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: 4.47). 08/2013; 24(19). DOI: 10.1091/mbc.E12-10-0765
Source: PubMed


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.

18 Reads
  • [Show abstract] [Hide abstract]
    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; 290(7). DOI:10.1074/jbc.M114.603977 · 4.57 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Insulin-stimulated delivery of glucose transporters (GLUT4) from specialized intracellular GLUT4 storage vesicles (GSVs) to the surface of fat and muscle cells is central to whole-body glucose. This translocation and subsequent internalization of GLUT4 back into intracellular stores transits numerous small membrane-bound compartments (internal GLUT4-containing vesicles; IGVs) including GSVs, but the function of these different compartments is not clear. Cellugyrin and sortilin define distinct populations of IGV; sortilin-positive IGVs represent GSVs, but the function of cellugyrin-containing IGVs is unknown. Here we demonstrate a role for cellugyrin in intracellular sequestration of GLUT4 in HeLa cells and have used a proximity ligation assay to follow changes in pairwise associations between cellugyrin, sortilin, GLUT4 and membrane trafficking machinery following insulin-stimulation of 3T3-L1 adipoctyes. Our data suggest that insulin stimulates traffic from cellugyrin- to sortilin- membranes, and that cellugyrin-IGVs provide an insulin-sensitive reservoir to replenish GSVs following insulin-stimulated exocytosis of GLUT4. Furthermore, our data support the existence of a pathway from cellugyrin-membranes to the surface of 3T3-L1 adipocytes that bypasses GSVs under basal conditions, and that insulin diverts traffic away from this into GSVs. © 2015. Published by The Company of Biologists Ltd.
    Journal of Cell Science 06/2015; 128(14). DOI:10.1242/jcs.166561 · 5.43 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Syntaxins are a family of membrane proteins involved in vesicle trafficking, such as synaptic vesicle exocytosis. Syntaxin 4 (Stx4) is expressed highly in skeletal muscle and plays a critical role in insulin-stimulated glucose uptake by promoting translocation of glucose transporter 4 (GLUT4) to the cell surface. A cell surface receptor cell adhesion molecule-related, down-regulated by oncogenes (Cdo) is a component of cell adhesion complexes and promotes myoblast differentiation via activation of key signalings, including p38MAPK and AKT. In this study, we investigate the function of Stx4 in myoblast differentiation and the crosstalk between Stx4 and Cdo in myoblast differentiation. The effects of overexpression or shRNA-based depletion of Stx4 and Cdo genes on C2C12 myoblast differentiation are assessed by Western blotting and immunofluorescence approaches. The interaction between Cdo and Stx4 and the responsible domain mapping are assessed by coimmunoprecipitation or pulldown assays. The effect of Stx4 depletion on cell surface localization of Cdo and GLUT4 in C2C12 myoblasts is assessed by surface biotinylation and Western blotting. Overexpression or knockdown of Stx4 enhances or inhibits myogenic differentiation, respectively. Stx4 binds to the cytoplasmic tail of Cdo, and this interaction seems to be critical for induction of p38MAPK activation and myotube formation. Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels. Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface. Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it. Stx4 promotes myoblast differentiation through interaction with Cdo and stimulation of its surface translocation. Both Cdo and Stx4 are required for GLUT4 translocation to cell surface and glucose uptake in myoblast differentiation.
    Skeletal Muscle 09/2015; 5(1):28. DOI:10.1186/s13395-015-0052-8


18 Reads
Available from