Cholesterol depletion modifies plasma membrane lipid raft integrity and affects glucose uptake in a human leukemic cell line C. Caliceti, L. Zambonin, C. Prata, F. Vieceli Dalla Sega, G. Hakim and D. Fiorentini Dipartimento di Biochimica “G. Moruzzi”, Università di Bologna - Italy In hemopoietic M07e cells, stem cell factor (SCF) activates glucose transport through GLUT1 translocation from intracellular stores. Recently, it has been reported that in many cell types GLUT1 is localized in part in detergent-resistant membrane (DRM) domains and a role for lipid rafts in GLUT1 stimulation has been suggested. We investigated whether a plasma membrane cholesterol depletion might play a role in GLUT1 activity. Cell exposure to 10 mM methyl--cyclodextrin (MBCD) for 20 min resulted in a loss of cholesterol from plasma membrane, without affecting viability. MBCD-treated cells exhibited a rise in glucose uptake, higher than that observed upon SCF treatment. The combined cell treatment with SCF and MBCD determined a further increase in glucose uptake, showing an additive effect. Membrane fractionation by flotation on sucrose density gradient showed that in untreated cells most of GLUT1 is distributed in the high-density region, but a little amount is colocalized with lipid raft marker proteins in low-density regions, corresponding to DRM. Upon MBCD treatment, GLUT1 resulted totally confined to the high-density region. DRM exhibited also the highest cholesterol content. The cholesterol distribution profile of the MBCD-treated samples shows that the cholesterol preferentially decreased in DRM, confirming its selective depletion. Cell membrane isolation by biotinylation and immunofluorescence staining indicated that MBCD treatment, like SCF stimulation, greatly enhanced the amount of GLUT1 at cell surface, promoting a translocation from intracellular stores. MBCD increases Akt-pS473, while SCF positively regulates PLCγ signaling cascade, both involved in GLUT translocation and in glucose uptake control. Therefore, these two stimuli could proceed through distinct signalling pathways. In summary, a mild lipid environment alteration in lipid rafts enhances GLUT1 activity in M07e cells via a cholesterol-dependent mechanism, possibly involving Akt.

Cholesterol depletion modifies plasma membrane lipid raft integrity and affects glucose uptake in a human leukemic cell line

CALICETI, CRISTIANA;ZAMBONIN, LAURA;PRATA, CECILIA;VIECELI DALLA SEGA, FRANCESCO;FIORENTINI, DIANA
2011

Abstract

Cholesterol depletion modifies plasma membrane lipid raft integrity and affects glucose uptake in a human leukemic cell line C. Caliceti, L. Zambonin, C. Prata, F. Vieceli Dalla Sega, G. Hakim and D. Fiorentini Dipartimento di Biochimica “G. Moruzzi”, Università di Bologna - Italy In hemopoietic M07e cells, stem cell factor (SCF) activates glucose transport through GLUT1 translocation from intracellular stores. Recently, it has been reported that in many cell types GLUT1 is localized in part in detergent-resistant membrane (DRM) domains and a role for lipid rafts in GLUT1 stimulation has been suggested. We investigated whether a plasma membrane cholesterol depletion might play a role in GLUT1 activity. Cell exposure to 10 mM methyl--cyclodextrin (MBCD) for 20 min resulted in a loss of cholesterol from plasma membrane, without affecting viability. MBCD-treated cells exhibited a rise in glucose uptake, higher than that observed upon SCF treatment. The combined cell treatment with SCF and MBCD determined a further increase in glucose uptake, showing an additive effect. Membrane fractionation by flotation on sucrose density gradient showed that in untreated cells most of GLUT1 is distributed in the high-density region, but a little amount is colocalized with lipid raft marker proteins in low-density regions, corresponding to DRM. Upon MBCD treatment, GLUT1 resulted totally confined to the high-density region. DRM exhibited also the highest cholesterol content. The cholesterol distribution profile of the MBCD-treated samples shows that the cholesterol preferentially decreased in DRM, confirming its selective depletion. Cell membrane isolation by biotinylation and immunofluorescence staining indicated that MBCD treatment, like SCF stimulation, greatly enhanced the amount of GLUT1 at cell surface, promoting a translocation from intracellular stores. MBCD increases Akt-pS473, while SCF positively regulates PLCγ signaling cascade, both involved in GLUT translocation and in glucose uptake control. Therefore, these two stimuli could proceed through distinct signalling pathways. In summary, a mild lipid environment alteration in lipid rafts enhances GLUT1 activity in M07e cells via a cholesterol-dependent mechanism, possibly involving Akt.
The FEBS Journal - Abstracts of the 36th FEBS CONGRESS - Biochemistry for tomorrow's medicine
352
352
THE FEBS JOURNAL
C. Caliceti; L. Zambonin; C. Prata; F. Vieceli Dalla Sega; G. Hakim; D. Fiorentini
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/112072
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