Dulbecco's modified Eagle's medium (DMEM), newborn calf serum (NBCS), fetal bovine serum (FBS), phosphate-buffered saline (PBS), trypsin, and penicillin–streptomycin were obtained from Hyclone (Logan, UT, USA). Dexamethasone (DEX), insulin, 3-isobutyl-1-methyxanthine (IBMX), β-actin antibody, 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT), N omega-Nitro-L-arginine methyl ester hydrochloride (L-NAME), and rosiglitazone were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)-2-deoxyglucose (2-NBDG) was purchased from Invitrogen (Carlsbad, CA, USA). Tris (hydroxymethyl) aminomethane (Tris) base, glycine, and sodium dodecyl sulfate (SDS) were purchased from the Promega Corporation (Madison, WI, USA), and nitrocellulose membranes were purchased from Bio-Rad (Hercules, CA, USA). The western blot detection kit and lysis buffer were purchased from iNtRON Biotechnology (Burlington, MA, USA). Insulin receptor β (IRβ;#3025), phospho-phosphoinositide 3-kinases (p-PI3K;#4228), PI3K (#4292), phospho-AKT (p-AKT;#9271), AKT(#9272), glucose transporter (GLUT4;#2213), phospho-p38 mitogen-activated protein kinase (p-p38 MAPK;#4511), p38 MAPK (#8690), phospho-AMP-activated protein kinase α (p-AMPKα;#2535), and AMP-activated protein kinase α (AMPKα;#2532) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). GLUT4, insulin receptor substrate-1 (IRS-1), and β-actin primers were purchased from Integrated DNA Technologies (IDT, Korea).
Extraction of C. unilocularis leaves
The leaves of the C. unilocularis Buch-Ham plant were harvested and dried in Baglung, Nepal in July 2014. Dried C. unilocularis leaves (400 g) and 2L of distilled water (DW) were added together, followed by hot water extraction at 100 °C. This process was repeated twice for 60 min to obtain an extract. The extract was filtered with a filter paper and concentrated on a rotary evaporator (EYELA, Tokyo, Japan) in a water bath below 45 °C. The concentrate was lyophilized and powdered and diluted to various concentrations for use in the experiments. The C. unilocularis leaf extract was named CULE. The yield of the extract was determined to be 12% (48 g). C. unicolularis was reviewed and certified by Dr. Kim Hui, Mokpo National University, Korea. The voucher specimens of the C. unicolularis plant were kept in the laboratory for further documentation .
High-performance liquid chromatography (HPLC) analysis
HPLC analysis was carried out in the Alliance HPLC system (Waters, MA, USA) with a PDA detector (190–500 nm) using a Capcell Pak C18 (250 × 4.6 mm, 5 μm, Shiseido, Japan). Samples and standard solutions were eluted by an isocratic solvent system (H2O: 5% acetic acid: acetonitrile = 40: 30: 30) with the flow rate of 0.8 mL/min at 35 °C. The standard solutions of quercetin and CULE were prepared as follows; 10 mg of quercetin was dissolved in methanol and prepared as a stock solution with the concentration of 1,000 ppm using a 10 mL volumetric flask. Then, it was diluted to prepare a standard solution (20 to 500 ppm). For the CULE sample, 1 g of CULE was extracted with 40 mL of 60% EtOH and 5 mL of 6 N HCl under reflux condition for 2 h at 95 °C, and made into 50 mL of final volume with 60% EtOH. The standard solutions and test solutions were filtered with 0.45 μm nylon filter and injected with a volume of 10 μL. All HPLC chromatograms were extracted under 370 nm. The calibration curve for quercetin was recorded using concentrations (X) and peak areas (Y).
Measurement of survival rate and 2-NBDG uptake analysis in zebrafish
Wild-type adult zebrafish received from the Seoul Institute of Science and Technology for Environmental Toxicity and Health Research Labs were maintained in 14 h light/10 h dark cycles, at a temperature of 28 °C and a pH of 7.0, circulating in a tank with a filtration system (Zebtec Stand Alone, Italy). Embryo collection was performed by placing adult male and female zebrafish into the embryo collection tank 24 h before the experiment and collecting the embryos within 2 h of the photoperiod of the next day. After 6 h post-fertilization (hpf), 100 μL of E2 medium (zebrafish embryo medium) was added to each well of each 96-well plates and one embryo was transferred in each well. On 7-days post-fertilization (dpf), zebrafish larvae were exposed to CULE for 24 h, and the survival and deformity rates were observed compared to the control group .
Embryos obtained through mating of male and female zebrafish were grown up for 7 days. Then, on day 7 dpf, zebrafish larvae were exposed to CULE for 24 h. After 24 h, zebrafish larvae were treated with 600 μM 2-NBDG for 3 h and washed 3 times with E2 medium. The zebrafish larvae after washing were observed and photographed under a fluorescence microscope (Leica, Wetzlar, Germany). The fluorescence intensity of the zebrafish larvae was quantified using the ImageJ program . All experiments were performed in accordance with the guidelines of American Veterinary Medical Association and Mokpo National University for laboratory animal.
Measurement of protein tyrosine phosphatase 1B (PTP-1B) and dipeptidyl peptidase IV (DPP-IV) enzyme activities
The activation of DPP-IV were made in 25 μL of 50 mM HEPES buffer (pH 7.4) containing Gly-Pro-AMC (1 mM) and 10 μL of rat serum to which CULE was added and reacted for 1 h. The reaction was terminated by adding 70 μL of 3 M acetic acid, and fluorescence was measured at an excitation (ex) wavelength of 370 nm and an emission (em) wavelength of 440 nm using a multilabel plate reader (VICTOR 3 V, PerkinElmer Inc., Waltham, MA, USA). The activation of PTP-1B was initiated by adding 10 μL of CULE, 3 μL of reaction buffer (50 mM citrate, pH 6.0) containing 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol (DTT), 35 μL of DW, 2 μL of PTP-1B enzyme, and 50 μL of 20 mM p-nitrophenyl phosphate (p-NPP) and incubated at 37 °C for 30 min. After terminating the reaction by adding 100 µL of 1 M NaOH, the absorbance was measured at 405 nm using Immuno-mini NJ-2300 microplate reader (Nalge Nunc, Tokyo, Japan) .
Cell culture and differentiation
RIN-m5F pancreatic beta cells, L6 myoblasts, and 3T3-L1 preadipocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). RIN-m5F pancreatic beta cells and L6 myoblasts were cultured in DMEM containing 10% FBS, 100 μg/mL streptomycin, and 100 units/mL penicillin and incubated in 95% oxygen and 5% CO2 at 37 ℃. 3T3-L1 preadipocytes were cultured in DMEM containing 10% NBCS. All cell lines were supplemented with fresh medium every 2 days, and used in the experiments.
The procedure for the differentiation of 3T3-L1 preadipocytes and L6 myoblast is as follows. 3T3-L1 preadipocytes were seeded in a 6-well plates at 1 × 105 cells/well and cultured in differentiation medium (5 μg/mL insulin, 1 μM DEX, and 0.5 mM IBMX in DMEM with 10% FBS) for 2 days when the cell growth reached 90–100% confluency. After 2 days of differentiation, the medium was exchanged with DMEM with 10% FBS containing 5 μg/mL insulin, and used for the experiment four days later. Fresh DMEM with 10% FBS was added every 2 days. L6 myoblasts were seeded into 6-well plates at 1.8 × 104 cells/well, when the cells reached about 80–90% confluency, the medium was changed to DMEM with 2% horse serum (HS) and the cells were allowed to differentiate for 7 days.
Insulin resistance cell model
Recombinant murine tumor necrosis factor-α (TNF-α, 315-01A; PeproTech, NJ, USA) at a concentration of 10 ng/mL was added to 3T3-L1 adipocytes, which were completely differentiated into adipocytes, and then used as a cell model in which insulin resistance was induced after 48 h with an exchange of fresh medium every 24 h .
RIN-m5F pancreatic beta cells were seeded in 96-well plates at 1 × 105 cells/well, incubated for 12 h, reacted for 3 h with various concentrations of CULE, then reacted with IL-1β (2 ng/mL) and IFN-γ (100 U/mL) for 48 h. 3T3-L1 adipocytes and L6 myotubes were aliquoted at 5 × 103 or 1 × 103 cells/well in 96-well plates and incubated for 24 h before adding various concentrations of CULE. After the reactions, MTT (1 mg/mL) solution was added for 3 h and the absorbance was measured at 540 nm (Immuno-mini NJ-2300 microplate reader, Nalge Nunc, Tokyo, Japan).
Measurement of NO and insulin
RIN-m5F pancreatic beta cells were seeded in 96-well plates at 1 × 105 cells/well and incubated for 12 h. Then, IL-1β (2 ng/mL) and IFN-γ (100 U/mL) were added along with various concentrations of CULE. After reacting for 48 h, the same amount of cell culture medium and Griess solution was added per well and the absorbance was measured at 540 nm after 5 min using the Immuno-mini NJ-2300 microplate reader (Nalge Nunc, Tokyo, Japan). NO production was calculated using a standard curve generated using NaNO2 standard solution.
Insulin secretion analysis was conducted under the same conditions and the insulin content secreted into the cell culture was measured using a Rat Insulin ELISA Kit (Mercodia, Uppsala, Sweden). L-NAME (1 mM) was used as a positive control.
2-NBDG uptake analysis
After differentiation into 3T3-L1 adipocytes and L6 myotubes in a black 96-well plates, the cells were treated with different concentrations of CULE and incubated for 24 h. Then, the cells were treated for 30 min with 80 μM (L6 myotubes) or 150 μg/mL (3T3-L1 adipocytes) 2-NBDG. After washing three times with PBS, fluorescence was measured at an excitation (ex) wavelength of 485 nm and an emission (em) wavelength of 535 nm using a Perkin-Elmer VICTOR 3 V, (PerkinElmer Inc.; Waltham, MA, USA). The fluorescence intensity reflected the glucose uptake of 2-NBDG into the 3T3-L1 adipocytes and L6 myotubes. Insulin (IN, 100 ng/mL) and rosiglitazone (RSG, 5 μM) were used as positive controls .
After differentiating 3T3-L1 preadipocytes and L6 myoblasts on a glass cover slide, cells were treated with various concentrations of CULE and incubated for 24 h. After the experiment was completed, the coverslip was transferred to a 6-well plates, washed with PBS, and a 4% paraformaldehyde solution was added to fix the cells. The fixed cells were washed with 0.2% Triton X-100 and washed again with PBS. The primary and secondary antibodies were added to the washed coverslip, incubated, and washed with PBS. Then, a small amount of mounting medium was sprayed on a glass cover slide, and the fluorescently stained cells were photographed using LMS-700 (Carl Zeiss, Gottingen, Germany).
Reverse transcription-PCR analysis
TRIzol reagent (1 mL) was added to L6 myotubes after the experimental treatments and the cells were homogenized. Then, 0.2 mL of chloroform was added and gently mixed, followed by centrifugation at 12,000 g × 4 ℃ for 15 min. Isopropanol (0.5 mL) was added to the supernatant and the mixture was vortexed and stored at 25 ℃ for 5–10 min before centrifuging at 12,000 g × 4 ℃ for 8 min. The supernatant was removed, 1 mL of 75% ethanol was added, vortexed, and washed twice at 12,000 g × 4 ℃ for 5 min. After washing, the ethanol was removed, the sample was air-dried for 3–5 min, and 30–50 μL of diethyl pyrocarbonate (DEPC) was added and stored at -20 °C until used for the experiments. RNA was quantified by measuring the absorbance at wavelengths of 260 and 280 nm using a Nanodrop spectrometer (St. Louis, MO, USA, Sigma-Aldrich). The DiastarTM2X OneStep RT-PCR Premix Kit (Biofact, Daejeon, Korea) was used to synthesize cDNA from total RNA (25 ng) by adding 3 μL of RNA and 1 μL of primer to a total 30 μL reverse transcription reaction. The base sequence of each primer was as follows: The forward sequence of GLUT4 was 5'-CCT GCC CGA AAG AGT CTA AAG C-3', and the reverse sequence was 5'-ACT AAG AGC ACC GAG ACC AAC G-3'; The forward sequence of IRS-1 was 5'-AGA GTG GTG GAG TTG AGT TG-3', and the reverse sequence was 5'-GGT GTA ACA GAA GCA GAA GC-3'; The forward sequence of β-actin was 5'-TGC CCA TCT ATG AGG GTT ACG-3' and the reverse sequence was 5'-TAG AAG CAT TTG CGG TGC ACG-3'. PCR analysis was carried out for 45 min by reacting for 30 min at 50 ℃, 15 min at 95 ℃ and denaturing for 1 min at 95 ℃, then attaching for 1 min at 55 ℃ and extending for 2 min at 72 ℃. The PCR products were electrophoresed on 1.5% agarose gel stained with RedSafe™ nucleic acid staining solution (iNtRON, Seongnam, Gyeonggi, Korea), and β-actin was used as a control for the amplified genes.
Western blot analysis
3T3-L1 adipocytes were washed twice with PBS. Lysis buffer was then added and dissolved at 4 °C for 10 min and the mixture was centrifuged at 13,000 rpm × 4 °C for 20 min to recover supernatant protein and the amount was quantified. The lysate (30 μg/mL) was mixed with SDS loading buffer (1 M Tris, 50% glycerol, 10% SDS, and 1% bromophenol blue), heated at 95 ℃ for 10 min, and electrophoresed on 10% SDS polyacrylamide gels. The gels were transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). Five percent BSA was added to TBS-T (1 M Tris, 5 M NaCl, and 0.05% Tween 20) buffer and used to block the membranes for 1 h. After washing three times with TBS-T buffer, primary antibody (IRβ 1: 1000, p-PI3K 1: 500, PI3K 1: 1000, p-AKT 1: 1000, AKT 1: 1000, GLUT4 1: 500, p-AMPKα 1: 500, AMPKα 1: 500, p-p38 1: 500, p38 1: 1000, β-actin 1: 5000) was added and incubated overnight, and the membranes were washed three times with TBS-T buffer. Then, they were reacted with a secondary antibody (goat anti-mouse 1: 1000 or anti-rabbit 1: 1000) containing peroxidase for 2 h and visualized using a western blot detection kit. The reactions were analyzed using UVP image analysis software (VisionworkTMLS, Fisher Scientific, UK) .
All experimental results are expressed as the mean ± standard error (SE). The difference between the groups was analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism and the Dunnett post-test test. Comparison of the means of two independent sample variables were analyzed using an unpaired t-test.