glucose co-transporters (i.e. jejunum of mice and alveolar epithelial cells of rats) where, as previously shown, addition of glucose to the apical surface results in a change in Isc [20,21]. Moreover, addition of phlorizin, an SGLT blocker, did not result in a decrease in Isc, further confirming absence of Na+-glucose co-transport activity. Complementary experiments were performed using well-differen- tiated cultures of human airway epithelia (Figure S1) and results are consistent with data from intact airway epithelia. These data suggest that electrogenic Na+-glucose co-transport is not the mechanism that generates the transepithelial glucose gradient in human airway epithelia. Polarized expression of glucose transporters in human airway epithelia To identify glucose transporters in human airway epithelia, we first performed a screening analysis of GLUT (glucose facilitated diffusion proteins) and SGLT mRNA expression. We analyzed a dataset (GEO Accession #GSE20502) derived from microarray hybridizations with human in vivo and in vitro airway tissue (Figure 2, C). The analysis revealed a high correlation between expression levels of glucose transporters in vitro and in vivo (R2 = 0.81). We noticed high-level expression of GLUT-10 (Entrez GeneID: 81031), which is encoded by a 5-exon gene, located on chromosome 20. GLUT-10 mRNA has previously been detected in lung tissue and is also expressed in the heart, brain, skeletal muscle and placenta [22,23]. We then investigated the expression and localization of glucose transporters in human airway epithelia by immunocytochemistry. Airway Glucose and Innate Immunity We found no expression of GLUT-2, GLUT-3, GLUT-4, GLUT- 8, SGLT-1 or SGLT-2. We detected GLUT-1 (Entrez GeneID: 6513) in the basolateral membrane (Figure 2, D), and GLUT-10 localized to the apical membrane of human airway epithelia (Figure 2, E). These data suggest that expression of different facilitated diffusion glucose transporters in the apical and basolateral membranes of human airway epithelia may play a role in generation of the transepithelial glucose concentration gradient. Bidirectional fluxes of glucose in human airway epithelia The existence of a transepithelial glucose concentration gradient in the absence of active sodium-coupled absorption suggests that either 1) basolateral glucose supplied to the epithelial cells is unable to reach the ASL through the paracellular or the transcellular pathway, unlikely given that even in ‘‘tight epithelia’’, paracellular permeability to small molecules is not 0 (the paracellular pathway is dynamic) or 2) At equilibrium, the transepithelial flux of glucose into ASL is balanced by an absorptive process. We first measured the bidirectional flux of 14C-L-glucose in human airway epithelia. L-glucose is a stereoisomer of D-glucose that allows determination of paracellular transport of glucose because it is not a substrate for glucose transporter proteins. The 14C-L-glucose transport rate (Figure 3, A) was similar in both directions (basolateral to apical = 1.9060.09 nmoles/cm2/h, apical to basolateral = 2.1960.24 nmoles/cm2/h, p = 0.24) indicating paracellular permeability to glucose but no net 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 6 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 mm. doi:10.1371/journal.pone.0016166.g002 PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16166