Cell culture.
H441 airway epithelial cells were cultured at 37°C, 5% CO2, in RPMI 1640 media containing 10 mM glucose and supplemented with 10% fetal calf serum (Sigma-Aldrich), 2 mM l-glutamine, 1 mM sodium pyruvate, 5 µg/mL insulin, 2.75 µg/mL penicillin, and 100 mg/mL streptomycin (Life Technologies). Human bronchial epithelial cells (HBECs) were originally purchased from Lonza and Epithelix before semi-immortalization with polycomb complex protein BMI-1 (BMI-1) transduction and were cultured in collagen-coated flasks (Corning) in bronchial epithelial growth media (BEGM; Lonza) in a humidified environment at 37°C, 5% CO2. Growth media was replaced every second day, and cells were passaged once 80% confluent.
Polarized monolayers were cultured on Transwells (Corning). H441 cells were plated onto the Transwell using the medium described above until confluent. The apical medium was then removed, and the basolateral medium was changed to RPMI 1640 media containing 10 mM glucose and supplemented with 4% charcoal stripped serum, 200 µM dexamethasone, 10 nM 3,3′-5-triiodothyronine, 2 mM l-glutamine, 1 mM sodium pyruvate, 5 µg/mL insulin, 2.75 µg/mL penicillin, and 100 mg/mL streptomycin. Cells were then cultured at air-liquid interface (ALI) for 10 days, changing the medium every other day until the cells formed a resistive monolayer. HBECs were seeded at a density of 200,000 cells/cm2 on Transwells. After confluence was achieved, media were removed from the apical surface, and the cells were fed on the basolateral side only with 50% BEGM and 50% high-glucose minimal essential medium containing 100 nM retinoic acid. The media were exchanged every 2–3 days, and the apical surface mucus was removed by gentle washing with phosphate-buffered saline once a week. Cultures were used for functional analysis 28–35 days after exposure to ALI. BMI-1-transduced cells exhibit normal cell morphology, karyotype, and doubling times despite extensive passaging. When cultured at ALI, they show normal ciliation, show normal production of MUC5AC and MUC5B, and have electrophysiological properties similar to primary cells (26). Transepithelial resistance was measured before use with the epithelial volt/ohmmeter EVOM (Word Precision Instruments), and at least 200 Ω·cm2 resistance was required before use in experiments. Eighteen hours before experiments, cell media were exchanged with growth medium containing 5 mM d-glucose (supplemented as listed above). To investigate the effect of hyperglycemia, cells were either exposed to 5 mM d-glucose + 10 mM l-glucose (an analog not transported or metabolized, to control for any osmotic effects of raising glucose) to mimic normoglycemia (5 mM glucose) or 15 mM d-glucose to mimic hyperglycemia (15 mM glucose). The apical surface of cell cultures was gently washed with 100 μL PBS to obtain airway surface liquid washes. Glucose in the washes was analyzed using an Amplex Red glucose oxidase kit (Thermo Fisher).

Cell transfection.
Cells were seeded at a density of 2 × 105 cells/cm2 onto glass coverslips coated in polylysine and, once at 50–65% confluency, were transiently transfected with 1 µg of the glucose-sensitive sensor FLII12Pglu-700µΔ6 (Addgene plasmid no. 17866) or cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) FRET positive control plasmid (a kind gift from R. Tarran, University of North Carolina at Chapel Hill, Chapel Hill, NC) using Lipofectamine 2000 (Thermo Fisher). Polarized monolayers were apically transfected in a similar fashion, with 1 µg of plasmid transfected using TransIT-X2 (Mirus) applied to the apical surface of the cells.

FRET microscopy.
Cells were imaged 48–72 h posttransfection in phosphate-buffered saline at 37°C, 95% air-5% CO2, supplemented with glucose and/or inhibitors using a Zeiss LSM 510 Meta confocal microscope with a ×20 Plan-Neofluar lens or a Leica SP8 with a ×20 PL APO CS2 lens. FLII12Pglu-700µΔ6 contains the FRET paired fluorophores enhanced CFP (eCFP; donor) and Citrine (acceptor), which report a reduced eCFP-to-Citrine FRET ratio with a binding of glucose. This was measured on the Zeiss LSM 510 by collecting emission data from eCFP (459–505 nm) and Citrine (525–600 nm) every 4 s over an 8-min time period while exciting eCFP at 458 nm. Settings were optimized for the growth conditions of each cell type, which took into account opacity of the substrate (i.e., glass coverslips and Transwells), cell height, and cell density. Thus, the output measurement was different for the three conditions studied.

Generating dose-response data for the sensor.
Glucose dose-response data were generated for each cell type and growth condition. Cells transfected with FLII12Pglu-700µΔ6 were treated with hexokinase inhibitor 3-bromopyruvic acid (BrPy, 100 µM) plus the respiratory chain complex I inhibitor rotenone (100 nM) for 30 min to inhibit glucose metabolism. During this time, cells were incubated with different glucose concentrations to equilibrate intracellular glucose with extracellular glucose before imaging as previously described to equilibrate intracellular and extracellular lactate for FRET measurement (33). FRET activity of FLII12Pglu-700µΔ6 was imaged as described above.

Hexokinase assay.
Cells were untreated or pretreated for 10 min with BrPy (0.1 µM to 1 mM) at 37°C, 95% air-5% CO2. Cell lysates were prepared, and a colorimetric hexokinase assay (ab-136957; Abcam), which measures the conversion of glucose to glucose-6-phosphate by hexokinase, was performed as per the manufacturer’s instructions.

Sorbitol assay.
Proliferating H441 cells were exposed to 5 mM d-glucose + 10 mM l-glucose or 15 mM d-glucose in the presence or absence of BrPy (100 µM) for 10 min before washing in ice-cold PBS. Cells were then lysed in 200 µL of assay buffer and centrifuged for 5 min at 4°C at 12,000 rpm. The lysate was decanted, and sorbitol concentrations were determined by sorbitol colorimetric assay (ab-118968; Abcam) as per the manufacturer’s protocol.

Seahorse glycolysis stress assay.
Human bronchiolar epithelial cells were seeded into a Seahorse XF96 plate and incubated at 37°C, 5% CO2 for 48 h. The medium was changed 24 h before the Seahorse experiment, and cells were exposed to 5 or 15 mM glucose with or without BrPy (100 μM) or epalrestat (1 or 10 μM) for the last 30 min before the Seahorse glycolysis stress assay was performed according to the manufacturer’s instructions followed by the sequential injection of oligomycin to inhibit ATP-linked reparation and 2-deoxy-d-glucose (2-DG) to inhibit glucose metabolism. The plate layout was separated into quadrants to reduce edge effects. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured. Glycolysis rate was calculated by subtracting the normalized ECAR values after 2-DG injection from the ECAR values after glucose injection to exclude the nonglycolytic acidification from the calculation. Glycolytic capacity was calculated by subtracting the nonglycolytic acidification rate (ECAR after 2-DG injection) from the maximum ECAR after 1 μM oligomycin injection.

Western blots.
Cells were lysed in RIPA buffer [20 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Nonidet P-40, and 1% sodium deoxycholate] plus protease inhibitor cocktail (Sigma) with gentle agitation at 4°C for 30 min. Protein concentration was calculated from a bicinchoninic acid assay (Thermo Fisher). Twenty micrograms of protein were electrophoresed through a 4–12% Bis-Tris gel. Gels were blotted onto a PVDF membrane and blocked with Odyssey blocking buffer (Li-Cor). Membranes were incubated in primary antibodies [hexokinase I (HKI), ab-65069, 1:500; hexokinase II (HKII), ab-37593, 1:250; hexokinase III (HKIII), ab-126217, 1:500; and β-actin, A-5441, 1:10,000] followed by secondary antibodies (goat anti-rabbit 680RD, 925-68071, 1:15,000; and donkey anti-mouse 800CW, 925-32212, 1:15,000). Blots were imaged using the Li-Cor Odyssey system.

Data analysis.
FRET eCFP/Citrine intensity and Western blot band intensity data were measured using ImageJ software. Data are displayed as means ± standard deviation and analyzed using GraphPad Prism 7 using ANOVA followed by a post hoc Tukey’s test unless otherwise stated.