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Dig Dis Sci. 2012 May;57(5):1203-12. doi: 10.1007/s10620-011-1984-4. Epub 2011 Nov 25.

Translocation of transfected GLUT2 to the apical membrane in rat intestinal IEC-6 cells.

Author information

1
Department of Surgery and The Gastroenterology Research Unit, Mayo Clinic (GU 10-01), 200 1st Street SW, Rochester, MN 55905, USA. zheng.ye@mayo.edu

Abstract

AIM:

In this study, we transfected the full length cDNA of glucose transporter 2 (GLUT2) into IEC-6 cells (which lack GLUT2 expression) to investigate GLUT2 translocation in enterocytes. The purpose of this study was to investigate cellular mechanisms of GLUT2 translocation and its signaling pathway.

METHODS:

Rat GLUT2 cDNA was transfected into IEC-6 cells. Glucose uptake was measured by incubating cell monolayers with glucose (0.5-50 mM), containing (14)C-D-glucose and (3)H-L-glucose, to measure stereospecific, carrier-mediated and passive uptake. We imaged GLUT2 immunoreactivity by confocal fluorescence microscopy. We evaluated the GLUT2 inhibitor (1 mM phloretin), SGLT1 inhibitor (0.5 mM phlorizin), disrupting microtubular integrity (2 μM nocodazole and 0.5 μM cytochalasin B), protein kinase C (PKC) inhibitors (50 nM calphostin C and 10 μM chelerythrine), and PKC activator (50 nM phorbol 12-myristate 13-acetate: PMA).

RESULTS:

In GLUT2-IEC cells, the K(m) (54.5 mM) increased compared with non-transfected IEC-6 cells (7.8 mM); phloretin (GLUT2 inhibitor) inhibited glucose uptake to that of non-transfected IEC-6 cells (P < 0.05). Nocodazole and cytochalasin B (microtubule disrupters) inhibited uptake by 43-58% only at glucose concentrations ≥25 and 50 mM and the 10-min incubations. Calphostin C (PKC inhibitor) reproduced the inhibition of nocodazole; PMA (a PKC activator) enhanced glucose uptake by 69%. Exposure to glucose increased the GFP signal at the apical membrane of GLUT-1EC cells.

CONCLUSION:

IEC-6 cells lacking GLUT2 translocate GLUT2 apically when transfected to express GLUT2. Translocation of GLUT2 occurs through glucose stimulation via a PKC-dependent signaling pathway and requires integrity of the microtubular skeletal structure.

PMID:
22116644
PMCID:
PMC3331913
DOI:
10.1007/s10620-011-1984-4
[Indexed for MEDLINE]
Free PMC Article
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