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Apical and basolateral localisation of GLUT2 transporters in human lung epithelial cells.

Kalsi KK, Baker EH, Medina RA, Rice S, Wood DM, Ratoff JC, Philips BJ, Baines DL - Pflugers Arch. (2008)

Bottom Line: In non-polarised H441 cells, uptake of D: -glucose and deoxyglucose was similar.We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells.We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway.

View Article: PubMed Central - PubMed

Affiliation: Centre for Ion Channel and Cell Signalling, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London, SW17 0RE, UK.

ABSTRACT
Glucose concentrations of normal human airway surface liquid are approximately 12.5 times lower than blood glucose concentrations indicating that glucose uptake by epithelial cells may play a role in maintaining lung glucose homeostasis. We have therefore investigated potential glucose uptake mechanisms in non-polarised and polarised H441 human airway epithelial cells and bronchial biopsies. We detected mRNA and protein for glucose transporter type 2 (GLUT2) and glucose transporter type 4 (GLUT4) in non-polarised cells but GLUT4 was not detected in the plasma membrane. In polarised cells, GLUT2 protein was detected in both apical and basolateral membranes. Furthermore, GLUT2 protein was localised to epithelial cells of human bronchial mucosa biopsies. In non-polarised H441 cells, uptake of D: -glucose and deoxyglucose was similar. Uptake of both was inhibited by phloretin indicating that glucose uptake was via GLUT-mediated transport. Phloretin-sensitive transport remained the predominant route for glucose uptake across apical and basolateral membranes of polarised cells and was maximal at 5-10 mM glucose. We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells. Our study provides the first evidence that glucose transport in human airway epithelial cells in vitro and in vivo utilises GLUT2 transporters. We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway.

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GLUT2-fluorescent immunohistochemistry of representative sequential frozen sections from two independent human bronchial mucosa biopsies (a–f) and (g–l). GLUT2–FITC fluorescence was localized to the epithelial cells present in the sections (a, c, g and i). DAPI staining of cellular nuclei was observed in all cells visible in the section (b, e, h and k). Merged fluorescence images of GLUT2–FITC and DAPI are shown in (c, f, i and l). No FITC fluorescence was observed in the epithelial cells in the absence of GLUT2 antiserum (d and f). Pre-absorbtion of GLUT 2 antiserum with GLUT2 antigenic peptide also inhibited FITC labelling of epithelial cells (j and l). White arrows indicate the position of epithelial cells. All images were obtained at ×40 magnification. Bar = 10 μm
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Fig9: GLUT2-fluorescent immunohistochemistry of representative sequential frozen sections from two independent human bronchial mucosa biopsies (a–f) and (g–l). GLUT2–FITC fluorescence was localized to the epithelial cells present in the sections (a, c, g and i). DAPI staining of cellular nuclei was observed in all cells visible in the section (b, e, h and k). Merged fluorescence images of GLUT2–FITC and DAPI are shown in (c, f, i and l). No FITC fluorescence was observed in the epithelial cells in the absence of GLUT2 antiserum (d and f). Pre-absorbtion of GLUT 2 antiserum with GLUT2 antigenic peptide also inhibited FITC labelling of epithelial cells (j and l). White arrows indicate the position of epithelial cells. All images were obtained at ×40 magnification. Bar = 10 μm

Mentions: Fluorescence microscopy of three independent biopsies from human bronchial mucosa including one control patient with no airway disease revealed specific staining of epithelial cells with GLUT2 antibody. Representative sequential sections from two biopsies immunostained with GLUT2 antiserum and counterstained with DAPI are shown in Fig. 9. Images a–f and images g–l are from two independent biopsies. GLUT2 immunofluorescence was present only in the epithelial cell layer (Fig. 9a,c,g and i). The epithelial cell membranes were clearly defined and we could not distinguish any differences in intensity between the apical and basolateral membranes indicating that GLUT2 was present in both (Fig. 9a,c). Non-specific binding of anti rabbit FITC was not observed in the absence of GLUT2 antibody (Fig. 9d,f). Furthermore, we could not detect FITC fluorescence in the epithelial cell membrane after pre-absorption with GLUT2 antigenic peptide (Fig. 9j,l).Fig. 9


Apical and basolateral localisation of GLUT2 transporters in human lung epithelial cells.

Kalsi KK, Baker EH, Medina RA, Rice S, Wood DM, Ratoff JC, Philips BJ, Baines DL - Pflugers Arch. (2008)

GLUT2-fluorescent immunohistochemistry of representative sequential frozen sections from two independent human bronchial mucosa biopsies (a–f) and (g–l). GLUT2–FITC fluorescence was localized to the epithelial cells present in the sections (a, c, g and i). DAPI staining of cellular nuclei was observed in all cells visible in the section (b, e, h and k). Merged fluorescence images of GLUT2–FITC and DAPI are shown in (c, f, i and l). No FITC fluorescence was observed in the epithelial cells in the absence of GLUT2 antiserum (d and f). Pre-absorbtion of GLUT 2 antiserum with GLUT2 antigenic peptide also inhibited FITC labelling of epithelial cells (j and l). White arrows indicate the position of epithelial cells. All images were obtained at ×40 magnification. Bar = 10 μm
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2480509&req=5

Fig9: GLUT2-fluorescent immunohistochemistry of representative sequential frozen sections from two independent human bronchial mucosa biopsies (a–f) and (g–l). GLUT2–FITC fluorescence was localized to the epithelial cells present in the sections (a, c, g and i). DAPI staining of cellular nuclei was observed in all cells visible in the section (b, e, h and k). Merged fluorescence images of GLUT2–FITC and DAPI are shown in (c, f, i and l). No FITC fluorescence was observed in the epithelial cells in the absence of GLUT2 antiserum (d and f). Pre-absorbtion of GLUT 2 antiserum with GLUT2 antigenic peptide also inhibited FITC labelling of epithelial cells (j and l). White arrows indicate the position of epithelial cells. All images were obtained at ×40 magnification. Bar = 10 μm
Mentions: Fluorescence microscopy of three independent biopsies from human bronchial mucosa including one control patient with no airway disease revealed specific staining of epithelial cells with GLUT2 antibody. Representative sequential sections from two biopsies immunostained with GLUT2 antiserum and counterstained with DAPI are shown in Fig. 9. Images a–f and images g–l are from two independent biopsies. GLUT2 immunofluorescence was present only in the epithelial cell layer (Fig. 9a,c,g and i). The epithelial cell membranes were clearly defined and we could not distinguish any differences in intensity between the apical and basolateral membranes indicating that GLUT2 was present in both (Fig. 9a,c). Non-specific binding of anti rabbit FITC was not observed in the absence of GLUT2 antibody (Fig. 9d,f). Furthermore, we could not detect FITC fluorescence in the epithelial cell membrane after pre-absorption with GLUT2 antigenic peptide (Fig. 9j,l).Fig. 9

Bottom Line: In non-polarised H441 cells, uptake of D: -glucose and deoxyglucose was similar.We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells.We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway.

View Article: PubMed Central - PubMed

Affiliation: Centre for Ion Channel and Cell Signalling, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London, SW17 0RE, UK.

ABSTRACT
Glucose concentrations of normal human airway surface liquid are approximately 12.5 times lower than blood glucose concentrations indicating that glucose uptake by epithelial cells may play a role in maintaining lung glucose homeostasis. We have therefore investigated potential glucose uptake mechanisms in non-polarised and polarised H441 human airway epithelial cells and bronchial biopsies. We detected mRNA and protein for glucose transporter type 2 (GLUT2) and glucose transporter type 4 (GLUT4) in non-polarised cells but GLUT4 was not detected in the plasma membrane. In polarised cells, GLUT2 protein was detected in both apical and basolateral membranes. Furthermore, GLUT2 protein was localised to epithelial cells of human bronchial mucosa biopsies. In non-polarised H441 cells, uptake of D: -glucose and deoxyglucose was similar. Uptake of both was inhibited by phloretin indicating that glucose uptake was via GLUT-mediated transport. Phloretin-sensitive transport remained the predominant route for glucose uptake across apical and basolateral membranes of polarised cells and was maximal at 5-10 mM glucose. We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells. Our study provides the first evidence that glucose transport in human airway epithelial cells in vitro and in vivo utilises GLUT2 transporters. We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway.

Show MeSH
Related in: MedlinePlus