Airway Epithelial Cell Regulation of Intracellular Glucose Concentrations: The Role of Glucose Homeostasis

Abstract

Maintaining low glucose concentrations in airway surface liquid (ASL) is essential in reducing the risk of airway infections. In normoglycaemic conditions (5mM) ASL glucose is maintained at ~0.4mM, but can rise up to 4mM during hyperglycaemia (15mM)1. Glucose crosses the airway epithelium from blood to ASL and evidence indicates that this can occur via paracellular and transcellular routes. For transcellular transport to occur, intracellular glucose concentrations must exceed that of ASL glucose. Intracellular glucose concentrations are determined by glucose metabolism. Upon entering the cell, hexokinases phosphorylate glucose to glucose‐6‐phosphate keeping glucose concentration low in the cell so that glucose uptake is promoted over efflux into ASL. However, what happens in hyperglycaemia is unknown.Therefore, we investigated the effect of extracellular glucose on intracellular glucose concentrations to determine whether phosphorylation of glucose by hexokinase is a potential rate‐determining step for glucose efflux and ASL glucose concentrations in normo and hyperglycaemic conditions. We used the intracellular Förster resonance energy transfer (FRET) sensor Gluconic2, a glucose binding protein flanked by donor and acceptor fluorophores cyan and yellow fluorescent proteins (CFP and YFP respectively). This was transfected into H441 airway epithelial cells and changes in intracellular glucose were measured using CFP/YFP FRET ratio, which decreases with an increase in glucose concentration. To create a dose response curve for Gluconic, intracellular glucose was equibrilated with extracellular glucose by inhibiting glucose metabolism with hexokinase II inhibitor 3‐Bromopyruvic acid (BrPy) (1mM) and respiratory chain complex I inhibitor Rotenone (100nM). All values for intracellular glucose were calculated from the dose response data. Values are shown as mean ±SEM and analysed using unpaired T‐test with Welch's correction.At an extracellular glucose concentration of 5mM D‐Glucose +10mM L‐Glucose (osmotic control) intracellular glucose as measured by FRET ratio was 3.3nM ± 1.1nM (n=4). An increase in extracellular D‐glucose to 15mM had no effect on the mean intracellular glucose concentrations (8.3nM ± 4.2nM, n=4), but intracellular glucose concentrations fluctuated widely over time in a cyclic manner.H441 cell hexokinase activity was 32.9 ± 5.5nmol/mg/min (n=5). BrPy (100μM) reduced activity by 23 ±5% (P≤0.01; n=5) in 5mM D +10mM L‐Glucose. This resulted in an increase of intracellular glucose to 73.0nM ± 15.4nM (P≤0.001; n=4).These data show that under normoglycaemic conditions, intracellular glucose is ~100 fold lower than ASL glucose and that elevating extracellular glucose concentrations had no effect on mean intracellular glucose. This would suggest that under both conditions, transcellular transport of glucose is unlikely to occur because the gradient would promote uptake from the ASL over efflux. These data also indicate that hexokinase II activity plays a role in maintaining low intracellular glucose in airway epithelia, but other factors may also play a part.Support or Funding InformationFunded by MRC‐CASE studentship MR/L013509/1.