DS20060511 (Fig. 1a, molecular formula C30H32O6·C4H11N, molecular weight 561.71) was obtained from the Medicinal Chemistry Research Laboratories, Daiichi Sankyo Co., Ltd.
2-Deoxy-D-[l,2-3H]-glucose ([3H]-2-DG, ART0103A) and D-[1-12C]-mannitol ([12C]-mannitol, ARC0127A) were purchased from American Radiolabeled Chemicals.
C57BL/6 and db/db mice, as well as CE-2 normal chow and high-fat diet 32 (HFD) were purchased from CLEA Japan. GLUT4 knockout mice were generated at RIKEN BioResource Center according to the previous report35. The mice were group-housed under a 12-h light-dark cycle and given free access to normal chow and water. The HFD-fed obese mice were prepared by feeding C57BL/6 mice a HFD for at least 8 weeks from 6 weeks of age. Male mice were used for all the experiments in this study. The animal care and experimental procedures used in the study were approved by The University of Tokyo Animal Care Committee, and the study was carried out in accordance with the Animal Experimentation Guidelines of Daiichi-Sankyo Co., Ltd.
Streptozotocin (STZ)-treated C57BL/6 mouse model
C57BL/6 mice deprived of access to food overnight received an intraperitoneal injection of STZ (125 mg/kg) and a repeat injection a week later. Thereafter, their blood glucose levels were monitored and insufficient mice (blood glucose levels of under 400 mg/dl and blood glucose levels after overnight food deprivation of under 200 mg/dl) were excluded from the experiment.
Cell Culture and Differentiation
The L6-GLUT4myc rat myoblast cell line was obtained from Dr. Amira Klip and Dr. Philip Bilan, through a license granted by The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1 × 8; or purchased from Kerafast (ESK202)36. The 3T3-L1-GLUT4myc fibroblast cell line was provided by Dr. Tomoyuki Yuasa, Tokushima University37. L6-GLUT4myc myoblasts were grown in MEMα supplemented with 10% FBS and 1% antibiotics, and then induced to differentiate into myotubes in MEMα supplemented with 2% FBS and 1% antibiotics for 5–8 days. 3T3-L1-GLUT4myc fibroblasts were induced to differentiate into adipocytes as described previously,38 with minor modifications. Briefly, cells were grown to confluence in growth medium: DMEM (Gibco) supplemented with 10% FBS and 1% antibiotics, and then induced to differentiate into adipocytes in growth medium supplemented with 1 µM dexamethasone, 2 µM rosiglitazone, 0.5 mM isobutylmethylxanthine, and 10 µg/mL insulin for two days. The adipocytes were then cultured in growth medium supplemented with 10 µg/mL insulin for a few days before being used for the experiments.
Detection and Quantitation of Cell Surface GLUT4 Using anti-Myc Antibody
Cell surface GLUT4 levels in the L6-GLUT4myc myoblasts and myotubes were determined by antibody binding assay as described previously,36,37 with minor modifications. Briefly, cells in a 96- or 24-well plate were starved in serum-free MEMα (0.1% BSA and 1% antibiotics) for 3 h and then treated with the indicated concentrations of DS20060511 and/or insulin for 30 min at 37 °C. After fixation with 4% paraformaldehyde, the cells were incubated with 1% glycine in PBS at 4 °C for 10 min, and blocked with 10% normal goat serum and 3% BSA in PBS at 4 °C for 30 min. The cells were then incubated with anti-c-Myc antibody (sc-40, 1:500) diluted with blocking buffer at 4 °C for 1 h, washed with cold PBS(+), incubated with HRP-conjugated anti-mouse IgG diluted with blocking buffer at 4 °C for 30 min, and then washed again. SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Scientific) was added and the luminescent signal was measured. To investigate GLUT4 translocation in the 3T3-L1-GLUT4myc adipocytes, the cells were prepared in a 24-well plate, and anti-c-Myc antibody (sc-789, 1:1000), HRP-conjugated anti-rabbit IgG and SIGMAFAST OPD regent (Sigma-Aldrich) were added for optical detection of the cell surface GLUT4myc levels. The GLUT4 translocation activity was normalized to that in the vehicle-treated group.
In vitro Cellular 2-DG Uptake
L6-GLUT4myc myotubes and 3T3-L1-GLUT4myc adipocytes were incubated in the wells of a 24-well plate containing serum-free medium for 3 h at 37 °C and then incubated in glucose-free medium for 30 min at 37 °C. The cells were treated with the indicated concentrations of DS20060511 or insulin for 30 min at 37 °C, followed by the addition of 1 mM 2-deoxy-D-glucose (2-DG) and 0.3 µCi/mL [3H]-2-DG for 10 min. 2-DG uptake was measured with a liquid scintillation counter and normalized to the level in the vehicle-treated group.
Glucose Tolerance Test (GTT)
Mice that had been denied access to food overnight received the indicated oral dose of DS20060511 or vehicle 15 min prior to the glucose load. Then, after the oral glucose load, the blood glucose levels were measured using Glutest Every (Sanwa Kagaku Kenkyusho) in samples obtained from the tail vein at the indicated time points. Plasma insulin concentrations were also measured with an ELISA kit (Morinaga) in blood samples collected from the tail vein at the indicated time points.
Insulin Tolerance Test (ITT)
Mice received the indicated oral dose of DS20060511 or vehicle at the same time as the intraperitoneal injection of insulin at the indicated dose. Blood glucose levels were measured using Glutest Every (Sanwa Kagaku Kenkyusho) in blood samples collected from the tail vein at the indicated time points.
In vivo 2-DG Uptake
Tissue glucose uptake was examined by measuring the uptake of [3H]-2-DG during an intraperitoneal GTT as described previously39,40, with minor modifications. Mice that had been denied access to food overnight received oral administration of 30 mg/kg of DS20060511 or vehicle 15 min prior to the glucose load. The mice then received intraperitoneal glucose administration (1 g/kg containing 100 µCi/kg [3H]-2-DG as tracer), followed by quick removal of the tissues 60 min later. Tissue samples were homogenized in 0.5% perchloric acid and centrifuged, and the supernatants were neutralized with KOH. One aliquot of the supernatants was used for measuring the total radioactivity ([3H]-2-DG and [3H]-2-DG 6-phosphate ([3H]-2-DGP)). A second aliquot of the supernatants was treated with 1N Ba(OH)2 and 1N ZnSO4 to remove [3H]-2-DGP, and the [3H]-2-DG count was measured. 2-DG uptake into the tissue, which is rapidly metabolized to 2-DGP in the tissue, was estimated by subtracting the count of [3H]-2-DG from the total count.
2-DG Uptake in Isolated Skeletal Muscle
Soleus or EDL muscle was removed from mice that had been denied access to food overnight under isoflurane anesthesia and incubated with oxygenated (95% O2/5% CO2) Krebs-Henseleit Bicarbonate (KHB) buffer (118.1 mM NaCl, 4.7 mM KCl, 1.1 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3, pH7.4) in the presence of 11.1 mM glucose. Muscles were then treated with the indicated concentrations of compounds in KHB buffer containing 11.1 mM glucose and 8 mM mannitol for 10 min. Thereafter, the muscles were rinsed with KHB buffer containing 8 mM mannitol and the compounds for 5 min. Lastly, the muscles were treated with compounds in KHB buffer containing 1 mM 2-DG (with 0.3 µCi/mL [3H]-2-DG) and 8 mM mannitol (with 0.03 µCi/mL [14C]-mannitol) for 10 min. The 14C and 3H specific activities were counted with a liquid scintillation counter. The specific uptake of 2-DG was calculated by subtracting the non-specific uptake of mannitol from the total 2-DG uptake. To investigate the effects of muscle contraction, muscle contraction was induced by electrical stimulation with 5 Hz (1 ms pulse duration, 100 V) for 10 min (SEN-5201, Nihonkoden).
Plasma Membrane Fractionation of the Skeletal Muscle
Mice that had been denied access to food overnight received DS20060511 (10 mg/body), insulin (5 unit/body) or saline (vehicle control) via the inferior vena cava under isoflurane anesthesia, and 10 min later, the hindlimb muscles were excised. The plasma membrane fraction of the skeletal muscle was prepared as described previously41,42. Briefly, the hindlimb muscles were homogenized in Buffer A (20 mM HEPES, 1 mM EDTA, 1 mM PMSF and protease inhibitor) containing 250 mM sucrose. The muscle homogenates were centrifuged at 2,000 × g for 10 min to remove any unhomogenized muscle fibers. The supernatants were then centrifuged at 19,000 × g for 20 min. The pellets were resuspended in 3 mL Buffer A, layered on a 6 mL sucrose cushion (38% sucrose in Buffer A) and centrifuged at 100,000 × g for 60 min. The membrane fraction recovered on top of the sucrose cushion was resuspended in Buffer A and centrifuged at 40,000 × g for 20 min. The pellet was designated as the plasma membrane fraction and subjected to immunoblotting.
Sample preparation for immunoassays of insulin- and AMPK-signaling molecules
For the analyses of insulin and AMPK signal transduction, mice that had been denied access to food overnight received DS20060511 (10 mg/body), insulin (5 unit/body) or saline (vehicle control) via the inferior vena cava under isoflurane anesthesia and 10 min later, the hindlimb muscles were excised for Western blot analysis. Tissue samples were lysed and the lysates were centrifuged at 15,000 rpm for 10 min at 4 °C. The supernatants were collected and the protein concentrations were determined by BCA assay. Immunoprecipitation of IRβ and IRS1 was performed as described previously10, using specific antibodies against IRβ (Santa Cruz, sc-711), IRS1 (Millipore, 06-248) and for detection of phosphotyrosine (Millipore, 05-321). Five milligrams the extracts were incubated with specific antibodies against IRS1 for 1 h at 4 °C. Then, protein G-Sepharose was added, followed by incubation for 2 h at 4 °C. After washing three times, the immunocomplexes were subjected to immunoblotting.
Phosphorylated or total protein of IRβ and IRS1 was resolved on 7% SDS PAGE and analyzed using specific antibodies against IRβ, IRS1, and phosphotyrosine. Analyses were also conducted for phosphorylated Akt (1:2000), Akt (1:2,000), AS160 (1:1,000), phospho-AS160 (1:1,000), AMPKα (1:1,000), phospho-AMPKα (1:1,000), GLUT4 (1:200), Na,K-ATPaseα (1:1,000), GAPDH (1:1,000) by immunoblotting with specific antibodies after the tissue lysates were resolved on SDS–PAGE and transferred to a PVDF membrane using Trans-Blot Turbo transfer system (Bio-Rad Laboratories). Bound antibodies were detected with HRP-conjugated secondary antibodies using ECL detection reagents (Amersham Biosciences).
Differentiated L6-GLUT4myc myotubes were prepared in a collagen-coated 4-well chamber slide. After serum starvation for 3 h, the cells were stimulated with insulin or DS20060511 for 15 min at 37ºC. Cells were rinsed with cold PBS(+), fixed with 4% paraformaldehyde for 30 min on ice and blocked with 1% BSA and 10% normal goat serum in PBS(+) for 30 min at room temperature. Surface GLUT4myc staining was carried out without cell membrane permeabilization. Cells were incubated with anti-c-Myc antibody for 30 min at room temperature, followed by treatment with 0.1% Triton-X. After blocking, the cells were incubated with Alexa 488-conjugated secondary antibody and Alexa 594-conjugated phalloidin for 30 min at room temperature. Fluorescence images were obtained with a Leica TSC-SP8 confocal microscope. Specimens were scanned along the z-axis and a single composite image was generated by the maximal projection method using the LAS software (Leica Microsystems).
Indirect calorimetry under treadmill running
The mice received oral administration of 30 mg/kg of DS20060511 or vehicle 15 min before they were made to run. At 0 min, the treadmill was started at a velocity of 10 m/min, with the speed increased by 2 m/min every 3 min. The respiratory quotient, and glucose and fat oxidation under treadmill running were analyzed by ARCO-2000 (ARCO system Inc.).
Repeated DS20060511 treatment of the db/db mice
db/db mice were acclimatized to a 6-h restricted feeding pattern from 10 am to 4 pm prior to the start of the experiment. For 28 days from 6 weeks of age, the mice received oral administration of 10 mg/kg of DS20060511 or vehicle once a day, 15 min prior to their feeding. On day 1 and day 28, the blood glucose levels were measured between 10 am to 4 pm. Daily food intake was monitored throughout the study, and the body weight, blood glucose and plasma insulin were measured on day 1 and day 28. The hemoglobin A1c (HbA1c) level was also measured with a HLC-723G8 Automated Glycohemoglobin Analyzer on day 1 and day 28 (Tosoh India Pvt. Ltd.).
Antibodies against c-Myc (9E10, sc-40 and A-14, sc-789), Glut4 (C-20, sc-1608) and IRβ (C-19, sc-711) were purchased from Santa Cruz Biotechnology. Antibodies against Akt (9272), phospho-Akt (9271), AMPKα (2532), phospho-AMPKα (2531), Na,K-ATPaseα (3010) and GAPDH (2118), and anti-mouse IgG (7076) and anti-rabbit IgG (7074) were purchased from Cell Signaling Technology. Rabbit polyclonal antibody against IRS1 (06-248), mouse monoclonal antibody against phosphotyrosine (05-321), rabbit antiserum against AS160 (07-741), and rabbit polyclonal antibody against phospho-AS160 (07-802) were purchased from Millipore. HRP-conjugated donkey anti-goat IgG (V8051) was purchased from Promega. HRP-conjugated goat anti-rabbit IgG (111-035-144) and goat anti-mouse IgG (115-035-003) were purchased from Jackson ImmunoResearch.
Values are expressed as the means ± SEM. Differences between two groups were assessed using Student’s t-tests. Statistical differences among multiple groups were evaluated by ANOVA followed by indicated post-hoc multiple comparison.