Effective glucose metabolism maintains low intracellular glucose in airway epithelial cells after exposure to hyperglycemia.

The airway epithelium maintains differential glucose concentrations between the airway surface liquid (ASL, ~0.4 mM) and the blood/interstitium (5-6 mM), which is important for defense against infection. Glucose primarily moves from the blood to the ASL via paracellular movement, down its concentration gradient, across the tight junctions. However, there is evidence that glucose can move transcellularly across epithelial cells. Using a Förster resonance energy transfer sensor for glucose, we investigated intracellular glucose concentrations in airway epithelial cells and the role of hexokinases in regulating intracellular glucose concentrations in normoglycemic and hyperglycemic conditions. Our findings indicated that in airway epithelial cells (H441 or primary human bronchial epithelial cells) exposed to 5 mM glucose (normoglycemia), intracellular glucose concentration is in the micromolar range. Inhibition of facilitative glucose transporters (GLUTs) with cytochalasin B reduced intracellular glucose concentration. When cells were exposed to 15 mM glucose (hyperglycemia), intracellular glucose concentration was reduced. Airway cells expressed hexokinases I, II, and III. Inhibition with 3-bromopyruvate decreased hexokinase activity by 25% and elevated intracellular glucose concentration, but levels remained in the micromolar range. Exposure to hyperglycemia increased glycolysis, glycogen, and sorbitol. Thus, glucose enters the airway cell via GLUTs and is then rapidly processed by hexokinase-dependent and hexokinase-independent metabolic pathways to maintain low intracellular glucose concentrations. We propose that this prevents transcellular transport and aids the removal of glucose from the ASL and that the main route of entry for glucose into the ASL is via the paracellular pathway.

A modified fluorescent sensor for reporting glucose concentration in the airway lumen.

We have modified the periplasmic Escherichia coli glucose/galactose binding protein (GBP) and labelled with environmentally sensitive fluorophores to further explore its potential as a sensor for the evaluation of glucose concentration in airway surface liquid (ASL). We identified E149C/A213R GBP labelled with N,N'-Dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD, emission wavelength maximum 536nm) with a Kd for D-glucose of 1.02mM and a fluorescence dynamic range of 5.8. This sensor was specific for D-glucose and exhibited fluorescence stability in experiments for several hours. The use of E149C/A213R GBP-IANBD in the ASL of airway cells grown at air-liquid-interface (ALI) detected an increase in glucose concentration 10 minutes after raising basolateral glucose from 5 to 15mM. This sensor also reported a greater change in ASL glucose concentration in response to increased basolateral glucose in H441 airway cells compared to human bronchial epithelial cells (HBEC) and there was less variability with HBEC data than that of H441 indicating that HBEC more effectively regulate glucose movement into the ASL. The sensor detected glucose in bronchoalveolar lavage fluid (BALf) from diabetic db/db mice but not normoglycaemic wildtype mice, indicating limited sensitivity of the sensor at glucose concentrations <50µM. Using nasal inhalation of the sensor and spectral unmixing to generate images, E149C/A213R GBP-IANBD fluorescence was detected in luminal regions of cryosections of the murine distal lung that was greater in db/db than wildtype mice. In conclusion, this sensor provides a useful tool for further development to measure luminal glucose concentration in models of lung/airway to explore how this may change in disease.

Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H+ secretion.

The cystic fibrosis (CF) airway surface liquid (ASL) provides a nutrient rich environment for bacterial growth including elevated glucose, which together with defective bacterial killing due to aberrant HCO3- transport and acidic ASL, make the CF airways susceptible to colonisation by respiratory pathogens such as Pseudomonas aeruginosa. Approximately half of adults with CF have CF related diabetes (CFRD) and this is associated with increased respiratory decline. CF ASL contains elevated lactate concentrations and hyperglycaemia can also increase ASL lactate. We show that primary human bronchial epithelial (HBE) cells secrete lactate into ASL, which is elevated in hyperglycaemia. This leads to ASL acidification in CFHBE, which could only be mimicked in non-CF HBE following HCO3- removal. Hyperglycaemia-induced changes in ASL lactate and pH were exacerbated by the presence of P. aeruginosa and were attenuated by inhibition of monocarboxylate lactate-H+ co-transporters (MCTs) with AR-C155858. We conclude that hyperglycaemia and P. aeruginosa induce a metabolic shift which increases lactate generation and efflux into ASL via epithelial MCT2 transporters. Normal airways compensate for MCT-driven H+ secretion by secreting HCO3-, a process which is dysfunctional in CF airway epithelium leading to ASL acidification and that these processes may contribute to worsening respiratory disease in CFRD.