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Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin.

Boston University School of Medicine, Boston, MA 02118.
Molecular biology of the cell (Impact Factor: 5.98). 08/2013; DOI: 10.1091/mbc.E12-10-0765
Source: PubMed

ABSTRACT In mammalian organism, insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles, the IRVs. However, it is not yet clear whether or not the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We have prepared two lines of 3T3-L1 cells with low and high expression of myc7-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 pre-adipocytes, translocation of myc7-Glut4 was low regardless of its expression levels. Co-expression of sortilin increased targeting of myc7-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc7-Glut4. AS160/TBC1D4 is expressed at low levels in pre-adipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.

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    ABSTRACT: Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C-terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C-terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA binding domain containing 3), and acetylation reduced binding of TUG to ACBD3, but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and IRAP, and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. SIRT2, a NAD+-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knockout mice, compared to wildtype controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells, and enhances insulin-stimulated glucose uptake. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 01/2015; DOI:10.1074/jbc.M114.603977 · 4.60 Impact Factor

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