Proceedings of The Physiological Society

Future Physiology (Leeds, UK) (2017) Proc Physiol Soc 39, C03

Oral Communications

Airway epithelial cell regulation of intracellular glucose concentrations

J. M. Bearham1, D. L. Baines1

1. Infection and immunity, St Georges University of London, Mitcham, United Kingdom.

Maintaining low glucose concentrations in airway surface liquid (ASL) is essential in reducing the risk of airway infections. In normoglycemia (5mM) ASL glucose is maintained at ~0.4mM, but can rise up to 4mM during hyperglycaemia (15mM) and inflammation1. Glucose crosses the airway epithelium from blood to ASL and evidence indicates this can occur via paracellular and transcellular routes. For transcellular transport to occur, intracellular glucose concentrations should 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, promoting glucose uptake over efflux into ASL. However, what happens in hyperglycaemia is unknown. We investigated the effect of extracellular glucose on intracellular glucose concentrations to determine whether phosphorylation of glucose is a potential rate-determining step for glucose efflux and ASL glucose concentrations. We used the intracellular Förster resonance energy transfer (FRET) sensor Gluconic2, a glucose binding protein with donor and acceptor fluorophores. This was transfected into H441 airway epithelial cells and changes in intracellular glucose were measured using FRET ratio. To create a dose response curve, intracellular glucose was equilibrated 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 this. Data are shown as mean±SD and analysed using unpaired T-test. At an extracellular glucose concentration of 5mM D-Glucose +10mM L-Glucose (osmotic control) intracellular glucose as measured by FRET ratio was 47.4±12.3nM (n=16). Increasing extracellular D-glucose to 15mM decreased mean intracellular glucose concentrations (17.8±4.8nM, n=16, p≤0.0001). A cyclic fluctuation in FRET ratio was observed in both instances, with a cycle taking 3.4±0.2 minutes or 4.5±0.08 minutes respectively (p<0.05; n=4). H441 cell hexokinase activity was 32.9 ± 5.5nmol/mg/min (n=5). BrPy (100µM) reduced activity by 25±11% (p≤0.01). In 5mM D +10mM L-Glucose this resulted in an increase of intracellular glucose to 273.0 ±51.9nM (p≤0.001; n=14), and in 15mM D-Glucose, an increase to 169.8 ±53.4nM (p≤0.001; n=15). These data show that under normoglycaemic conditions, intracellular glucose is ~10-fold lower than ASL glucose and that elevating extracellular glucose concentrations decreased 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.

Where applicable, experiments conform with Society ethical requirements