Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C035

Oral Communications

FRET-ting about intracellular airway epithelial glucose concentrations and metabolism

J. M. Bearham1, D. L. Baines1

1. Institute for Infection and Immunity, St Georges University of London, London, United Kingdom.

Normally glucose in the liquid lining the lumen of the airway (airway surface liquid; ASL) is maintained at ~0.4mM, and this is thought to promote sterility of the airways. However elevation of blood/basolateral glucose and inflammation increase glucose in the ASL both in vitro and in vivo1. Glucose crosses the airway epithelium from blood to ASL via paracellular and transcellular routes. For transcellular transport to occur, we hypothesised that intracellular glucose concentrations must exceed that of ASL glucose. Intracellular glucose concentrations are determined by glucose metabolism. We investigated the effect of extracellular glucose on intracellular glucose concentrations and metabolism to determine whether hexokinase activity is a rate determining step for ASL glucose concentrations. We used the intracellular FRET sensor Gluconic2, a glucose binding protein with donor and acceptor fluorophores cyan and yellow fluorescent proteins (CFP and YFP respectively) transfected into H441 airway epithelial cells. Changes in intracellular glucose were measured using CFP/YFP FRET ratio, which decreases with an increase in glucose concentration. A glucose dose response curve for Gluconic, was generated, and values for intracellular glucose were calculated from this. Sorbitol concentrations were measured (a product from the polyol metabolic pathway) by colorimetric assay. Data are shown as mean±S.D. In H441 cells exposed to an extracellular glucose concentration of 5mM D-Glucose +10mM L-Glucose (osmotic control), intracellular glucose, as measured by FRET ratio was 4.57±1.46µM (n=83). An increase in extracellular D-glucose to 15mM decreased intracellular glucose to 2.17±0.54µM (n=83, p≤0.001). H441 cell hexokinase activity was 32.9±5.5nmol/mg/min (n=5). Bromopyruvic acid (100µM) reduced total hexokinase activity by 23±5% (P≤0.01; n=5) in 5mM glucose, which resulted in an increase of intracellular glucose to 0.031±0.0047mM (n=83, p≤0.001) in H441 cells. In 15mM extracellular D-glucose, there was a trend towards increased intracellular sorbitol concentrations compared to 5mM (n=3) and inhibition of hexokinase had no effect on these values. These data indicate that under normoglycaemic conditions, intracellular glucose is ~100 fold lower than ASL glucose and that elevating extracellular glucose concentrations decreased intracellular glucose. This would suggest that under both conditions, transcellular transport of glucose is unlikely because the glucose gradients would favour uptake from the ASL over efflux into the ASL, and that the primary flux of glucose into the ASL occurs via the paracellular route. In addition, we previously showed that hyperglycaemia increased lactate production3. This study has provided preliminary data indicating that the polyol pathway may also be activated in hyperglycaemia. We therefore propose that the decrease in intracellular glucose in hyperglycaemia is due to a shift in metabolic processing of glucose.

Where applicable, experiments conform with Society ethical requirements