title: Glut-1 translocation in FRTL-5 thyroid cells: role of phosphatidylinositol 3-kinase and N-glycosylation.
abstract: 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.
authors.affiliation: INSERM Unité 260, Faculté de Médecine, Université de la Mediterranée, Marseille, France.
authors.forename: N
authors.initials: N
authors.lastname: Samih
authors.forename: S
authors.initials: S
authors.lastname: Hovsepian
authors.forename: A
authors.initials: A
authors.lastname: Aouani
authors.forename: D
authors.initials: D
authors.lastname: Lombardo
authors.forename: G
authors.initials: G
authors.lastname: Fayet
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1; D006031:Glycosylation; D007328:Insulin / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051381:Rats; D013449:Sulfonamides; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D003994:Bucladesine; D002867:Chromones; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005947:Glucose; D051272:Glucose Transporter Type 1; D007328:Insulin; D007546:Isoquinolines; D009004:Monosaccharide Transport Proteins; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D013449:Sulfonamides; D013972:Thyrotropin; D014415:Tunicamycin; D000077191:Wortmannin; C495679:Slc2a1 protein, rat; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide