Preparation of ARA extract
The roots of A. japonica Koidzumi (ARA) were purchased from the Kwanmyungdang Medicinal Herbs (Ulsan, South Korea) and authenticated by Prof. Yong-Ki Park, a medicinal botanist, as an author in this study. Voucher specimens (ARA-W-1201) have been deposited in the herbarium of Oriental Medicine R&D Center, Dongguk University, South Korea. The dried roots (210 g) were extracted by boiling in water for 3 h, filtered through a two-layer mesh and concentrated in a boiling water bath to obtain the residue (yields of 26%). ARA extract was stored at −20°C until the experiment was performed.
3T3-L1 cell culture and differentiation into adipocytes
3T3-L1 cells, murine preadipocytes (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated bovine calf serum (BCS; Hyclone, Logan, UT) in a 5% CO2 humidified atmosphere at 37°C. To differentiate preadipocytes into adipocytes, the cells were seeded in 60-mm dishes at a density of 1 × 106/mL cells, and then cultured to confluence for 8 days while changing the medium every 2 days, followed by culturing for 2 days in medium supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone and 5 μg/mL insulin. The cells were cultured for an additional 1 day in medium containing 5 μg/mL insulin. ARA extract was dissolved in adipocyte-induction media and filtered through 0.2 μm-pore syringe filters. The cells were treated every 2 days with ARA extract at concentrations of 100, 250 and 500 μg/mL in adipocyte-induction media for 6 days.
Cell viability was assessed by the conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT; Roche, Mannheim, Germany) to formazan. 3T3-L1 cells were pre-treated with ARA extract at different concentrations for 8 days. At the termination of culture, 10 μL of MTT solution was added to each well, and the cells were then cultured for 4 h. 100 μL of DMSO was added to each well, and then the optical density (OD) was measured at 550 nm by a microplate reader (GENios, TEKAN Instruments, Inc., Austria).
Oil Red O staining
3T3-L1 cells were washed with 1× phosphate-buffered saline (PBS) and fixed with 10% formalin-PBS solution for 1 h. After removing this solution, the differentiated cells were stained with Oil Red O dye (Sigma Aldrich, St. Louis, MO) for 30 min at room temperature. The cells were washed four times with distilled water. Images were collected using an Olympus microscope (Tokyo, Japan). Stained oil droplets were dissolved in isopropyl alcohol and quantified at 520 nm using a microplate reader (GENios, TEKAN Instruments, Inc., Austria).
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA from the cells was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was reverse transcribed for 1 h at 42°C in a reaction mixture containing RNA, 1× reverse transcriptase buffer (Promega, Madison, WI), 0.5 mM of dNTP (deoxynucleotide triphosphate), 3 mM MgCl2, 5 U RNase inhibitor (Amersham, Piscataway, NJ), 0.5 μM oligo-dT primer, and 5 U of Superscript Reverse Transcriptase (Promega, Madison, WI) in a total volume of 20 μL. The PCR was performed using the prepared cDNA as a template with the following cycle parameters: 94°C, 2 min, 30–35 cycles; 94°C, 30s; 56 ~ 59°C, 30s; 72°C, 1 min; 92°C, 10 min. PCR products were then resolved on 1% agarose gels at 100 V. Specific genes were verified by assessing their predicted sizes under UV light. The primer sequences for PPARγ and GAPDH were as follows: PPARγ [accession no. NM 011146] Fw: 5′-GAA AGA CAA CGG ACA AAT CAC C-3′ and Rv: 5′-GGG GGT GAT ATG TTT GAA CTT G-3′, and GAPDH [accession no. XM 994067.2] Fw: 5′-CTC CTG GAG TCT ACT GGT GT-3′ and Rv: 5′-GTC ATC ATA CTT GGC AGG TT-3′. GAPDH was used as an internal control for PCR.
The cells were lysed with lysis buffer containing10 mM Tris–HCl, pH 7.9, 10 mM NaCl, 3 mM MgCl2, and 1% NP-40. After centrifugation at 12,000 rpm for 10 min, the supernatant was stored at −80°C until use. The protein concentration was determined by Bradford’s assay. 30 μg/mL of protein were separated by 8% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk (BD, Franklin Lakes, NJ, USA) in TBS-T buffer (10 mM Tris–HCl, 150 mM NaCl, and 0.5% Tween-20) for 1 h. The membranes were incubated overnight with primary antibodies at 4°C and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The blots were developed with ECL Western detection reagents (Amersham Bioscience, Piscataway, NJ). The antibodies used in this study were anti-PPARγ (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-GLUT4 (1:500, Santa Cruz Biotechnology), anti-PI3K (1:1000, Cell Signaling Technology, Beverly, MA), anti-IRS-1 (1:500, Cell Signaling Technology), anti-phospho-IRS-1 (1:500, Cell Signaling Technology), anti-β-actin (1:1000, Sigma Aldrich) and HRP-labeled anti-rabbit or mouse IgG (1:5000; Santa Cruz Biotechnology).
The cells were grown on glass coverslips in 2-well culture plates. After 8-day treatment with ARA extract, the cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS, and then permeabilized with 0.2% Triton X-100 in 1× PBS for 15 min at room temperature. After blocking in 1% BSA in 1× PBS for 1 h, the cells were incubated overnight with anti-GLUT-4 (1:50, Cell Santa Cruz Biotechnology) antibody at 4°C. Coverslips were washed and incubated with Alexa Fluor-conjugated goat anti-rabbit antibody (1:50, Santa Cruz Biotechnology) for 2 h at RT. After DAPI staining, the coverslips were mounted on glass slides and examined under a fluorescence microscope (Olympus, Japan).
Data of all experiments are expressed as the mean ± S.D. and are representative of three independent experiments. Statistical analysis was carried out by one-way ANOVA with the Post-Hoc test using Graphpad Prism 5.0 statistical analysis software (GraphPad Software, Inc., San Diego, CA). Values of p < 0.05 were considered significant.