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: 8.74). 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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Available from: Jonathan S Bogan, Aug 10, 2015
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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 · 9.69 Impact Factor
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    ABSTRACT: Insulin stimulates glucose transport in fat cells by inducing the movement of glucose transporters (Glucose transporter-4) from specialised storage vesicles to the plasma membrane. Insulin resistant individuals and those with Type II Diabetes exhibit impairment in the ability of insulin to stimulate glucose transport. The molecular mechanisms of glucose transporter-4 trafficking in adipocytes are an important focus in understanding the underlying etiology of this disease. Glucose transporter-4 (GLUT4) recycles between the plasma membrane and intracellular stores in the absence of insulin using a complex intracellular pathway. This involves two intracellular cycles: one is the prototypical endosomal system, the other a specialised cycle involving the trans-Golgi network and a sub-set of intracellular vesicles called GSVs (the slow cycle). Understanding the control of the entry into this second cycle is the subject of this thesis. In particular, the work in this thesis will examine the role of Syntaxin 16 and its cognate Sec1/Munc18 protein mammalian Vps45 (mVps45). The regulation of Syntaxin 16 has not been fully elucidated and understanding the role of Syntaxin 16 in SNARE complex regulation and subsequent control of GLUT4 traffic into the slow cycle requires an understanding of its cognate binding partner Sec1/Munc18 (SM) protein, mammalian Vps45 (mVps45). The absolute levels of both Syntaxin 16 and mVps45 were quantified and found to be present in 3T3-L1 adipocytes in roughly stoichiomeric amounts. IP experiments also showed the ability of mVps45 to interact with Syntaxin 16 in the absence of insulin. Using the model eukaryote Saccharomyces cerevisiae, we found that mVps45 could complement for the deletion of Vps45p. Assays for CPY secretion showed that mVps45 is able to complement for the loss of Vps45p function in the trafficking of carboxypeptidase Y (CPY). Additionally, mVps45 mutants were made that correspond to yeast mutants made previously in the lab and were tested for homology of function. Depleting 3T3-L1 adipocytes of mVps45 showed alterations in the levels of GLUT4 protein as well as the protein levels of Syntaxin 16, IRAP, and Rabenosyn. Insulin-stimulated deoxyglucose uptake was also profoundly decreased upon depletion of mVps45. Further experiments using mVps45 depleted cells show that these cells lose their sensitivity to insulin and that the loss of mVps45 in these cells causes GLUT4 to have the inability to enter the slow cycle. Taken together, these findings demonstrate that mVps45 has a role in allowing GLUT4 entry into the slow cycle.
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    ABSTRACT: Glucose transporter 4 (GLUT4) is an insulin facilitated glucose transporter that plays an important role in maintaining blood glucose homeostasis. GLUT4 is sequestered into intracellular vesicles in unstimulated cells and translocated to the plasma membrane by various stimuli. Understanding the structural details of GLUT4 will provide insights into the mechanism of glucose transport and its regulation. To date, a crystal structure for GLUT4 is not available. However, earlier work from our laboratory proposed a well validated homology model for GLUT4 based on the experimental data available on GLUT1 and the crystal structure data obtained from the glycerol 3-phosphate transporter. In the present study, the dynamic behavior of GLUT4 in a membrane environment was analyzed using three forms of GLUT4 (apo, substrate and ATP-substrate bound states). Apo form simulation analysis revealed an extracellular open conformation of GLUT4 in the membrane favoring easy exofacial binding of substrate. Simulation studies with the substrate bound form proposed a stable state of GLUT4 with glucose, which can be a substrate-occluded state of the transporter. Principal component analysis suggested a clockwise movement for the domains in the apo form, whereas ATP substrate-bound form induced an anti-clockwise rotation. Simulation studies suggested distinct conformational changes for the GLUT4 domains in the ATP substrate-bound form and favor a constricted behavior for the transport channel. Various inter-domain hydrogen bonds and switching of a salt-bridge network from E345-R350-E409 to E345-R169-E409 contributed to this ATP-mediated channel constriction favoring substrate occlusion and prevention of its release into cytoplasm. These data are consistent with the biochemical studies, suggesting an inhibitory role for ATP in GLUT-mediated glucose transport. In the absence of a crystal structure for any glucose transporter, this study provides mechanistic details of the conformational changes in GLUT4 induced by substrate and its regulator.
    PLoS ONE 12/2010; 5(12):e14217. DOI:10.1371/journal.pone.0014217 · 3.53 Impact Factor
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