Dulbecco’s Modified Eagle medium (DMEM) (0 mM and 25 mM glucose) was from GIBCO BRL, Grand Island, USA. SB431542 and DEAE-sephacel were purchased from Sigma-Aldrich, MO, USA. GKA (RO28-1675) was from Axon Medchem, The Netherlands. Anti-rabbit IgG HRP, GAPDH, anti-phospho-Smad2 (Ser465/467) rabbit monoclonal antibody and human transforming growth factor beta-1 (TGF-β) was from Cell Signalling Technology, Danvers, USA. Glucokinase activator Compound A was from Merck, Darmstadt, Germany. [35S]-sulfate was from MP Biomedicals, Irvine, CA. Cetylpyidinium chloride (CPC) was from Uni-Lab Chemicals and Pharmaceuticals, India. 3MM Whatman chromatography paper was from Whatman International, Ltd., Maldstone, UK. YSI 2300 Stat plus glucose and lactate analyser kindly provided by Professor Stephen Bird, Exercise and Metabolism Research Laboratory, RMIT University. YSI buffer concentrated kit, YSI glucose standard were from YSI Inc, Yellow Spring, USA.
Culture of bovine aortic endothelial cells and rat MIN6 pancreatic beta cells
Primary cultured bovine aortic endothelial cells (BAEC) were prepared by collagenase treatments of the aortas acquired aseptically from the abattoirs . The cells were passaged to provide sufficient cells for frozen stocks and experimentation. Stock cultures were thawed from the frozen stocks in liquid nitrogen and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) 5 mM glucose, 10% foetal bovine serum (FBS) and 1% antibiotics (streptomycin and penicillin) and incubated in 5% CO2 at 37°C.
MIN6 pancreatic beta cells were provided by Professor Jun-ichi Miyazaki, Osaka University Medical School Japan. The cells were maintained in high glucose 25 mM DMEM containing 10% FBS (FBS heat inactivated for 30 min at 60°C) and 2.5 μl of 2-mercaptoethanol.
Cell culture protocol for experimentation
For experimentation, BAEC between passages 20–50 were subcultured in 60 mm diameter dishes and 24 well plates at a density of 200,000 and 50,000 cells/well until they were confluent cultures. Cells were serum deprived in DMEM, 5 mM glucose, 0% FBS for 24 h. For glucose utilisation experiments quiescent BAEC were treated with different glucose medium concentration 2.5, 5, 15, 25 mM containing 0% FBS in the presence and absence of glucokinase activator (GKA) RO28-1675 (10 μM) and with TGF-β (2 ng/mL) as positive control and medium was collect at different time points 0, 4, 8, 24 h. For phosphorylated Smad2C protein detection experiments, BAEC were treated with RO28-1675 (0.1, 0.3, 1, 3, 10 μM) in the presence of high glucose (15 mM) for 1 h. For a time course BAEC were treated with 15 mM glucose medium at different time points 0, 15, 30, 60, 120, 240 min. For GKA dose response on phosphorylated Smad, BAEC were treated with different glucose medium concentrations 5, 10, 15, 20, 25 mM containing 0% FBS. For proteoglycan experiments cells were treated with GKA RO28-1675 (1, 3, 10 μM), SB431542 (3 μM) and GKA, Compound A (1, 3, 10 μM) in presence of TGF-β (2 ng/ml) and then radiolabeled with [35S]-sulfate (50 μCi/ml) for 24 hours.
MIN6 pancreatic beta cells were used as positive controls for glucose and GKA responses. These cells were subcultured between passages 30–45 in 24 well plates until they reached 80% confluence. MIN6 cells were then washed twice with DMEM containing no glucose and glucose deprived for 24 hours in DMEM containing no glucose, 10% FBS and 2.5 μl of 2-mercaptoethanol. Cells were then treated with either 5 or 25 mM glucose in the presence and the absence of two GKA compounds, RO28-1675 (10 μM) and Compound A (10 μM).
Glucose utilisation studies
To determine cellular glucose consumption, glucose concentrations in the medium were measured pre- and post-treatment using YSI Stat plus 2300 glucose and lactate analyser. Confluent cultures were treated as described, incubated at 37°C then the media (500 μl) from each well of each treatment was collected in 1 ml eppendorf tubes and centrifuged for 10 min at 5000 rpm. Glucose concentrations were measured by placing the eppendorf tube in the sipper tube of the YSI analyser; the sipper automatically aspirates 25 μl of the sample. Glucose concentration measurements were displayed on screen. The YSI system buffer in the instrument contained sodium dihydrogen phosphate (25 mM), sodium hydrogen phosphate (45 mM), and sodium chloride (31 mM) used to flush the sample chamber through the system.
Total cells lysates were resolved on 10% acrylamide gels by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein was then transferred to a PVDF membrane in transfer buffer (3.7% SDS, 20% methanol, 48 mM Tris base, 39 mM Glycine) at 4°C. Membranes were blocked in 5% skim milk powder and then incubated with primary rabbit monoclonal antibodies (anti-phospho-Smad2C (Ser465/467) (1:1000), anti-Smad2 (1:1000) and anti-GAPDH (1:4000)) followed by a secondary horseradish peroxidase-anti-rabbit IgG (1:1000) and ECL detection. Blots were imaged using the Bio-Rad gel documentation system and densitometry analysis was performed on blots from at least three experiments with Quantity One imaging software.
Quantitation of proteoglycans synthesis
After cells were treated as mentioned in tissue culture for experimentation section, media from each of 24 wells containing secreted proteoglycans was collected and protease inhibitors (100 mM 6-amino caproic acid, 5 mM benzamidine hydrochloride) added. Radiolabel incorporation into proteoglycans were measured by cetylpyridium chloride (CPC) precipitation assay as previously described in detail .
Assessing proteoglycan size by SDS-PAGE
Proteoglycans labelled with [35S]-sulfate were prepared for SDS-PAGE by isolation through the DEAE-sephacel anionic exchange mini columns. Samples were added to pre-equilibrated columns and then washed extensively with low salt buffer (8 M urea, 0.25 M NaCl, 2 mM disodium EDTA, 0.5% Triton X-100). Proteoglycans were eluted using high salt buffer (8 M urea, 3 M NaCl, 0.02 M EDTA, Triton-X).
Equal counts of proteoglycans (20,000 - 50,000 cpm) were precipitated by ethanol solution (1.3% potassium acetate in 95% ethanol) chondroitin sulfate was added as a cold carrier. Samples were suspended in 20 μl of buffer (8 M urea, 2 mM disodium EDTA, at pH 7.5) and 20 μl sample buffer (0.5 M Tris–HCl pH 6.8, 10% SDS, 50% glycerol, 2-mercaptoethanol, and 0.1% bromophenol blue). Proteoglycans were separated on gradient separating gel  with 4-13% acrylamide separating gels and 3% acrylamide stacking gels and run overnight at 60 V. Methylated protein molecular weight marker (Rainbow™ [14C]) was used. Processed and dried gels were exposed to an imaging plate (Fujifilm BAS-MS 2040 imaging plate) for approximately 4 days. Images were developed on a phosphoimager (Fujix BAS 1000 image plate scanner) and viewed using imaging software (Fujifilm Multi-Gauge).
Cannulated arteriole assessment of vascular reactivity
All animal procedures were approved by the Animal Experimentation Ethics Committee of RMIT University. Adult Wistar rats (n = 6, 281 ± 13 g body weight) were killed by CO2 asphyxiation and cervical dislocation and the cremaster muscles were surgically removed to ice cold Kreb’s solution. The primary arteriole was micro dissected, cannulated with glass pipettes, placed on the stage of an inverted microscope, pressurised at 70 mmHg (lumen pressure) and superfused with Krebs/HEPES buffer at 34°C. Arterioles were equilibrated for 60 min, until pressure-induced constriction (myogenic tone) developed, usually about 50% of the maximum diameter measured in calcium free Krebs (2 mM, EGTA) and the diameter (μm) was measured by video microscopy using either an automated computer program (DiamTrak) or video callipers as previously described . Changes in diameter were measured in response to increasing concentrations of phenylephrine or acetylcholine (10 nM to 10 μM Sigma, Australia) in the presence and absence of the glucokinase activator RO28-1675 (10 μM). In addition changes in arteriole diameter were recorded in response to increasing concentrations of RO28-1675 or DMSO (vehicle).
Data were collected using MacLab Chart (ADInstruments, v 4.2) and analysed using GraphPad Prism. Results are expressed as the mean ± SEM, n represents the number of arterioles and animals and ANOVA was used to determine the effect of RO28-1675 treatment relative to vehicle (DMSO). Arteriole diameter in the figures is expressed as percentage of the maximum diameter, measured in 0 mM calcium, 2 mM EGTA containing Kreb’s solution.
Data was normalised and shown as the mean ± standard error of the mean (SEM) of three independent experiments performed in triplicate, unless stated otherwise. Western blotting and proteoglycan experiments were analysed by a 1-way ANOVA and glucose utilisation experiments were analysed by 2-way ANOVA. Results were considered significant when the probability was less than 0.05 (*P ≤ 0.05), 0.01 (**P ≤ 0.01) and 0.001(***P ≤ 0.001).