Figure 1.
Human airway epithelia maintain a transepithelial glucose gradient.
(A) Transmission electron microscopy of perfluorocarbon-fixed cultures of human airway epithelia. Figure indicates airway surface liquid (ASL), epithelial surface (ES) and filter membrane support (FM). (B) Glucose concentration in ASL collected from cultures of human airway epithelia was compared to glucose concentration in basolateral nutrient media. Data shown as mean ± s.e.m. n = 6 samples per group. (***: p<0.0001).
Figure 2.
Human airway epithelia express GLUT-1 and GLUT-10.
(A) Short-circuit current (Isc) in human bronchial epithelia was studied in Ussing chambers. To maximize detection of SGLT-mediated sodium absorption, amiloride and GlyH were added to the apical buffer to block ENaC and CFTR-mediated absorption of sodium and secretion of chloride, respectively, followed by glucose in increasing concentrations and phlorizin. Each circle corresponds to one sample (11 total) obtained from 3 donors. Mean ± s.e.m are shown (B) Representative Isc tracing from intact human bronchial epithelia. (C) Microarray analysis of mRNA from in vivo and in vitro human airway tissue. Normalized expression levels of GLUT and SGLT gene family members are shown. n = 16 samples per group. (D and E) Cultures of human airway epithelia were immunostained using a GLUT-1 (D green) or GLUT-10 (E, green) antibody. XY (top image) and XZ (bottom image) projections are shown. DAPI (nuclei) is shown in blue, E-cadherin (intercellular junction) is shown in D (red), and acetylated alpha tubulin (cilia) is shown in E (red). Representative images from 3 experiments are shown. Isotype-matched antibody and no-primary controls did not show fluorescence after staining. Scale bars, 10 µm.
Figure 3.
Airway epithelia can deplete airway surface liquid glucose.
(A and B) Basolateral to apical (BL to AP) and apical to basolateral (AP to BL) fluxes of L-[1-14C]glucose (A) or 2-deoxy-d-[1-14C]glucose (B) were measured in cultures of human airway epithelia over 1 hour. Data shown as mean ± s.e.m. n = 6 samples per group. (***: p<0.0001, ns: p ≥ 0.05). (C) Basolateral (BL) and apical (AP) uptake of 2-deoxy-d-[1-14C]glucose (2-DOG) in cultures of human airway epithelia were measured over 1 hour. Data shown as mean ± s.e.m. n = 6 samples per group. (***: p<0.0001). (D) Data from Figures 3 A, B and C are integrated into a model of glucose transport in human airway epithelia. Data in nmol/h. Black solid arrow represents paracellular diffusion of glucose. Gray solid arrows represent 2-DOG uptake capacity across the apical (top) and basolateral (bottom) membranes. Dotted arrows represent transcellular bidirectional fluxes of 2-DOG (adjusted by subtracting corresponding L-glucose fluxes). GLUT-10 is present in the apical membrane and GLUT-1 is present in the basolateral membrane. Intracellular glucose (G) is phosphorylated by hexokinase (HK) for subsequent glycolysis. G6P = glucose-6-phosphate.
Figure 4.
Low glucose concentration impairs growth of P. aeruginosa in vitro.
(A) Glucose concentration-dependent growth of P. aeruginosa. M9 medium with glucose concentrations as shown (0, 0.5, 2, 8 and 24 mM) was inoculated with P. aeruginosa (PAO1) and OD600 was followed. Data shown as mean ± s.e.m. n = 3. (B) Growth of P. aeruginosa in ASL is nutrient-limited. ASL and M9 medium with and without added glucose (20 mM) as shown were inoculated with PAO1. OD600 was followed over time. Data shown as mean ± s.e.m. n = 3. (***: p<0.0001 for ASL vs. ASL + glucose). (C) Growth of P. aeruginosa in human airway epithelia is limited by ASL glucose concentration. Cultures of human airway epithelia were inoculated apically with 2.3 or 3.3 log CFU of PAO1 in the absence (white bars) or presence (gray bars) of added glucose (20 mM). After 24 h, apical bacteria were quantified. Percentage of samples in which growth of bacteria occurred are shown. n = 5. (D) Facilitated diffusion transport in airway epithelia in vitro impairs growth of P. aeruginosa. Cultures of human airway epithelia were inoculated apically with 0.5, 1.5 or 2.5 log CFU of PAO1 after incubation with apical vehicle (white bars) or phloretin (gray bars). After 24 h, apical bacteria were quantified. Percentage of samples in which growth of bacteria occurred are shown. n = 12. (*: p<0.05 for vehicle vs. phloretin).
Figure 5.
Hyperglycemia promotes growth of P. aeruginosa in the lungs of mice.
(A and B) An intranasal inoculum (7.69 log CFU) of PAO1 (A) or edd− (B) was given to ob/ob and db/db mice and their respective littermate controls (ob/ob ctrl and db/db ctrl). After 6 hours, lungs were homogenized for bacterial counts. n = 5 mice per group. (*: p<0.05, ns: p ≥ 0.05).
Figure 6.
Alterations in transepithelial glucose transport.
Diagram of transepithelial glucose transport under normal conditions (A), hyperglycemia (B), increased tight junction permeability (C) or impaired apical glucose transporter function (D). Gray solid arrows represent glucose uptake capacity across the apical (top) and basolateral (bottom) membranes. Dotted arrows represent transcellular bidirectional fluxes of glucose. Arrow width and font size are modified in each figure according to the primary alteration in glucose transport rate and resulting basolateral and apical concentration, respectively. GLUT-10 is present in the apical membrane and GLUT-1 is present in the basolateral membrane. Intracellular glucose (G) is phosphorylated by hexokinase (HK) for subsequent glycolysis. G6P = glucose-6-phosphate.