Glut-1 Translocation in FRTL-5 Thyroid Cells: Role of Phosphatidylinositol 3-Kinase and N-Glycosylation

It was previously demonstrated that insulin or TSH treatment of FRTL-5 cells resulted in an elevation of glucose transport and in an increase of cell surface expression of the glucose transporter Glut-1. However, the signaling mechanisms leading to the insulin or TSH-induced increase in the cell sur...

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Bibliographic Details
Published in:Endocrinology (Philadelphia) 2000-11, Vol.141 (11), p.4146-4155
Main Authors: Samih, Nezha, Hovsepian, Sonia, Aouani, Azedine, Lombardo, Dominique, Fayet, Guy
Format: Article
Language:English
Subjects:
Androstadienes - pharmacology
Animals
Biological Transport - drug effects
Bucladesine - pharmacology
Cell Line
Chromones - pharmacology
Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors
Enzyme Inhibitors - pharmacology
Gene Expression - drug effects
Glucose - metabolism
Glucose Transporter Type 1
Glycosylation
Insulin - pharmacology
Isoquinolines - pharmacology
Monosaccharide Transport Proteins - genetics
Monosaccharide Transport Proteins - metabolism
Morpholines - pharmacology
Phosphatidylinositol 3-Kinases - antagonists & inhibitors
Phosphatidylinositol 3-Kinases - metabolism
Rats
Sulfonamides
Thyroid Gland - drug effects
Thyroid Gland - metabolism
Thyrotropin - pharmacology
Tunicamycin - pharmacology
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Summary:It was previously demonstrated that insulin or TSH treatment of FRTL-5 cells resulted in an elevation of glucose transport and in an increase of cell surface expression of the glucose transporter Glut-1. However, the signaling mechanisms leading to the insulin or TSH-induced increase in the cell surface expression of Glut-1 were not investigated. In the present study, we demonstrated that wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), interfere both in the signaling pathways of insulin and TSH leading to glucose consumption enhancement and Glut-1 translocation. Two hours after insulin treatment, TSH or cAMP analog (Bu)2cAMP stimulation, glucose transport was increased and most of the intracellular Glut-1 pool was translocated to plasma membranes. Wortmannin or LY294002 blocked the insulin, (Bu)2cAMP, and the TSH-induced translocation of Glut-1. Wortmannin or LY294002 alone did not alter the basal ratio between intracellular and cell surface Glut-1 molecules. These results suggest that in FRTL-5 cells wortmannin and LY294002 inhibited the insulin, (Bu)2cAMP and TSH events leading to Glut-1 translocation from an intracellular compartment to the plasma membrane. Likewise, (Bu)2cAMP effects on glucose transport and Glut-1 translocation to plasma membrane were repressed by PI3-kinase inhibitors but not by the protein kinase A (PKA) inhibitor H89. We suggest that (Bu)2cAMP stimulates Glut-1 translocation to plasma membrane through PI3-kinase-dependent and PKA-independent signaling pathways. To further elucidate mechanisms that regulate the translocation of Glut-1 to cell membrane, we extended this study to the role played by the N-glycosylation in the translocation and in the biological activity of Glut-1 in FRTL-5 cells. For this purpose we used tunicamycin, an inhibitor of the N-glycosylation. Our experiments with tunicamycin clearly showed that both the glycosylated and unglycosylated forms of the transporter reached the cell surface. Likewise, a decrease in glucose consumption (−50%) after treatment of cells with tunicamycin was accompanied by a decrease (−70% vs. control) in the membrane expression of a 50-kDa form of Glut-1 and an increase in its unglycosylated 41-kDa form. These results suggest that carbohydrate moiety is essential for the biological activity of glucose transport but is not required for the translocation of Glut-1 from the intracellular membrane pool to the plasma membrane.
ISSN:0013-7227
1945-7170
DOI:10.1210/endo.141.11.7793