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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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a RT-PCR of GLUT1–GLUT4 mRNA sequences from non-polarised H441 cells. Products were resolved on ethidium bromide-stained agarose gels. RT-PCR products corresponding to the correct size for GLUT1 (400 bp), GLUT2 (425 bp) and GLUT4 (299 bp) were amplified. No products corresponding to GLUT3 (411 bp) were detected in H441 cells but were detected in a positive control, Ishikawa cell line (data not shown). Amplification of β-actin was used as a control for the reaction. b Western blot analysis of glucose transporter expression in non-polarised H441 cells. Total protein (Total), intracellular proteins (Intracellular) and plasma membrane protein (Plasma membrane) (50 μg) were resolved on acrylamide gels. Immunostained products corresponding to GLUT2 (∼60 kDa) and GLUT4 (∼45 kDa) were detected. Positive controls (+C) used: GLUT1, skeletal muscle; GLUT2, liver; GLUT3, Ishikawa cells; GLUT4, adipose tissue
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Fig3: a RT-PCR of GLUT1–GLUT4 mRNA sequences from non-polarised H441 cells. Products were resolved on ethidium bromide-stained agarose gels. RT-PCR products corresponding to the correct size for GLUT1 (400 bp), GLUT2 (425 bp) and GLUT4 (299 bp) were amplified. No products corresponding to GLUT3 (411 bp) were detected in H441 cells but were detected in a positive control, Ishikawa cell line (data not shown). Amplification of β-actin was used as a control for the reaction. b Western blot analysis of glucose transporter expression in non-polarised H441 cells. Total protein (Total), intracellular proteins (Intracellular) and plasma membrane protein (Plasma membrane) (50 μg) were resolved on acrylamide gels. Immunostained products corresponding to GLUT2 (∼60 kDa) and GLUT4 (∼45 kDa) were detected. Positive controls (+C) used: GLUT1, skeletal muscle; GLUT2, liver; GLUT3, Ishikawa cells; GLUT4, adipose tissue

Mentions: We detected mRNA for GLUT1, GLUT2 and GLUT4 in non-polarised H441 cells (Fig. 3a). In non-polarised cells cultured at 10 mM glucose, a protein of predicted size for GLUT2 (60 kDa) was detected in separated plasma membrane and intracellular protein fractions in approximately equal quantities (Fig. 3b). A 45-kDa product corresponding to GLUT4 protein was detected in total cell protein and the intracellular fraction but was absent from the plasma membrane protein fraction. It is interesting to note that although GLUT1 mRNA was detected, translated protein could not be detected by western blotting in these cells (Fig. 3b). Neither GLUT3 mRNA nor protein was detected (Fig. 3a,b).Fig. 3


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)

a RT-PCR of GLUT1–GLUT4 mRNA sequences from non-polarised H441 cells. Products were resolved on ethidium bromide-stained agarose gels. RT-PCR products corresponding to the correct size for GLUT1 (400 bp), GLUT2 (425 bp) and GLUT4 (299 bp) were amplified. No products corresponding to GLUT3 (411 bp) were detected in H441 cells but were detected in a positive control, Ishikawa cell line (data not shown). Amplification of β-actin was used as a control for the reaction. b Western blot analysis of glucose transporter expression in non-polarised H441 cells. Total protein (Total), intracellular proteins (Intracellular) and plasma membrane protein (Plasma membrane) (50 μg) were resolved on acrylamide gels. Immunostained products corresponding to GLUT2 (∼60 kDa) and GLUT4 (∼45 kDa) were detected. Positive controls (+C) used: GLUT1, skeletal muscle; GLUT2, liver; GLUT3, Ishikawa cells; GLUT4, adipose tissue
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2480509&req=5

Fig3: a RT-PCR of GLUT1–GLUT4 mRNA sequences from non-polarised H441 cells. Products were resolved on ethidium bromide-stained agarose gels. RT-PCR products corresponding to the correct size for GLUT1 (400 bp), GLUT2 (425 bp) and GLUT4 (299 bp) were amplified. No products corresponding to GLUT3 (411 bp) were detected in H441 cells but were detected in a positive control, Ishikawa cell line (data not shown). Amplification of β-actin was used as a control for the reaction. b Western blot analysis of glucose transporter expression in non-polarised H441 cells. Total protein (Total), intracellular proteins (Intracellular) and plasma membrane protein (Plasma membrane) (50 μg) were resolved on acrylamide gels. Immunostained products corresponding to GLUT2 (∼60 kDa) and GLUT4 (∼45 kDa) were detected. Positive controls (+C) used: GLUT1, skeletal muscle; GLUT2, liver; GLUT3, Ishikawa cells; GLUT4, adipose tissue
Mentions: We detected mRNA for GLUT1, GLUT2 and GLUT4 in non-polarised H441 cells (Fig. 3a). In non-polarised cells cultured at 10 mM glucose, a protein of predicted size for GLUT2 (60 kDa) was detected in separated plasma membrane and intracellular protein fractions in approximately equal quantities (Fig. 3b). A 45-kDa product corresponding to GLUT4 protein was detected in total cell protein and the intracellular fraction but was absent from the plasma membrane protein fraction. It is interesting to note that although GLUT1 mRNA was detected, translated protein could not be detected by western blotting in these cells (Fig. 3b). Neither GLUT3 mRNA nor protein was detected (Fig. 3a,b).Fig. 3

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