3T3-L1 mouse fibroblast cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Tissue culture reagents, including Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum (CS) and fetal bovine serum (FBS), were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Antibodies against adiponectin, phospho-c-Jun N-terminal kinases (p-JNK; Thr183/Tyr185), total JNK (T-JNK), phospho-p38 mitogen-activated protein kinases (p-p38 MAPK; Thr180/Tyr182), total p38 MAPK (T-p38 MAPK), phospho-p44/42 MAPK [phospho-extracellular signal-related kinases 1 and 2 (p-ERK1/2); Thr202/Tyr204] and total p44/42 MAPK (T-ERK1/2) were rabbit monoclonal antibodies and were obtained from Cell Signaling Technology (Beverly, MA, USA). Unless otherwise noted, all other chemicals were purchased from Sigma (St. Louis, MO, USA).

Murine 3T3-L1 cells were plated in six-well plates and maintained in high glucose (HG)-DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Two days following 100% confluence, preadipocytes were induced to differentiate by using MDI media containing 520 μM isobutylmethylxanthine, 1 μM dexamethasone and 1 μg/ml insulin in DMEM containing 10% fetal bovine serum (FBS). After two days, the medium was replaced with DMEM containing 10% FBS, antibiotics and insulin (insulin media). Until >95% of the cells contained lipid droplets, HG-DMEM with 10% FBS was replaced every two days. Differentiated 3T3-L1 adipocytes were serum-starved in serum-free DMEM (free media) prior to the TNF-α treatment. Fig. 1 illustrates the experimental design for TNF-α treatment and the subsequent wash-off period with/without the inhibitor. The cells were treated with 10 ng/ml TNF-α for the indicated incubation times [16, 32 h or 48 h (Fig. 1A, C and F, respectively)], rinsed and then incubated with DMEM containing 10% FBS (complete media) for the indicated periods according to TNF-α incubation time. In the adipocytes that received 16 h TNF-α treatment, the wash-off incubation was 16 h (Fig. 1B). TNF-α-treated 3T3-L1 adipocytes for 32 h were cultured with complete media for either an additional 16 h (Fig. 1D) or 32 h (Fig. 1E). DMEM containing 10% FBS was used for either 16 h (Fig. 1G) or 48 h (Fig. 1H) following 48 h of TNF-α incubation.

Total RNA was isolated using the RNA Mini kit (Invitrogen Life Technologies) and cDNA was prepared using the high-capacity cDNA kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. The reactions were incubated initially at 37°C for 60 min and subsequently at 95°C for 5 min. The PCR primer design for qPCR was performed using the Universal Probe Library (UPL) software (Roche Applied Science, Mannheim, Germany). qPCR was conducted using the Roche real-time PCR master mix in combination with UPL in at least triplicate using a Roche Lightcycler 480 (Roche Applied Science) as follows: one cycle of pre-denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 sec and annealing at 60°C for 20 sec, followed by one cycle of extension at 40°C for 30 sec. The expression levels were determined using the ΔΔCt method. The following sense and antisense primers were used for adiponectin: 5′-GGAGAGAAAGGAGATGCAGGT-3′ and 5′-CTTTCCTGCCAGGGGTTC-3′; and for β-actin: 5′-ACTGCTCTGGCTCCTAGCAC-3′ and 5′-CCACCGATCCACACAGAGTA-3′.

Differentiated 3T3-L1 adipocytes were serum-starved for 6 h prior to treatment. Cells were incubated with TNF-α for 16, 32 or 48 h. After the indicated time periods, the cells were washed with serum-free DMEM and re-fed with HG-DMEM containing the ERK1/2 inhibitor (PD98059; 50 μM) for the indicated times (Fig. 1). The cells were washed in ice-cold PBS and lysed in radioimmunoprecipitation assay lysis buffer (Amresco, Solon, OH, USA) containing freshly added protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma). The protein concentrations were measured using the bicinchoninic acid assay (Pierce Biotechnology, Inc., Rockford, IL, USA). Bromophenol blue and NuPage reducing agent (Invitrogen Life Technologies) were added to the cell lysates and this mixture was heated at 95°C for 5 min. Equal protein levels were loaded into each lane of a 4–20% SDS polyacrylamide gel. The proteins were separated by electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) with the Trans-Blot apparatus (Bio-Rad, Hercules, CA, USA). For immunoblotting, the membranes were blocked in 5% non-fat dry milk in TBST (0.05% Tween 20, 50 mM Tris-HCl, pH 7.5 and 150 mM NaCl), washed twice and incubated overnight with primary antibodies. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The signal was detected using the Amersham enhanced chemiluminescence plus system (Amersham-Pharmacia Biotech, Arlington Heights, IL, USA). Densitometric analysis of the individual bands was performed with Geliance Imaging software (PerkinElmer Life and Analytical Sciences, Boston, MA, USA). The data presented are representative of at least three independent experiments with similar results.

Data are expressed as mean ± standard error (SE). Statistical significance was assessed using PASW Statistics 18 (SPSS, Inc., Chicago, IL, USA). Student’s t-test or analysis of variance was performed to compare between the groups. P<0.05 was considered to indicate a statistically significant difference between values.

To investigate the impact of TNF-α on adiponectin, the mRNA expression was first measured in differentiated 3T3-L1 adipocytes. TNF-α significantly suppressed adiponectin gene expression in a dose- and time-dependent manner as compared with the control group. There was a significant reduction in mRNA levels by 37% following 8 h incubation at 10 ng/ml (Fig. 2). The maximal inhibition of adiponectin mRNA expression was 62%, observed after 16 h incubation at a TNF-α concentration of 10 ng/ml. There was no significant difference between the expression from the 16 h and 24 h incubation periods.

To investigate the reversibility in the inhibitory effects of the proinflammatory adipokine, TNF-α, on adiponectin mRNA expression, the effect of TNF-α at different exposure and removal time periods in differentiated 3T3-L1 adipocytes was examined. After three different TNF-α incubation periods (16, 32 and 48 h), the cells were rinsed and re-fed with HG-DMEM containing 10% FBS for an additional time as follows: i) 16 h wash-off incubation following 16 h TNF-α treatment; ii) 16 or 32 h recovery period following 32 h TNF-α treatment and iii) 16 or 48 h TNF-α removal following 48 h TNF-α exposure (Fig. 1). Chronic TNF-α incubation significantly reduced adiponectin gene expression by 60% compared with the untreated control cells (Fig. 3). Following removal of TNF-α from the medium, there was a time-dependent reversal of the TNF-α-mediated reduction in adiponectin levels. In Fig. 3, column 3, the 16 h wash-off phase completely reversed the decrease in adiponectin levels following 16 h of TNF-α treatment, returning to the levels observed in the control. The 16 h TNF-α withdrawal did not induce a significant effect (column 6), but the 32 h wash-off period fully reversed the decrease in adiponectin expression following the 32 h TNF-α treatment (column 7). Similar results were observed for 48 h TNF-α incubation, a 16 h restoration did not have a significant impact (column 10); however, a 48 h wash-off period fully reversed the negative effect of TNF-α on adiponectin mRNA expression (column 11). These findings suggested that the inhibitory effect of TNF-α on adiponectin is reversible and the adverse effects are dependent on the length of TNF-α exposure and the subsequent recovery period.

There is a positive correlation between TNF-α and activation of three different MAPKs, JNK, ERK1/2 and p38 MAPK, in the development of obesity-associated insulin resistance. Therefore, in the present study, the effect of a wash-off period following exposure to the proinflammatory cytokine TNF-α and the subsequent activation of MAPKs was examined. Similar to the pattern of adiponectin mRNA expression (Fig. 4A), the decrease in adiponectin protein expression after 32 h treatment with TNF-α was completely reversed following an equivalent (32 h) wash-out period (Fig. 4B and C). In addition, the reduction in adiponectin levels following 32 h TNF-α treatment was concomitantly accompanied by activation of JNK, ERK1/2 and p38 MAPK, expressed as phospho-protein/total protein. A 16 h wash-off phase following 32 h TNF-α-treatment reversed the TNF-α-activated JNK and p38 MAPK, but not ERK1/2 (Fig. 4B). ERK1/2 activation was attenuated following a 32 h wash-off period (Fig. 4B and D). This restoration pattern of ERK1/2 in response to chronic TNF-α exposure and wash-off was similar to that of adiponectin protein and mRNA expression. These results suggested that the effect of TNF-α on adiponectin expression was reversible through ERK activation.

To delineate the effects of the ERK pathway on the recovery of adiponectin expression following TNF-α exposure, 3T3-L1 adipocytes were treated with TNF-α (32 h, 10 ng/ml) and co-treated with the ERK inhibitor PD98059 (50 μM) in HG-DMEM containing 10% FBS for an additional restoration time period (16 or 32 h) (Fig. 5). The 16 h recovery period did not completely reverse the inhibitory effects of 32 h TNF-α treatment, whereas the use of PD98059 during this recovery period fully restored adiponectin levels to the control levels. The recovery effect of the ERK inhibitor on adiponectin levels was equivalent to the effect of the 32 h wash-off time without the inhibitor. However, there were no additional effects on restoration in the 32 h restoration phase with PD98059. Furthermore, the addition of the ERK1/2 inhibitor during the 16 h restoration period significantly increased the protein expression of adiponectin compared with the control and concomitantly reduced ERK activation.