H441 cells obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 media + 10% foetal bovine serum (FBS) (Invitrogen, UK); glucose (10 mM); l-glutamine (2 mM); sodium pyruvate (1 mM); insulin (10 μg/ml); transferrin (5 μg/ml); sodium selenite (7 ng/ml); penicillin (100 U/ml); and streptomycin (100 μg/ml). Non-polarised cells were grown in 12-well tissue culture plates. Polarised monolayers were produced by culturing cells for 7 days on permeable 4-µm pore polyester membrane supports (Transwells, Corning, MA, USA). The basolateral membrane was exposed to RPMI media [4% charcoal stripped serum; glucose (10 mM); dexamethasone (200 μM); 3,3′-5-Triiodothyronine (10 nM); l-glutamine (2 mM); sodium pyruvate (1 mM); insulin (10 μg/ml); transferrin (5 μg/ml); sodium selenite (7 ng/ml); penicillin (100 U/ml); and streptomycin (100 μg/ml)] and the apical membrane was at air interface. The day before the experiment, cells were washed with glucose-free RPMI media and incubated in medium with the required glucose concentration.
Glucose uptake experiments
Cultured cells were washed twice with glucose-free transport medium [15 mM HEPES buffer (pH 7.6), 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2 and 0.8 mM MgCl2] to remove culture medium, then incubated for 15 min at room temperature in the same solution to deplete the cells of intracellular glucose (± inhibitors).
Uptake experiments were performed as described previously . In non-polarised cells, the experiment was initiated by replacing the medium with 0.5 ml transport medium containing 1.0 μCi of radiolabelled glucose or glucose analogue plus 10 mM of non-radiolabelled equivalent glucose or glucose analogue (tracer mix), followed by incubation at room temperature for 10 min. Preliminary experiments indicated that uptake was linear between 0 and 10 min (data not shown). Uptake was terminated by adding 2 ml ice-cold stop solution [15 mM HEPES buffer (pH 7.6); 135 mM choline Cl; 5 mM KCl; 0.8 mM MgSO4; 1.8 mM CaCl2 and 0.2 mM HgCl2]. The cells were then rinsed twice with 2 ml stop solution and lysed in 0.5 ml of 10 mM Tris–HCl (pH 8.0) with 0.2% SDS. Lysed samples were added to 2 ml scintillation cocktail and radio-active emissions determined using a scintillation counter to quantify glucose uptake. Each uptake experiment was performed three times (n = 3) unless otherwise stated. The same basic protocol was used for all experiments with the following modifications: In polarised monolayers, the radioactive tracer mix was added either to the basolateral or apical side of the monolayer.
Most glucose uptake studies were carried out using [3H]-d-glucose, which is transported by all glucose transporters. Radiolabelled glucose analogues [3H]-deoxyglucose (DOG) (Sigma, UK) and [14C]-α-methyl-d-glucopyranoside (AMG) (Amersham, UK) were used to study GLUT- and SGLT-mediated glucose transport, respectively.
The effect of different glucose concentrations on glucose transport was determined by culturing cells at different glucose concentrations (1, 5, 10, and 15 mM) the day before the experiment. Cells from the same passage were used for all uptake experiments at different glucose concentrations to avoid discrepancies in results.
Several inhibitors were used to block glucose transport. Phlorizin (500 μM dissolved in ethanol) is a high affinity inhibitor of Na+–d-glucose co-transport via SGLT in whole tissue [13, 14]. Phloretin (1 mM dissolved in ethanol) and cytochalasin B (10 μM) inhibit glucose transport mediated by GLUTs in whole tissue . Ouabain (1 mM dissolved in ethanol), which inhibits Na+K+ATPase activity, and Na+ substitution with choline were also used in some experiments to determine if glucose transport was coupled to the Na+ gradient (SGLT-mediated). The amount of ethanol used to dissolve inhibitors (5 μl/well, 1% of total volume) was added to cells in control groups (vehicle control, 1% of total volume). Where inhibitors were used in experiments, they were added as soon as cells were transferred from culture to transport medium.
Reverse transcriptase polymerase chain reaction
Total RNA was isolated from H441 cells using the RNeasy kit (Qiagen). cDNA was generated using reverse transcriptase (Superscript II, Invitrogen, Carlsbad, CA, USA). Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using primer sequences derived from regions within GLUT1–GLUT4 and SGLT1 coding regions in a PCR reaction mix [1.5 mM MgCl2, 250 nM of each primer and 0.2 U of AGS gold Taq polymerase (Hybaid, Ashford, Middx, UK)]. PCR products were fractionated on 1.2% agarose gels and visualized by ethidium bromide staining and UV fluorescence. The following primer pairs were used in the reactions. GLUT1 sense: TCCACGGAGCATCTTCGAGA; GLUT1 anti-sense: ATACTGGAAGCACATGCCC (cycling protocol of 94°C for 1 min, 56°C for 1 min and 72°C for 90 s for 40 cycles); GLUT2 sense: CACTGATGGCTGCATGTGGC; GLUT2 anti-sense: ATGTGAACAGGGTAAAGGCC (cycling protocol of 94°C for 1 min, 56°C for 1 min and 72°C for 90 s for 40 cycles); GLUT2 nested sense: CTACTCAACCAGCATTTTTC; GLUT2 nested anti-sense: AACACATAAGGTCCACAGAA (cycling protocol of 94°C for 1 min, 52°C for 1 min and 72°C for 90 s for 40 cycles); GLUT3 sense: AAGGATAACTATAATGG; GLUT3 anti-sense: GGTCTCCTTAGCAGGCT (cycling protocol of 95°C for 45 s, 46°C for 30 s and 72°C for 45 s for 37 cycles); GLUT4 sense: GCCATTGTTATCGGCATTCT; GLUT4 anti-sense: GAGCTGGAGCAGGGACAGT (cycling protocol of 94°C for 1 min, 51°C for 1 min and 72°C for 1 min for 35 cycles). Primers amplifying a region of β-actin were used as an internal control. β-actin sense: CGGGACCTGACTGACTACC; β-actin anti-sense: TGAAGGTAGTTTCGTGGATGC (cycling protocol of 94°C for 3 min, 53°C for 1 min and 72°C for 1 min for 35 cycles). Cycle curves for all sets of PCR primers were performed. The number of cycles performed for each primer set was in the linear range of the curve.
Total cell protein was prepared from cells scraped from flasks and homogenised in tissue lysis buffer [100 mM Tris pH, 6.8, 1 mM EDTA pH 8.0, 1% NP-40, 10% v/v glycerol and 10 ml/l protease inhibitor cocktail] followed by centrifugation (5 min, 250×g) to remove nuclei. Plasma membranes were isolated as previously described . Briefly, cells were grown in six T75 flasks, lysed and the membranes separated from the intracellular fraction by centrifugation (60,000×g, 4°C, 30 min). Membrane and intracellular fractions were suspended in lysis buffer as above.
Membrane protein biotinylation
Apical or basolateral membrane proteins were biotinylated as previously described . Briefly, after 6 days at air interface, polarised cells were washed with ice-cold PBS. Sulfo-NHS-biotin (0.5 mg/ml) was applied to the apical or basolateral membrane. Cells were then lysed, proteins solubilised and incubated overnight with streptavidin agarose beads. The following day, biotinylated proteins bound to beads were separated from non-biotinylated proteins by centrifugation and samples prepared for immunoblotting.
Membrane protein fractions (50 μg) were separated on 4–12% Bis-Tris acrylamide gels, transferred to polyvinylidine difluoride membranes and incubated overnight at 4oC with anti GLUT1–GLUT4 affinity-purified antisera (1:200; Alpha Diagnostic, San Antonio, TX and Millipore, UK) or mouse monoclonal anti β-actin (1:500, AbCam, Cambridge, UK) or mouse monoclonal anti α1-Na+K+ATPase (1:1,000, Developmental Studies Hybridoma Bank, University of Iowa, USA). Blots were washed three times in PBS + 0.01% TWEEN 20 then incubated with either biotinylated donkey anti-rabbit IgG or rabbit anti-mouse secondary antiserum (1:200) (GE Healthcare, UK) followed by streptavidin horseradish peroxidase (HRP) conjugate (1:200) (GE Healthcare, UK) for 1 h each at room temperature. Immunostained proteins were visualised using enhanced chemiluminescence (ECL) western blot analysis system (NEN Life Science Products, Boston, MA; Western lightning, PerkinElmer, Norwalk, CT, USA) and exposure to X-ray film. All western blots were repeated at least twice. Blots of protein from cells grown at different glucose concentrations were processed simultaneously to allow visual comparisons to be made. Binding specificity of GLUT2 antibodies was confirmed in a repeat experiment where membranes were pre-incubated with antigenic peptides.
Measurement of short circuit current (I
Polarised monolayers were mounted in Ussing chambers with both apical and basolateral surfaces bathed in a physiological salt solution [117 mM NaCl; 25 mM NaHCO3; 4.7 mM KCl; 1.2 mM MgSO4; 1.2 mM KH2PO4; 2.5 mM CaCl2; 11 mM d-glucose, equilibrated with 5% CO2 to pH 7.3–7.4]. The solution was maintained at 37°C, bubbled with premixed gas [21% O2 + 5% CO2] and circulated continuously throughout the experiment. Monolayers were maintained under open circuit conditions whilst transepithelial potential difference (V
t) and resistance (R
t) were monitored and observed to reach a stable level. The cells were then short circuited by clamping V
t at 0 mV using a DVC-4000 voltage/current clamp. The current required to maintain this condition (I
sc) was measured and recorded using a PowerLab computer interface. Every 30 s throughout each experiment, the preparations were returned to open circuit conditions for 3 s so that the spontaneous V
t could be measured and R
t could be calculated as previously described .
The contribution of SGLT and epithelial sodium channels (ENaC) to I
sc were measured by adding 500 μM phlorizin or vehicle control to the apical or basolateral solution and 10 μM amiloride to the apical bath, respectively. Ion transport across these cells is dependent on Na+K+ATPase activity. Thus, ouabain (1 mM) was added to the basolateral compartment to calculate values for total I
Immunofluorescence staining of human bronchial epithelium
Endobronchial biopsies were obtained at bronchoscopy from two patients with chronic obstructive pulmonary disease and a patient with no airway disease participating in a study approved by King’s College Research Ethics Committee. Participants gave written informed consent for inclusion in the study. Biopsies were frozen immediately at optimal cutting temperature and stored at −80°C. Cryostat sections of 7 μm were mounted on polysine-coated slides. Sections were blocked with 10% chicken serum (ISL) in 0.1% Triton X-100-PBS. Sections were incubated with rabbit polyclonal antibodies raised against the human liver GLUT2 transporter (1:100 dilution in 0.1%Triton-PBS; Chemicon, Hampshire, UK) at room temperature for 1 h. After washing 3 × 10 min in PBS, sections were incubated in FITC-conjugated goat anti-rabbit IgG 1:100 diluted in 0.1% Triton-PBS for 30 min at room temperature. Slides were washed three times for 10 min in PBS, sections were then counterstained with 4′,6-diamidino-2-phenyindole (DAPI; 1:1,000 diluted in 0.1% Triton-PBS) to localise cell nuclei. Sections were also incubated with FITC-conjugated goat anti-rabbit IgG alone or GLUT2 antiserum pre-absorbed with a 10-fold excess of antigenic peptide for 20 min. Images were observed with a fluorescence microscope Zeiss with Axioplan 2 analysis system using the Axiovision 4.5 software. Cell fluorescence was recorded by excitation at 450–490 nm.
Chemicals and reagents
All chemicals and reagents were obtained from Sigma, Poole, UK unless otherwise stated.
Values are reported as mean ± SEM. Statistical analysis was performed using paired Student’s t tests or one-way ANOVA tests (where appropriate). P values of <0.05 were considered statistically significant.