High-glucose DMEM, BCS, and FBS were purchased from Hyclon (Logan, Utah, USA). Trypsin/EDTA, insulin, dexamethasone, IBMX, and MTT which is used in cell culture and sample treatment were purchased from Sigma (St. Louis, MO, USA).

Trizol for RNA extraction was purchased from Invitrogen (Carlsbad, CA, USA), Superscript II reverse transcriptase was purchased from BD Bioscience (San Jose, CA, USA), 2 X SYBR green Mix was purchased from ABI Applied Bioscience (Foster City, CA, USA) and all primers and oligo dT primer were purchased from Bioneer (Daejeon, Korea).

Antibody against ERK1/2, phosphor-ERK1/2, β-actin, anti-rabbit IgG-horseradish peroxidase-conjugated, anti-mouse IgG-horseradish peroxidase-conjugated were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-STAT3 and anti-phospho-STAT3 antibodies were purchased from Biosource (Camarillo, CA, USA) and anti-IκB and anti-phospho-IκB were from Cell Signaling (Danvers, MA, USA).

Mouse 3T3-L1 pre-adipocytes were grown to confluence in DMEM with 10% BCS at 37℃ in a humidified atmosphere of 5% CO₂. At 1 day post-confluence (designated "day 0"), cell differentiation was induced with a mixture of methylisobutylxanthine (0.5 mM), dexamethasone (0.25 mM), and insulin (5 mg/ml) in DMEM containing 10% FBS. On day 2 and thereafter, DMEM containing 10% FBS and insulin (5 mg/ml) only was subsequently replaced every 2 days.

Berberine was dissolved in DMSO, divided into small quantities, and stored in -20℃. At day 8, after inducing differentiation of adipocytes, the sample was dissolved in DMEM at 10 mM concentration, which was treated into adipocytes for 4 hours. The final concentration of DMSO was less than 0.1%. After treating the samples for 4 hours, cells were used for RNA extraction, as described below.

Total RNA was isolated from 3T3-L1 adipocytes using Trizol reagent. First strand cDNA synthesis was performed with 2 mg of total RNA using Superscript II reverse transcriptase (BD bioscience, Palo Alto, CA, USA).

Real-time RT-PCR analysis was performed by Applied Biosystems 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Samples (mixture that 2 X SYBR green Mix, 0.5 µM each appropriate primers, and cDNA) were incubated in the Applied Biosystems 7500 Real Time PCR system for an initial denaturation at 94℃ for 10 min, followed by 40 PCR cycles. Each cycle consisted of 95℃ for 15s, 60℃ for 1minute. To confirm amplification of specific transcripts, melting curve profiles (cooling the sample to 65℃ for 15s and heating slowly to 95℃ with continuous measurement of fluorescence) were produced at the end of each PCR. The oligonucleotide primers for the experiments are: beta-actin; 5'-AGCCATGTACGTAGCCATCC-3' and 5'-TCCCTCTCAGCTGTGGTGAA-3', leptin; 5'-AGGATCTGAGGGGTGATGTGA-3' and 5'-TGAGGTGACCAAGGTGGCATAG-3', MCP-1; 5'-TTAAAAACCTGGATC GGAACCAA-3' and 5'-GCATTAGCTTCAGATTTACGGGT-3', adiponectin; 5'-GTC TCAGCTGTCGGTCTTCCCCT-3' and 5'-CCCTGGCTTTATGCTCTT TGC-3'.

After experimental treatment, cells were washed twice with ice-cold phosphate-buffered saline and lysed with extraction buffer (containing 50 mM Tris-HCl, pH 7.5), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 100 mM NaF, 1 mM PMSF and protease inhibitor cocktail on ice for 30 min. Insoluble materials were removed by centrifugation at 15,000g for 10 min at 4℃. The total concentration of extracted proteins was determined using the method of Bradford. The proteins in the supernatants were separated by 10% SDS-PAGE and transferred into nitrocellulose membranes. After blocking with TBS-T (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.6) containing 5% nonfat milk for 1 h at room temperature, the membranes were incubated with the appropriate first antibodies. To detect the antigen-bound antibodies, the blots were treated with secondary antibodies conjugated with horseradish-peroxidase. Immuno-reactivity was detected by using the ECL Western blotting detection system.

The data was analyzed by the ANOVA procedure of Statistical Analysis System (SAS Institute, 1999-2001). Significant differences between groups were determined using Duncan's multiple range tests at the p<0.05 level.

Previously, we showed that the mRNA expressions of inflammatory molecules such as TNF-α, IL-6, CRP, and Haptoglobin are decreased by berberine treatment in 3T3-L1 adipocytes. Here we focused more on adipokines and studied how their mRNA expressions are influenced by berberine treatment. We treated berberine for 18 hrs on fully differentiated adipocytes and examined mRNA expression levels of leptin, adiponectin, and MCP-1. Leptin and MCP-1 mRNA expressions were greatly decreased about 80-90%, but adiponectin which has a special role in anti-inflammation was increased by 35% after treatment of berberine (Fig. 1-a). In order to investigate when the inflammatory molecules are reduced mostly after a treatment of berberine, we examined the time course of TNF-α and leptin mRNA expressions in berberine-treated 3T3-L1 adipocytes. The mRNA expressions of TNF-α and leptin were estimated by real time RT-PCR. The mRNA expression of TNF-α was decreased mostly 4 hours later, then increased by 6 hours of treatment and went back to the original level of expression at 24 hours of treatment (Fig. 1-b). The mRNA expression of leptin was decreased more than 40% during all the times of berberine treatment (Fig. 1-c). Therefore, we chose 4 hours berberine-treated samples for the further investigations.

Anti-adipogenic and anti-inflammatory effects of berberine were shown in Fig. 1 and our previous report (Choi et al., 2006). Through these data, we assumed that reduced adipogenisity by berberine induces down-regulation of inflammatory molecules. We chose the leptin signaling as the best candidate to explore and have examined the effect of berberine on STAT-3 phosphorylation and ERK phosphorylation. STAT-3 and ERK phosphorylations were measured by western blotting and STAT-3 and ERK were used as internal control, respectively. However, contrary to our expectation, the phosphorylations of STAT-3 and ERK were not significantly changed after berberine treatment. (Fig. 2-a, b).

According to the previous experiments, berberine does not seem to inhibit the leptin signaling pathway. Therefore, we investigated the anti-inflammatory effect of berberine itself instead of examining berberine's anti-inflammatory effect in accordance with its anti-adipogenic effect. In this regard, we have investigated the activity of NF-κB which is the representative transcription factor of inflammation. Inactive NF-κB exists as a dimer with I-κB and along with the I-κB phosphorylation, NF-κB becomes activated (Ghosh et al., 1998). So we have examined the changes in I-κB phosphorylation by berberine in 3T3-L1 adipocytes. And we have found approximately a 57% decrease of I-κB phosphorylation (Fig. 3).