CGA, DMEM, Krebs-Ringer bicarbonate buffer (KRBB), antibiotic/antimycotic, insulin, wortmannin, cytochalasin B, MTT, AMP, PI/RNase were obtained from Sigma (St. Louis, MO, USA). Rat L6 skeletal muscle myoblasts were obtained from ATCC (Manassas, VA, USA). FBS was from Hyclone (Cramlington, UK). DMSO was purchased from MP Biomedicals (Illkirch, France). Glucose oxidase kits were obtained from Thermo Scientific (Waltham, MA, USA). Compound c and NP 40 were obtained from Merck (Darmstadt, Germany). 2-Deoxy-[3H]D-glucose and [γ-32P]-ATP were purchased from PerkinElmer (Waltham, MA, USA). Protease inhibitor cocktail was purchased from Abcam (Cambridge, UK). AMPK α1/2 and an unrelated siRNA (control siRNA-A) were purchased from Santa Cruz (Santa Cruz, CA). Antibodies like anti-IRS-1, anti-PI3-kinase p85α, anti-GLUT 4, anti-GLUT 1, anti-CAMKKβ, anti-phospho-AMPK α1/2, anti-phospho-Akt1 were obtained from Santa Cruz (Santa Cruz, CA) too. anti-GAPDH and anti-phospho-ACC were from Cell Signaling Technology (Danvers, MA, USA) and Milipore (Billerica, MA, USA) respectively. Oligofectamine and OPTI-MEM were purchased from Invitrogen (Carlsbad, CA, USA). Bradford protein estimation kit was from Bio-Rad (Hercules, CA, USA). G-Sepharose beads and ECL detection kit were obtained from GE Healthcare (Piscataway, NJ, USA). SAMS peptide was purchased from Tocris Bioscience (Minneapolis, MN, USA).
Thirty male db/db mice homozygous for diabetes spontaneous mutation (Leprdb) were obtained from The Jackson Laboratory (Sacramento, CA, USA). Ten C57BL/6 mice were purchased from Centre for Animal Resources (CARE), National University of Singapore. They were allowed to acclimatize to conditions in the Animal Holding Unit (AHU), NUS. They were housed throughout the experiment on a 12-hour light/dark cycle. Water and feeds were available to the animals ad libitum.
The Principles of Laboratory Animal Care (NIH, 1985) were followed throughout the duration of experiment. The experimental protocol for animal study was approved by NUS Institutional Animal Care and Use Committee (IACUC) (Protocol No: 085/07(A3)10).
Oral glucose tolerance test
mice were randomly assigned into four groups (n
4) and four C57BL/6 mice were assigned as lean control group. They were fasted for six hours before the test. Blood samples were collected from the tail vein for fasting glucose measurement using glucose oxidase method before treatments (vehicle, ip 250 mg/kg CGA, oral 250 mg/kg metformin). Ten minutes after the treatments, blood samples were collected again followed by oral gavaging of 2 g/kg glucose. Blood samples were collected 15, 30, 60 and 120 minutes after the glucose challenge.
2DG transport in soleus muscle
Soleus muscle was isolated from db/db mice as described previously 
. It was then incubated with treatments (vehicle, metformin or CGA) in KRBB for 30 minutes at 37°C. Treated muscle strips were subsequently incubated with 0.5 ml KRBB containing 1 µCi/ml 2-Deoxy-[3
H]D-glucose for 30 minutes at 37°C. Reaction was terminated by immediately blotting the tissues and dissolving them in 0.5 N NaOH for an hour followed by neutralization with equal amount of 0.5 N HCL. After centrifugation, supernatant was collected for quatitation of 2DG taken up by the tissue using liquid scintillation counter (Beckman Coulter LS6500 Multi-Purpose Scintillation Counter, Fullerton, CA, USA). Non-specific uptake was measured in the presence of 10 µmol/l cytochalasin B and subtracted from the total uptake. 2-DG uptake was expressed as a percentage of the basal uptake of cells incubated with KRBB buffer only.
Cell culture and differentiation of L6 skeletal muscle
The culture was maintained in DMEM containing 10% FBS and 1% antibiotic/antimycotic in a humidified atmosphere of 5% CO2
at 37°C. Differentiation of myoblasts into myotubes was carried out as described by Klip et al. 
. L6 myoblast was seeded in 10% FBS-DMEM until it reached 80–90% confluence; the FBS content was reduced to 2% for a further 5–7 days to induce myotube formation. The degree of differentiation was determined as the percentage of nuclei present in the multinucleated myotubes under a phase-contrast microscope. Before all experimental manipulations, L6 myotubes were deprived of serum for 4 hours to render the cells quiescent.
2-Deoxy-[3H]D-glucose (2-DG) transport in L6 skeletal muscle
The cells were grown and differentiated in 96-well plates. After the indicated periods of incubation with different treatments, the cells were rinsed with KRPH (HEPES-buffered Krebs-Ringer phosphate) buffer, consisting of 118 mmol/l NaCl, 5 mmol/l KCl, 1.3 mmol/l CaCl2, 1.2 mmol/l MgSO4, 1.2 mmol/l KH2PO4 and 30 mmol/l HEPES (pH 7.4). CGA was prepared in 5% DMSO and diluted with appropriate amount of DMEM to obtain different concentrations. 5% DMSO was used for all drug preparations. For the study with CGA+insulin, myotubes were treated with 2 mmol/l CGA for 24 hours and stimulated with 100 nmol/l insulin for 30 mins before 2DG uptake measurement. For the study involving inhibitors, myotubes were pre-incubated with 100 nmol/l wortmannin or 10 µmol/l compound c for 30 mins before adding CGA. Washed cells were incubated with 10 µmol/l 2-Deoxy-[3H]D-glucose (1 µCi/ml) in KRPH buffer for 30 mins at 37°C. The 2-DG uptake was terminated immediately by aspirating the radioactive incubation solution and washing three times by ice-cold phosphate-buffered saline (PBS). The cells were then lysed with 0.5 N NaOH, followed by 0.5 N HCL for neutralization. The quantity of 2-DG taken up by the cells was measured with liquid scintillation counter. Non-specific uptake was measured in the presence of 10 µmol/l cytochalasin B and subtracted from the total uptake. 2-DG uptake was expressed as a percentage of the basal uptake of cells incubated with KRPH buffer only.
Myotube subcellular fractionation
The subcellular fractionation of myotubes was done as previously described 
. Treated cells were scraped gently from 10 cm dishes and centrifuged at 700 g, 4°C for 10 mins. The pellet was then resuspended in sucrose buffer, consisting of 250 mmol/l sucrose, 5 mmol/l sodium azide, 2 mmol/l EGTA, 20 mmlol/l HEPES (pH 7.4) and protease inhibitor cocktail, and homogenized with 20 strokes using a Dounce homogenizer. The homogenate was centrifuged at 760 g, 4°C for 5 mins to remove nuclei and cell debris. The supernatant was collected as total cell lysate. For plasma membrane isolation, the supernatant was removed and centrifuged at 31,000 g, 4°C for 60 mins to pellet the crude plasma membrane (PM). PM fraction was resuspended in sucrose buffer and stored at −80°C.
siRNA transfection of myotubes
Transfection of siRNA into myotubes was done as described previously with a minute modification 
. Cells were seeded and grown in 6-well plates as described earlier (see section on cell culture and differentiation). After cells were treated with 2% FBS for 3 days, siRNAs were transfected with oligofectamine, acoording to the instructions of the manufacturer. Briefly, the complex mixture in serum- and antibiotic-free OPTI-MEM was layered onto the cells and incubated for 4 hours at 37°C. The serum content was then brought up to 2% by adding half volume of antibiotic-free DMEM supplemented with 6% FBS and incubated for an additional 20 hours. Another volume of 2%-FBS DMEM with antibiotic was then added and incubated for further 24 hours to allow the siRNA to remain on the cells for a total of 48 hours.
Immunoprecipitation and detection of association between IRS-1 and p85 subunit of PI3K
Treated cells were scraped gently from 6-well plate and pelleted with ice-cold PBS at 3,000 rpm, 4°C for 5 minutes. Cell pellet was then lysed in lysis buffer (50 mmol/l Tris [pH 8], 170 mmol/l NaCl, 1 mmol/l DTT, 0.5% NP40 and protease inhibitor cocktail for 30 minutes at 4°C. Cell lysate was then centrifuged at 13,000 rpm for 10 minutes to remove cell debris. 2 µl of the cell lysate was used for Bradford protein estimation. 1 mg of total cellular protein was immunoprecipitated with 1 µg of anti-IRS-1 antibody coupled to 40 µl of 100 mg/ml protein G-Sepharose beads. Immunoprecipitated proteins, together with the beads, were separated with SDS-PAGE and blotted as mentioned in the western blot analysis below. Blotted proteins were probed with anti-PI3-kinase p85α antibody.
Western blot analysis
Treated cells were scraped from 6-well plates and pelleted with ice-cold PBS at 3,000 rpm, 4°C for 5 minutes. Cell pellets were then lysed in lysis buffer containing 250 mmol/l sucrose, 150 mmol/l NaCl, 50 mmol/l HEPES (pH 7.4) 10 mmol/l sodium fluoride, 1 mmol/l sodium pyrophosphate, 1 mmol/l sodium orthovanadate, 1 mmol/l DTT, 0.5 mmol/l EDTA, 1% Triton-X 100 and proteases inhibitors cocktail. Cell lysates were separated via SDS-PAGE and the separated proteins were blotted onto nitrocellulose membrane. Membranes were probed with anti-GAPDH, anti-GLUT 4, anti-GLUT 1, anti-phospho-AMPK α1/2, anti-phospho-Akt1 and anti-phospho-ACC. They were then developed using the ECL detection kit.
Cellular viability and proliferation analysis
Myotube viability and proliferation were assayed with MTT and propidium iodode (PI) staining. For the MTT assay, cells were treated with different concentrations of CGA for 24 hours and then incubated with 5 mg/ml MTT for a fruther 4 hours at 37°C. Medium was then removed and 100 µl of DMSO was added to dissolve the formazan crystal formed. Absorbance was measured at a wavelength of 500 nm with a microplate reader (Tecan Infinite m200, Mannedorf, Switzerland). Similarly, for the PI staining, cells were trypsinized and washed with PBS after incubation with different concentrations of CGA for 24 hours. The cells were then fixed in 70% ethanol at 4°C for at least 2 hours. After fixation, the cells were stained with PI/RNase in PBS supplemented with 1% FBS at room temperature for 20 mins before proceeding to flow cytometry analysis, using cyAn ADP Flow Cytometer (Beckman Coulter, Brea, CA).
AMPK activity assay
Myotubes were treated with CGA of different concentrations for indicated periods of time. Treated cells were scraped and pelleted with ice-cold PBS at 3,000 rpm, 4°C for 5 minutes. Cell pellets were then lysed in lysis buffer containing 250 mmol/l sucrose, 150 mmol/l NaCl, 50 mmol/l HEPES (pH 7.4) 10 mmol/l sodium fluoride, 1 mmol/l sodium pyrophosphate, 1 mmol/l sodium orthovanadate, 1 mmol/l DTT, 0.5 mmol/l EDTA, 1% Triton-X 100 and proteases inhibitors cocktail. 2 µl of the cell lysate was used for Bradford protein estimation. 1 mg of total cellular protein was immunoprecipitated with 1 µg of anti-AMPK α1/2 antibody coupled to 40 µl of 100 mg/ml protein G-Sepharose beads. Kinase reaction was carried out on washed immunoprecipitate in 40 mmol/l HEPES (pH 7.0), 0.2 mmol/l AMP, 80 mmol/l NaCl, 0.8 mmol/l DTT, 5 mmol/l MgCl2, 0.2 mmol/l ATP (2 mCi [γ-ATP]) and 0.1 mmol/l SAMS peptide for 20 minutes at 37°C. Reaction mixture was then spotted on P81 Whatman filter paper and washed extensively using phosphoric acid and acetone. Radioactivity on the filter paper was measured using liquid scintillation. Kinase activity was expressed as incorporated ATP/mg protein/minute.
Experiments were repeated three times, each time in triplicate. Values are expressed as mean ± SE. One-way ANOVA followed by Tukey's t test was used to determine significant differences between groups. P values<0.05 were interpreted as statistically significant.