p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation

Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1-109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site.     

ArPIKfyve-PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes

Insulin activates glucose transport by promoting translocation of the insulin-sensitive fat/muscle-specific glucose transporter GLUT4 from an intracellular storage compartment to the cell surface. Here we report that an optimal insulin effect on glucose uptake in 3T3-L1 adipocytes is dependent upon expression of both PIKfyve, the sole enzyme for PtdIns 3,5-P(2) biosynthesis, and the PIKfyve activator, ArPIKfyve. Small-interfering RNAs that selectively ablated PIKfyve or ArPIKfyve in this cell type depleted the PtdIns 3,5-P(2) pool and reduced insulin-activated glucose uptake to a comparable degree. Combined loss of PIKfyve and ArPIKfyve caused further PtdIns 3,5-P(2) ablation that correlated with greater attenuation in insulin responsiveness. Loss of PIKfyve-ArPIKfyve reduced insulin-stimulated Akt phosphorylation and the cell surface accumulation of GLUT4 or IRAP, but not GLUT1-containing vesicles without affecting overall expression of these proteins. ArPIKfyve and PIKfyve were found to physically associate in 3T3-L1 adipocytes and this was insulin independent. In vitro labeling of membranes isolated from basal or insulin-stimulated 3T3-L1 adipocytes documented substantial insulin-dependent increases of PtdIns 3,5-P(2) production on intracellular membranes. Together, the data demonstrate for the first time a physical association between functionally related PIKfyve and ArPIKfyve in 3T3-L1 adipocytes and indicate that the novel ArPIKfyve-PIKfyve-PtdIns 3,5-P(2) pathway is physiologically linked to insulin-activated GLUT4 translocation and glucose transport.     

VAMP2, but not VAMP3/cellubrevin, mediates insulin-dependent incorporation of GLUT4 into the plasma membrane of L6 myoblasts

Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.     

A specific dileucine motif is required for the GGA-dependent entry of newly synthesized insulin-responsive aminopeptidase into the insulin-responsive compartment

In muscle and adipose cells, the insulin-responsive aminopeptidase (IRAP) is localized to intracellular storage sites and undergoes insulin-dependent redistribution to the cell surface. Following expression, the newly synthesized IRAP protein traffics to the perinuclear insulin-sensitive compartment and acquires insulin sensitivity 6-9 h following biosynthesis. Knockdown of GGA1 by RNA interference prevented IRAP from entering, but not exiting, the insulin-responsive compartment. Mutation of the dileucine motif at positions 76 and 77 (EGFP-IRAP/AA(76,77)), but not the dileucine motif at positions 53 and 54, resulted in the rapid default of the reporter to the cell surface beginning at 3 h following biosynthesis. Alanine substitution of 9 residues amino- or carboxyl-terminal to LL(76,77) did not perturb basal intracellular sequestration or abrogate insulin-stimulated IRAP translocation. Moreover, a dominant interfering GGA mutant (VHS-GAT) potently inhibited insulin-stimulated translocation of EGFP-IRAP/WT but did not block the constitutive exocytotic trafficking of EGFP-IRAP/AA(76,77). In addition, the EGFP-IRAP/WT and EGFP-IRAP/AA(76,77) constructs occupied morphologically distinct tubulovesicular compartments in the perinuclear region. Taken together, these data indicate that LL(76,77) functions during the GGA-dependent sorting of newly made IRAP into the insulin-responsive storage compartment.     

Expression and compartmentalization of caveolin in adipose cells: coordinate regulation with and structural segregation from GLUT4

Native rat adipocytes and the mouse adipocyte cell line, 3T3-L1, possess transport vesicles of apparently uniform composition and size which translocate the tissue-specific glucose transporter isoform, GLUT4, from an intracellular pool to the cell surface in an insulin- sensitive fashion. Caveolin, the presumed structural protein of caveolae, has also been proposed to function in vesicular transport. Thus, we studied the expression and subcellular distribution of caveolin in adipocytes. We found that rat fat cells express the highest level of caveolin protein of any tissue studied, and caveolin is also expressed at high levels in cardiac muscle, another tissue possessing insulin responsive GLUT4 translocation. Both proteins are absent from 3T3-L1 fibroblasts and undergo a dramatic coordinate increase in expression upon differentiation of these cells into adipocytes. However, unlike GLUT4 in rat adipocytes not exposed to insulin, the majority of caveolin is present in the plasma membrane. In native rat adipocytes, intracellular GLUT4 and caveolin reside in vesicles practically indistinguishable by their size and buoyant density in sucrose gradients, and both proteins show insulin-dependent translocation to the cell surface. However, by immunoadsorption of GLUT4-containing vesicles with anti-GLUT4 antibody, we show that these vesicles have no detectable caveolin, and therefore, this protein is present in a distinct vesicle population. Thus, caveolin has no direct structural relation to the organization of the intracellular glucose transporting machinery in fat cells.     

A Glut4-vesicle marker protein, insulin-responsive aminopeptidase, is localized in a novel vesicular compartment in PC12 cells

Glut4-containing vesicles represent a regulated recycling compartment in insulin-sensitive fat and skeletal muscle cells, the nature and origin of which are not fully understood. In addition to Glut4 itself, these vesicles compartmentalize a number of proteins, at least one of which, insulin-responsive aminopeptidase, or IRAP, is completely colocalized with Glut4 in insulin-sensitive tissues. However, unlike Glut4, IRAP is expressed in a variety of other tissues and cell lines. Here, we explored the intracellular localization of IRAP in the rat pheochromocytoma cell line PC12. We found that this protein is present in a distinct population of slowly recycling light vesicles. By gradient centrifugations, immunoadsorption and double immunofluorescent staining, these vesicles are different from transferrin-containing endosomes, small synaptic vesicles and secretory granules and may thus represent a novel compartment in PC12 cells. Glut4-GFP chimera transiently expressed in PC12 cells is targeted to IRAP-containing vesicles indicating that cotargeting of Glut4 and IRAP is not specific for adipocytes and myocytes, but is faithful in a foreign cell type. We suggest that PC12 cells may possess a novel type of a vesicular carrier that may represent the homolog of Glut4-vesicles.     

Regulation of insulin-responsive aminopeptidase expression and targeting in the insulin-responsive vesicle compartment of glucose transporter isoform 4-deficient cardiomyocytes

In adipocytes and cardiac or skeletal muscle, glucose transporter isoform 4 (GLUT4) is targeted to insulin-responsive intracellular membrane vesicles (IRVs) that contain several membrane proteins, including insulin-responsive aminopeptidase (IRAP) that completely colocalizes with GLUT4 in basal and insulin-treated cells. Cardiac GLUT4 content is reduced by 65-85% in IRAP knockout mice, suggesting that IRAP may regulate the targeting or degradation of GLUT4. To determine whether GLUT4 is required for maintenance of IRAP content within IRVs, we studied the expression and cellular localization of IRAP and other GLUT4 vesicle-associated proteins, in hearts of mice with cardiac-specific deletion of GLUT4 (G4H-/-). In G4H-/- hearts, IRAP content was reduced by 60%, but the expression of other vesicle-associated proteins, namely cellugyrin, IGF-II/mannose-6-phosphate, and transferrin receptors, secretory carrier-associated membrane proteins and vesicle-associated membrane protein were unchanged. Using sucrose gradient centrifugation and cell surface biotinylation, we found that IRAP content in 50-80S vesicles where GLUT4 vesicles normally sediment was markedly depleted in G4H-/- hearts, and the remaining IRAP was found in the heavy membrane fraction. Although insulin caused a discernible increase in cell surface IRAP content of G4H-/- cardiomyocytes, cell surface IRAP remained 70% lower than insulin-stimulated controls. Immunoabsorption of intracellular vesicles with anticellugyrin antibodies revealed that IRAP content was reduced by 70% in both cellugyrin-positive and cellugyrin-negative vesicles. Endosomal recycling, as measured by transferrin receptor recycling was normal. Thus, GLUT4 and IRAP content of early endosome-derived sorting vesicles and of IRVs are coordinately regulated, and both proteins are required for maintenance of key constituents of these compartments in cardiac muscle cells in vivo.     

Insulin stimulation of GLUT-4 translocation: a model for regulated recycling

Insulin stimulates glucose transport in muscle and fat cells by causing the redistribution of a facilitative glucose transporter, GLUT-4, from an intracellular compartment to the cell surface. But what is this intracellular GLUT-4 compartment? It may be a specialized compartment, perhaps analogous to synaptic vesicles, or may simply be part of the endosomal system. Other constituents of this compartment might be regulators of GLUT-4 movement to the cell surface, and their identification should make it possible to find the link between the insulin signal transduction pathway and GLUT-4 translocation.     

Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria

Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes.     

Interaction of the Akt substrate, AS160, with the glucose transporter 4 vesicle marker protein, insulin-regulated aminopeptidase

Insulin-regulated aminopeptidase (IRAP), a marker of glucose transporter 4 (GLUT4) storage vesicles (GSVs), is the only protein known to traffic with GLUT4. In the basal state, GSVs are sequestered from the constitutively recycling endosomal system to an insulin-responsive, intracellular pool. Insulin induces a rapid translocation of GSVs to the cell surface from this pool, resulting in the incorporation of IRAP and GLUT4 into the plasma membrane. We sought to identify proteins that interact with IRAP to further understand this GSV trafficking process. This study describes our identification of a novel interaction between the amino terminus of IRAP and the Akt substrate, AS160 (Akt substrate of 160 kDa). The validity of this interaction was confirmed by coimmunoprecipitation of both overexpressed and endogenous proteins. Moreover, confocal microscopy demonstrated colocalization of these proteins. In addition, we demonstrate that the IRAP-binding domain of AS160 falls within its second phosphotyrosine-binding domain and the interaction is not regulated by AS160 phosphorylation. We hypothesize that AS160 is localized to GLUT4-containing vesicles via its interaction with IRAP where it inhibits the activity of Rab substrates in its vicinity, effectively tethering the vesicles intracellularly.     

Vimentin binds IRAP and is involved in GLUT4 vesicle trafficking

Insulin-responsive aminopeptidase (IRAP) and GLUT4 are two major cargo proteins of GLUT4 storage vesicles (GSVs) that are translocated from a postendosomal storage compartment to the plasma membrane (PM) in response to insulin. The cytoplasmic region of IRAP is reportedly involved in retention of GSVs. In this study, vimentin was identified using the cytoplasmic domain of IRAP as bait. The validity of this interaction was confirmed by pull-down assays and immunoprecipitation in 3T3-L1 adipocytes. In addition, it was shown that GLUT4 translocation to the PM by insulin was decreased in vimentin-depleted adipocytes, presumably due to dispersing GSVs away from the cytoskeleton. These findings suggest that the IRAP binding protein, vimentin, plays an important role in retention of GSVs.     

Reduced insulin-stimulated glucose transport in denervated muscle is associated with impaired Akt-alpha activation

Insulin signaling was examined in muscle made insulin resistant by short-term (24-h) denervation. Insulin-stimulated glucose transport in vitro was reduced by 28% (P < 0.05) in denervated muscle (DEN). In control muscle (SHAM), insulin increased levels of surface-detectable GLUT-4 (i.e., translocated GLUT-4) 1.8-fold (P < 0.05), whereas DEN surface GLUT-4 was not increased by insulin (P > 0.05). Insulin treatment in vivo induced a rapid appearance of phospho[Ser(473)]Akt-alpha in SHAM 3 min after insulin injection. In DEN, phospho[Ser(473)]Akt-alpha also appeared at 3 min, but Ser(473)-phosphorylated Akt-alpha was 36% lower than in SHAM (P < 0. 05). In addition, total Akt-alpha protein in DEN was 37% lower than in SHAM (P < 0.05). Akt-alpha kinase activity was lower in DEN at two insulin levels tested: 0.1 U insulin/rat (-22%, P < 0.05) and 1 U insulin/rat (-26%, P < 0.01). These data indicate that short-term (24-h) denervation, which lowers insulin-stimulated glucose transport, is associated with decreased Akt-alpha activation and impaired insulin-stimulated GLUT-4 appearance at the muscle surface.     

Insulin-regulated aminopeptidase marks an antigen-stimulated recycling compartment in mast cells

Insulin-regulated aminopeptidase (IRAP) is a marker for insulin-sensitive recycling compartments of fat and muscle cells that contain the glucose transporter isoform GLUT4. Unlike GLUT4, IRAP is expressed in many other cell types. Thus, it is a potential marker for regulated recycling compartments that are analogous to GLUT4 vesicles. In bone marrow-derived mast cells, IRAP is highly expressed and localizes to an intracellular compartment different from secretory granules. Using cell-surface biotinylation, we determined that IRAP underwent rapid redistribution to the plasma membrane on antigen/immunoglobulin E (IgE) stimulation and was re-internalized within 30 min. When granule exocytosis was inhibited, by removing extracellular calcium, adding the protein kinase C inhibitor bisindolylmaleimide or the phosphatidylinositol 3-kinase inhibitor wortmannin, IRAP redistribution was still detected in stimulated cells. However, the redistribution of IRAP required intracellular calcium. By immunofluorescence, IRAP significantly co-localized with the transferrin receptor (TfR), a marker for constitutively recycling endosomes. However, antigen/IgE stimulation did not increase TfR on the cell surface, indicating that IRAP and TfR may follow different pathways to the plasma membrane. In rat peritoneal mast cells, the distributions of IRAP and TfR overlapped to only a limited extent, indicating that overlap may decrease with cell differentiation. We propose that IRAP vesicles represent a second IgE-sensitive exocytotic compartment in mast cells, which is regulated differently from secretory granules, and that these vesicles may be similar to GLUT4 vesicles.     

GLUT4 ablation in mice results in redistribution of IRAP to the plasma membrane

Glucose transporter (GLUT) 4 is the insulin responsive glucose transporter in adipose tissue, skeletal muscle, and heart. Insulin elicits increased glucose uptake by recruiting GLUT4 from a specialized intracellular storage site to the cell surface. Expression of various proteins that colocalize with GLUT4 and/or are involved in insulin-stimulated GLUT4 translocation was examined in adipocytes as well as skeletal and cardiac muscles from GLUT4 null mice. Our data demonstrate that expression of insulin-regulated aminopeptidase (IRAP) is divergently regulated in GLUT4 null tissues, e.g., upregulated 1.6-fold in GLUT4 null adipocytes and downregulated in GLUT4 null skeletal muscle (40%) and heart (60%). IRAP exhibited abnormal subcellular distribution and impaired insulin-stimulated translocation in GLUT4-deficient tissues. We propose the compartment containing IRAP and proteins normally associated with GLUT4 vesicle traffics constitutively to the cell surface in GLUT4 null adipocytes and skeletal muscle.     

Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase

The glucose transporter GLUT4 and the aminopeptidase IRAP (insulin-responsive aminopeptidase) are the major cargo proteins of GSVs (GLUT4 storage vesicles) in adipocytes and myocytes. In the basal state, most GSVs are sequestered in perinuclear and other cytosolic compartments. Following insulin stimulation, GSVs undergo exocytic translocation to insert GLUT4 and IRAP into the plasma membrane. The mechanisms regulating GSV trafficking are not fully defined. In the present study, using 3T3-L1 adipocytes transfected with siRNAs (small interfering RNAs), we show that insulin-stimulated IRAP translocation remained intact despite substantial GLUT4 knockdown. By contrast, insulin-stimulated GLUT4 translocation was impaired upon IRAP knockdown, indicating that IRAP plays a role in GSV trafficking. We also show that knockdown of tankyrase, a Golgi-associated IRAP-binding protein that co-localizes with perinuclear GSVs, attenuated insulin-stimulated GSV translocation and glucose uptake without disrupting insulin-induced phosphorylation cascades. Moreover, iodixanol density gradient analyses revealed that tankyrase knockdown altered the basal-state partitioning of GLUT4 and IRAP within endosomal compartments, apparently by shifting both proteins toward less buoyant compartments. Importantly, the afore-mentioned effects of tankyrase knockdown were reproduced by treating adipocytes with PJ34, a general PARP (poly-ADP-ribose polymerase) inhibitor that abrogated tankyrase-mediated protein modification known as poly-ADP-ribosylation. Collectively, these findings suggest that physiological GSV trafficking depends in part on the presence of IRAP in these vesicles, and that this process is regulated by tankyrase and probably its PARP activity.     

Coordinated Regulation of Vasopressin Inactivation and Glucose Uptake by Action of TUG Protein in Muscle

In adipose and muscle cells, insulin stimulates the exocytic translocation of vesicles containing GLUT4, a glucose transporter, and insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase. A substrate of IRAP is vasopressin, which controls water homeostasis. The physiological importance of IRAP translocation to inactivate vasopressin remains uncertain. We previously showed that in skeletal muscle, insulin stimulates proteolytic processing of the GLUT4 retention protein, TUG, to promote GLUT4 translocation and glucose uptake. Here we show that TUG proteolysis also controls IRAP targeting and regulates vasopressin action in vivo. Transgenic mice with constitutive TUG proteolysis in muscle consumed much more water than wild-type control mice. The transgenic mice lost more body weight during water restriction, and the abundance of renal AQP2 water channels was reduced, implying that vasopressin activity is decreased. To compensate for accelerated vasopressin degradation, vasopressin secretion was increased, as assessed by the cosecreted protein copeptin. IRAP abundance was increased in T-tubule fractions of fasting transgenic mice, when compared with controls. Recombinant IRAP bound to TUG, and this interaction was mapped to a short peptide in IRAP that was previously shown to be critical for GLUT4 intracellular retention. In cultured 3T3-L1 adipocytes, IRAP was present in TUG-bound membranes and was released by insulin stimulation. Together with previous results, these data support a model in which TUG controls vesicle translocation by interacting with IRAP as well as GLUT4. Furthermore, the effect of IRAP to reduce vasopressin activity is a physiologically important consequence of vesicle translocation, which is coordinated with the stimulation of glucose uptake.     

The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin- sensitive cells

Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle- associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP- 2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.     

Translocation of small preformed vesicles is responsible for the insulin activation of glucose transport in adipose cells. Evidence from the in vitro reconstitution assay

Insulin stimulates translocation of the glucose transporter isoform 4 (Glut4) from an intracellular storage compartment to the plasma membrane in fat and skeletal muscle cells. At present, the nature of the Glut4 storage compartment is unclear. According to one model, this compartment represents a population of preformed small vesicles that fuse with the plasma membrane in response to insulin stimulation. Alternatively, Glut4 may be retained in large donor membranes, and insulin stimulates the formation of transport vesicles that deliver Glut4 to the cell surface. Finally, insulin can induce plasma membrane fusion of the preformed vesicles and, also, stimulate the formation of new vesicles. In extracts of fat and skeletal muscle cells, Glut4 is predominantly found in small insulin-sensitive 60-70 S membrane vesicles that may or may not artificially derive from large donor membranes during cell homogenization. Here, we use a cell-free reconstitution assay to demonstrate that small Glut4-containing vesicles are formed from large rapidly sedimenting donor membranes in a cytosol-, ATP-, time-, and temperature-dependent fashion and, therefore, do not represent an artifact of homogenization. Thus, small insulin-responsive vesicles represent the major form of Glut4 storage in the living adipose cell. Fusion of these vesicles with the plasma membrane may be largely responsible for the primary effect of insulin on glucose transport in fat tissue. In addition, our results suggest that insulin may also stimulate the formation of Glut4 vesicles and accelerate Glut4 recycling to the plasma membrane.     

C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression

Insulin regulates the uptake of glucose into skeletal muscle and adipocytes by redistributing the tissue-specific glucose transporter GLUT4 from intracellular vesicles to the cell surface. To date, GLUT4 is the only protein involved in insulin-regulated vesicular traffic that has this tissue distribution, thus raising the possibility that its expression alone may allow formation of an insulin-responsive vesicular compartment. We show here that treatment of differentiating C2C12 myoblasts with dexamethasone, acting via the glucocorticoid receptor, causes a >or=10-fold increase in GLUT4 expression but results in no significant change in insulin-stimulated glucose transport. Signaling from the insulin receptor to its target, Akt2, and expression of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor, or SNARE, proteins syntaxin 4 and vesicle-associated membrane protein are normal in dexamethasone-treated C2C12 cells. However, these cells show no insulin-dependent trafficking of the insulin-responsive aminopeptidase or the transferrin receptor, respective markers for intracellular GLUT4-rich compartments and endosomes that are insulin responsive in mature muscle and adipose cells. Therefore, these data support the hypothesis that GLUT4 expression by itself is insufficient to establish an insulin-sensitive vesicular compartment.