Viscothionin isolated from Korean mistletoe was prepared with a slight modification of a previously reported method, and was used with purity over 97% . The insulin, dexamethasone (purity over 97%), 3-isobutyl-1-methylxanthine (purity over 99%), dimethyl sulfoxide (DMSO), and compound C (AMPK inhibitor) were procured from Sigma (St. Louis, MO, USA). Simvastatin as a standard anti-obesity drug was purchased from Sigma (St. Louis, MO, USA). A kit from Oil Red O staining was purchased from Lifeline Cell Technology (Carlsbad, CA, USA). Primary antibodies for AMPK, GSK3β, p-ACC, p-AKT, p-GSK3β, ACC, AKT, phosphorylate (p)-AMPK, and β-actin were procured from Cell Signaling (Danvers, MA, USA). Anti-rabbit, or anti-mouse IgG secondary antibodies were purchased from Millipore (Bedford, MA, USA). Other ingredients and cell culture medium were obtained from Hyclone Ltd., Logan, UT, USA.
Cell culture and adipocyte differentiation
Mouse 3T3-L1 pre-adipocytes (American Type Culture Collection, Manassas, VA, USA) were cultured at 37 °C, 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (Gibco BRL, NY, USA) supplemented with 10% FBS and penicillin/streptomycin (Gibco BRL). The 3T3-L1 pre-adipocytes were incubated for 2 days until confluence. Here the cells were exposed to an adipogenic differentiation medium (DMI; DMEM containing 5% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone, and 10 μg/mL insulin) for 4 days. After 2 days, the DMI was changed with DMEM containing 5% FBS and 10 μg/mL insulin, and then changed with DMEM containing 5% FBS every other day. The viscothionin was dissolved in phosphate buffered saline (PBS) at 100 mM, and then reconstituted in each medium at the working concentration. Additionally, cells were pre-treated with compound C (20 μM) for one hour, then treated with viscothionin (5 μM).
Oil Red O staining
Oil Red O staining was performed using the staining kit protocol described elsewhere. Oil Red O stained cultures were photographed using an Observer A1 microscope at magnification 100× (Carl Zeiss, Jena, Germany). After taking the photographs, the Oil Red O stained culture dye retained in the cells was eluted with isopropanol and quantified using spectrophotometer (absorbance at 540 nm, PowerWave 2, Bio-Tek Instruments, Winooski, VT, USA).
For RT-PCR analysis, A Mastercycler Gradient 5331 Thermal Cycler (Eppendorf, Germany) was used for the amplification of cDNA. Fluorescence signals were measured after each cycle for RT-PCR analysis using an ABI Step One Plus Sequence Detection System (Applied Biosystems, Branchburg, NJ, USA). A Primer Express Software (Applied Biosystems) was employed for the designing of specific sense and antisense RT-PCR primers which were as follows: forward 5′-GAAGTGGTGGAGAGACGCTTAC-3′ and reverse 5′-TATCCTCAAAGGGCTGGACTG-3′ for SREBP-1; forward 5′-CCCAGCCCATAAGAGTTACA-3′ and 5′-ATCGGGAAGTCAGCACAA-3′ for FAS; forward 5′-GCATGGCCTTCCGTGTTC-3′ and reverse 5′- GATGTCATCATACTTGGCAGGTTT-3′ for GAPDH, housekeeping gene.
Determination of intracellular triglyceride
Intracellular triglyceride content was determined using a commercial triglyceride assay kit system (BioAssay, Hayward, CA, USA). Aliquots of various concentrations of viscothionin, with or without compound C were used for the treatment of adipocyte cells in a 12-well plate during their differentiation. Then, the cells were washed with PBS and the cell suspension was homogenized after the addition of homogenizing solution (154 mM KCl, 1 mM EDTA, 50 mM Tris, pH 7.4) followed by centrifugation (3000 rpm/5 min) of cell lysate for the removal of fat layer. The supernatants were then assessed for triglyceride content. Triglyceride content was normalized using the bovine serum albumin (BSA) as a standard.
Proteins originated from the cell lysates were subjected to SDS-PAGE using 8%, 10% and 14% gel, and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Laboratories, Hercules, CA, USA). 5% skim milk was used for the blocking of the membranes, followed by incubation with each primary antibody in 1% skim milk at 4 °C overnight. Then, the blots were incubated with each secondary antibody for one hour, and signals were detected using a SuperSignal West Dura (Thermo Scientific, CA, USA). A ChemiImager (Alpha Innotech, San Leandro, CA, USA) was employed for analyzing the results.
Animals and in vivo experimental design
Forty male C57BL/6J mice, about 6 weeks old, were purchased from Dae-Han Biolink (Daejeon, Korea) and were maintained under pathogen-free conditions on a 12-h light/dark. The mice were randomly divided into two groups and were fed either a normal-diet (n = 8) or an HFD (n = 32) with 45% of the calorie fat. The HFD mice were fed for 6-week in order to induce obesity. After 6-week, the HFD-fed mice were randomly divided into four sub-groups and each sub-group was orally administered either 0.1 mL saline (n = 8), 1 mg/kg viscothionin in 0.1 mL saline (n = 8), 10 mg/kg viscothionin in 0.1 mL saline (n = 8), or 10 mg/kg simvastatin (Sigma; purity over 97%) in 0.1 mL saline daily for 3 weeks. The mice had free access to food and water and their food consumption was measured daily, while their weight gain was measured weekly. The weight gain/feed ratio was calculated as weight gain divided by total food intake. After fasting for 12 h and before collecting blood, all mice were scanned using micro-CT for abdominal visceral fat analysis. All mice were maintained according to the Chonbuk National University Institutional Guidelines for the care and use of experimental animals (Approval number: CBU 2018-0070; Approval date: November 28, 2018).
All biochemical tests for obesity (triglyceride and total cholesterol concentrations) were performed on a VetTest 8008 Chemistry Analyzer from IDEXX, Seoul, South Korea. Levels of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were determined according to the instructions of kit manufacturer (Cell Biolabs, San Diego, CA, USA).
Abdominal visceral fat analyses
The mouse visceral fat distribution in the abdominal cavity was determined by micro-CT scanner (Skyscan-1072, TomoNT, Aartselaar, Belgium) between frames at a 10 μm slice thickness. The visceral fat distribution was determined using dedicated software (CTAn v.126.96.36.199).
All the results are presented as mean ± SE, and analyzed by the Student’s t-test, followed by one-way ANOVA analysis and Tukey’s test. Values were considered significant at p < 0.05.