 99°C for 5 min.

To determine the expression of target genes, a primer pair (0.4 µM) and SYBR-Green Real-time PCR Maser Mix (Toyobo Co., Ltd.) were mixed with the prepared cDNA. The primer sequences are listed in Table I. Amplification was performed in duplicate by 40 cycles of 15 sec denaturation step at 95°C and a 1 min amplification and signal acquisition step at 60°C using StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Cycle threshold (Cq) values were obtained, and the relative expression level of a target gene was determined as 2 ^{(cyclophilin Cq-target gene Cq)} (25).

Whole protein lysates were isolated from mouse eAT or 3T3-L1 adipocytes (9.5×10⁵) using PRO-PREP Protein Extraction Solution (Intron Biotechnology, Inc., Seongnam, Korea). Protein concentrations were determined using the Bradford assay with the Bio-Rad Protein assay solution (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Samples (4 µg for tissue lysates and 10 µg for cell lysates) were resolved by 10% SDS-PAGE and transferred to Immobilon-P polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk in TBS containing 0.1% Tween-20 (TBS-T) solution for 1 h at room temperature and were subsequently incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Adiponectin (1:1,000, diluted in 5% skim milk containing TBST; cat. no. ab25891; rabbit; Abcam, Cambridge, UK); and β-actin [1:2,000, diluted in 2% BSA (Bovogen Biologicals, Pty, Ltd., East Keilor, Victoria, Australia) containing TBST; cat. no. A1978; mouse; Sigma-Aldrich; Merck KGaA]. The membranes were washed 3 times for 10 min in TBST and incubated with anti-rabbit (cat. no. BR170-6515; Bio-Rad Laboratories, Inc.) or anti-mouse (cat. no. BR170-6516; Bio-Rad Laboratories, Inc.) horseradish peroxidase-conjugated secondary antibodies (1:3,000, diluted in TBST) for 1 h at room temperature. Following incubation, membranes were washed 3 times for 10 min in TBST and developed using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce; Thermo Fisher Scientific, Inc.). Images were obtained using ImageQuant LAS 4000 (GE Healthcare Life Sciences, Little Chalfont, UK).

To detect secreted adiponectin in cell culture medium, conditioned medium was prepared by incubating differentiated 3T3-L1 adipocytes (3.8×10⁵) in serum-free DMEM (500 µl) for 18 h. Conditioned medium was concentrated 10-fold using 3-kDa cut-off Amicon Ultra centrifugal filter units (Merck KGaA) and centrifugation at 14,000 × g for 15 min at room temperature. Concentrated medium (50 µl) was prepared in sample buffer (0.5 M Tris/HCl pH 6.8, 25% glycerol, 10% SDS, 500 mM DTT, 1% bromophenol blue; Sigma-Aldrich; Merck KGaA) and 12 µl samples were separated on 10% SDS-PAGE for the detection of total secreted adiponectin. To detect adiponectin in multimer forms, concentrated medium was prepared in non-reducing and non-heat-denaturing conditions using sample buffer lacking reducing agents, and 12 µl samples were subsequently separated on 4–15% precast polyacrylamide gradient gels (Bio-Rad Laboratories, Inc.). Blots were semi-quantified by densitometric analysis using UN-SCAN-IT gel analysis software version 6.1 (Silk Scientific, Inc., Orem, UT, USA).

ROS and RNS production in total cell lysates or culture medium was measured using OxiSelect^™ In Vitro ROS/RNS assay kit (Cell Biolabs, Inc., San Diego, CA, USA), according to the manufacturer's protocol. Cellular ROS/RNS levels were normalized to total protein measured by the BCA Protein assay kit (Pierce; Thermo Fisher Scientific, Inc.).

Student's t-test or one-way analysis of variance with Dunnett's multiple comparisons test was performed for statistical analysis using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as the mean ± standard error of the mean from 3–4 independent experiments. P<0.05 was considered to indicate a statistically significant difference.

In our previous study, we demonstrated that T-MSC CM produces anti-adipogenic effects. In the present study, these in vitro findings were further evaluated in a mouse model of accelerated senescence (SPAM6 mice), as these mice have been reported to exhibit an obese phenotype (26). Control culture medium or T-MSC CM was injected into 7-month-old SAMP6 mice, and body weight changes were examined 2 months later. A significant decrease in the weight of mice was observed at 9 months in mice injected with T-MSC CM, whereas the mice injected with the control medium did not exhibit alterations in weight (Fig. 1A). Isolated organs were weighed and normalized to the body weight. T-MSC CM injection led to reduced eAT mass compared with control mice, whereas the inguinal adipose tissue, liver and muscle mass were not affected by injection of T-MSC CM (Fig. 1B). Histological analysis of eAT demonstrated a reduction in the size of adipocytes in the T-MSC CM-injected mice compared with control mice (Fig. 1C). To determine the mRNA expression of adipogenic markers in eAT adipocytes, collagen digestion was performed, followed by floating adipocyte fraction separation. Together with the reduction in eAT mass and adipocyte size, the mRNA expression of the adipogenic markers peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and leptin were significantly decreased, while adiponectin expression was increased in eAT adipocytes, compared with control-treated mice (Fig. 1D).

As weight reduction is associated with an increase in circulating adiponectin levels, the present study investigated total and HMW adiponectin in mouse plasma prior to (7 months) and following (9 months) treatment with T-MSC CM. A significant increase in total adiponectin following T-MSC CM treatment was observed, while levels of adiponectin were significantly decreased in the mice injected with control medium (Fig. 2A). In addition, levels of HMW adiponectin were increased following T-MSC CM injection, although this was not statistically significant (Fig. 2B). Total and HMW adiponectin levels in mouse plasma were normalized to the eAT mass and the results demonstrated a significant increase in the levels of total and HMW adiponectin following treatment with T-MSC CM, compared with control-treated mice (Fig. 2C). Furthermore, adiponectin protein levels in eAT lysates were significantly increased by T-MSC CM treatment compared with control treatment (Fig. 2D). Non-reduced and non-denatured tissue lysates were used to determine adiponectin multimerization. These experiments indicated that T-MSC CM enhances formation of adiponectin multimers (Fig. 2E). In addition, the present study investigated whether oxidative stress in the adipose tissue, an established negative regulator of adiponectin expression in aging, is modulated in the presence or absence of T-MSC CM injection (27). The mRNA expression of subunits of NADPH oxidase was investigated by RT-qPCR, and the results demonstrated a significant reduction of p40^phox and gp91^phox in eAT from mice injected with T-MSC CM compared with controls (Fig. 2F).

The current study confirmed these in vivo findings in vitro by performing similar experiments with the murine adipocyte cell line 3T3-L1. Preadipocytes were induced to differentiate to adipocytes for 7 days and subsequently treated with control medium or T-MSC CM for an additional 3 days. Total RNA or whole cell lysates were collected, and adiponectin expression was examined by RT-qPCR and western blot analysis. No significant changes in the cellular levels of adiponectin mRNA or protein were observed following treatment with T-MSC CM, compared with controls (Fig. 3A and B), however, adiponectin secretion into the culture medium was significantly increased in cells treated with T-MSC CM compared with controls (Fig. 3C). Adiponectin multimer formation was also investigated under non-reduced and non-heat-denatured conditions. T-MSC CM treatment slightly increased the formation of HMW adiponectin by differentiated 3T3-L1 adipocytes (Fig. 3D).

In order to extend the in vivo findings of the reduction in NADPH oxidase subunit transcription, we induced oxidative stress in mature 3T3-L1 adipocytes using glucose oxidase (28). Cells were differentiated for 10 days, challenged with glucose oxidase for 2 h and treated with control medium or T-MSC CM for the following 24 h. Cell culture medium and cells were collected and ROS/RNS production was measured. The results demonstrated that enhanced ROS/RNS generation in glucose oxidase-treated cells was significantly ameliorated in cells incubated with T-MSC CM, in culture medium and cells (Fig. 4A and B). Adiponectin expression was also determined, and T-MSC CM treatment was not able to reverse reduced mRNA expression induced by glucose oxidase (Fig. 4C), however, HMW adiponectin formation was restored to a certain extent in cells treated with T-MSC CM (Fig. 4D).