Biogenesis and regulation of insulin-responsive vesicles containing GLUT4

Section of Endocrinology and Metabolism, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA.
Current opinion in cell biology (Impact Factor: 8.47). 04/2010; 22(4):506-12. DOI: 10.1016/
Source: PubMed

ABSTRACT Insulin regulates the trafficking of GLUT4 glucose transporters in fat and muscle cells. In unstimulated cells, GLUT4 is sequestered intracellularly in small, insulin-responsive vesicles. Insulin stimulates the translocation of these vesicles to the cell surface, inserting the transporters into the plasma membrane to enhance glucose uptake. Formation of the insulin-responsive vesicles requires multiple interactions among GLUT4, IRAP, LRP1, and sortilin, as well as recruitment of GGA and ACAP1 adaptors and clathrin. Once formed, the vesicles are retained within unstimulated cells by the action of TUG, Ubc9, and other proteins. In addition to acting at other steps in vesicle recycling, insulin releases this retention mechanism to promote the translocation and fusion of the vesicles at the cell surface.

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    • "Because of the clinical importance in type II diabetes of the activity of GLUT4, the glucose transporter expressed primarily in insulin responsive tissues [6], [7], it is one of the best-studied regulatory systems for a transporter. There are excellent recent reviews that summarize up-to-date knowledge about the biochemistry of this regulation [8], [9], [10] and discuss in detail the biogenesis of specialized GLUT4 storage vesicles (GSV) [10], insulin signaling cascades involved in the regulation of GSV exocytosis and GLUT4 translocation to the plasma membrane (PM) [8], [9], and mechanisms of GLUT4 endocytosis, and sorting back to GSV [9], [10]. However, comparatively less is known about dynamics of GLUT4 already present in the PM, where it actually performs its function of facilitating the transport of glucose. "
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    ABSTRACT: Insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) is the hallmark of glucose metabolism. In this study we examined insulin's effects on GLUT4 organization in PM of adipose cells by direct microscopic observation of single monomers tagged with photoswitchable fluorescent protein. In the basal state, after exocytotic delivery only a fraction of GLUT4 is dispersed into the PM as monomers, while most of the GLUT4 stays at the site of fusion and forms elongated clusters (60-240 nm). GLUT4 monomers outside clusters diffuse freely and do not aggregate with other monomers. In contrast, GLUT4 molecule collision with an existing cluster can lead to immediate confinement and association with that cluster. Insulin has three effects: it shifts the fraction of dispersed GLUT4 upon delivery, it augments the dissociation of GLUT4 monomers from clusters ∼3-fold and it decreases the rate of endocytic uptake. All together these three effects of insulin shift most of the PM GLUT4 from clustered to dispersed states. GLUT4 confinement in clusters represents a novel kinetic mechanism for insulin regulation of glucose homeostasis.
    PLoS ONE 03/2013; 8(3):e57559. DOI:10.1371/journal.pone.0057559 · 3.23 Impact Factor
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    • "Additional proteins associated with this compartment include TUG (Tether containing UBX domain for GLUT4). UBX (ubiquitin associated) domains of TUG (which are similar in structure to ubiquitin) are thought to retain the GSV and a construct of the UBX domain can lead to release of the GSVs from an intracellular storage location to the cell surface [43], [44]. However, the mechanism by which insulin action leads to a change in the GSV-TUG interaction is currently unknown. "
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    ABSTRACT: In insulin target tissues, GLUT4 is known to traffic through multiple compartments that may involve ubiquitin- and/or SUMO-dependent targeting. During these trafficking steps, GLUT4 is sorted into a storage reservoir compartment that is acutely released by insulin signalling processes that are downstream of PI 3-kinase associated changes in inositol phospholipids. As ESCRT components have recently been found to influence cellular sorting processes that are related to changes in both ubiquitination and inositol phospholipids, we have examined whether GLUT4 traffic is routed through ESCRT dependent sorting steps. Introduction of the dominant negative inhibitory constructs of the ESCRT-III components CHMP3 (CHMP3(1-179)) and Vps4 (GFP-Vps4(E235Q)) into rat adipocytes leads to the accumulation of GLUT4 in large, coalesced and extended vesicles structures that co-localise with the inhibitory constructs over large parts of the extended structure. A new swollen hybrid and extensively ubiquitinated compartment is produced in which GLUT4 co-localises more extensively with the endosomal markers including EEA1 and transferrin receptors but also with the TGN marker syntaxin6. These perturbations are associated with failure of insulin action on GLUT4 traffic to the cell surface and suggest impairment in an ESCRT-dependent sorting step used for GLUT4 traffic to its specialised reservoir compartment.
    PLoS ONE 09/2012; 7(9):e44141. DOI:10.1371/journal.pone.0044141 · 3.23 Impact Factor
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    • "To better identify GSV exocytosis, we used VAMP2-pHluorin. VAMP2 is an established component of GSVs (Ramm et al., 2000; Williams and Pessin, 2008; Bogan and Kandror, 2010), and pHluorin is a pH-sensitive fluorescent protein that becomes Figure 1. 3T3-L1 adipocyte differentiation induces a change in the size of GLUT4 vesicles. "
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    ABSTRACT: Insulin stimulates translocation of GLUT4 storage vesicles (GSVs) to the surface of adipocytes, but precisely where insulin acts is controversial. Here we quantify the size, dynamics, and frequency of single vesicle exocytosis in 3T3-L1 adipocytes. We use a new GSV reporter, VAMP2-pHluorin, and bypass insulin signaling by disrupting the GLUT4-retention protein TUG. Remarkably, in unstimulated TUG-depleted cells, the exocytic rate is similar to that in insulin-stimulated control cells. In TUG-depleted cells, insulin triggers a transient, twofold burst of exocytosis. Surprisingly, insulin promotes fusion pore expansion, blocked by acute perturbation of phospholipase D, which reflects both properties intrinsic to the mobilized vesicles and a novel regulatory site at the fusion pore itself. Prolonged stimulation causes cargo to switch from approximately 60 nm GSVs to larger exocytic vesicles characteristic of endosomes. Our results support a model whereby insulin promotes exocytic flux primarily by releasing an intracellular brake, but also by accelerating plasma membrane fusion and switching vesicle traffic between two distinct circuits.
    The Journal of Cell Biology 05/2011; 193(4):643-53. DOI:10.1083/jcb.201008135 · 9.83 Impact Factor
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