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

ArticleinMolecular biology of the cell 24(19) · August 2013with22 Reads
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.
    • "Surprisingly, lack of sortilin also leads to decreased LDLcholesterol levels in blood through decreased VLDL secre- tion [26]. To complicate matters further, sortilin has been identified as a facilitator of proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion, a protein that reduces LDL-receptor expression [19, 27]. Consequently, the exact role of sortilin in cholesterol metabolism remains a matter of debate. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Sortilin and SorCS2 are part of the Vps10p receptor family. They have both been studied in nervous tissue with several important functions revealed, while their expression and possible functions in developing peripheral tissue remain poorly understood. Here we deliver a thorough characterization of the prenatal localization of sortilin and SorCS2 in mouse peripheral tissue. Results Sortilin is highly expressed in epithelial tissues of the developing lung, nasal cavity, kidney, pancreas, salivary gland and developing intrahepatic bile ducts. Furthermore tissues such as the thyroid gland, developing cartilage and ossifying bone also show high expression of sortilin together with cell types such as megakaryocytes in the liver. SorCS2 is primarily expressed in mesodermally derived tissues such as striated muscle, adipose tissue, ossifying bone and general connective tissue throughout the body, as well as in lung epithelia. Furthermore, the adrenal gland and liver show high expression of SorCS2 in embryos 13.5 days old. Conclusions The possible functions relating to the expression patterns of Sortilin and SorCS2 in development are numerous and hopefully this paper will help to generate new hypotheses to further our understanding of the Vps10p receptor family.
    Full-text · Article · Dec 2016
    • "Cell surface biotinylation was performed essentially as described previously [27]. Briefly, C2C12 cells were induced to differentiate for indicated time points by switching to DM and incubating in phosphatebuffered saline (PBS) containing Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, Rockford, IL) with the final concentration of 1 mg/ml for 30 min on ice. "
    [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.
    Full-text · Article · Sep 2015
    • "Both contain GLUT4 and other proteins that translocate to the PM of adipocytes in an insulin-dependent manner, including VAMP2 and the insulin-responsive aminopeptidase, but can be distinguished by the presence/absence of the multi-ligand sorting receptor sortilin and the protein cellugyrin (Jedrychowski et al., 2010; Kupriyanova and Kandror, 2000; Kupriyanova et al., 2002). Sortilin plays a key role in GSV biogenesis (Huang et al., 2013; Shi and Kandror, 2005) and like other GSV-residents translocates to the PM in response to insulin; supporting the notion that sortilin-positive (cellugyrin-negative) IGVs represent GSVs (Jedrychowski et al., 2010; Kupriyanova and Kandror, 2000; Journal of Cell Science Accepted manuscript 2002). In contrast, cellugyrin does not translocate to the PM in response to insulin and the function of cellugyrin-positive (sotillin-negative) IGVs is unknown (Jedrychowski et al., 2010; Kim and Kandror, 2012; Kupriyanova and Kandror, 2000; Kupriyanova et al., 2002; Li et al., 2009). "
    [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.
    Full-text · Article · Jun 2015
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