Clathrin-dependent and independent endocytosis of glucose transporter 4 (GLUT4) in myoblasts: regulation by mitochondrial uncoupling |
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Authors: | Antonescu Costin N Díaz Mònica Femia Guiseppe Planas Josep V Klip Amira |
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Affiliation: | Program in Cell Biology, The Hospital For Sick Children, Toronto, Ontario, Canada, M5G 1X8; Department of Biochemistry, University of Toronto, Ontario, Canada, M5S 1A8; Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona (IBUB), 08028 Barcelona, Spain |
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Abstract: | In myocytes and adipocytes, insulin increases glucose transporter 4 (GLUT4) exocytosis by promoting GLUT4 vesicle docking/fusion with the membrane. Less is known about the mechanism and regulation of GLUT4 endocytosis, particularly in myocytes. Here, we show that GLUT4 internalization in L6 myoblasts was inhibited in part by hypertonicity or clathrin heavy chain knockdown and in part by cholesterol depletion. Both strategies had additive effects, abolishing GLUT4 endocytosis. GLUT4 internalization was abrogated by expressing dominant-negative dynamin-2 but unaffected by inhibiting caveolar-dependent endocytosis through syntaxin-6 knockdown or caveolin mutants (which reduced lactosylceramide endocytosis). Insulin did not affect GLUT4 internalization rate or sensitivity to clathrin or cholesterol depletion. In contrast, the mitochondrial uncoupler dinitrophenol (DNP), which like insulin increases surface GLUT4, reduced GLUT4 (but not transferrin) internalization, an effect additive to that of depleting clathrin but not cholesterol. Trout GLUT4 (a natural variant of GLUT4 bearing different endocytic motifs) exogenously expressed in mammalian L6 cells internalized only through the cholesterol-dependent route that also included the non-clathrin-dependent cargo interleukin-2 receptor β, and DNP reduced internalization of both proteins. These results suggest that in muscle cells, GLUT4 internalizes simultaneously through clathrin-mediated endocytosis and a caveolae-independent but cholesterol- and dynamin-dependent route. Manipulating GLUT4 endocytosis to maintain surface GLUT4 may bypass insulin resistance. |
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Keywords: | caveolae cholesterol clathrin dinitrophenol dynamin endocytosis energy depletion GLUT4 insulin membrane traffic |
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