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.     

Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment

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 insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (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 knock-out mice, compared with wild type 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.     

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.     

Proteomic Analysis of GLUT4 Storage Vesicles Reveals LRP1 to Be an Important Vesicle Component and Target of Insulin Signaling

Insulin stimulates the translocation of intracellular GLUT4 to the plasma membrane where it functions in adipose and muscle tissue to clear glucose from circulation. The pathway and regulation of GLUT4 trafficking are complicated and incompletely understood and are likely to be contingent upon the various proteins other than GLUT4 that comprise and interact with GLUT4-containing vesicles. Moreover, not all GLUT4 intracellular pools are insulin-responsive as some represent precursor compartments, thus posing a biochemical challenge to the purification and characterization of their content. To address these issues, we immunodepleted precursor GLUT4-rich vesicles and then immunopurified GLUT4 storage vesicle (GSVs) from primary rat adipocytes and subjected them to semi-quantitative and quantitative proteomic analysis. The purified vesicles translocate to the cell surface almost completely in response to insulin, the expected behavior for bona fide GSVs. In total, over 100 proteins were identified, about 50 of which are novel in this experimental context. LRP1 (low density lipoprotein receptor-related protein 1) was identified as a major constituent of GSVs, and we show it interacts with the lumenal domains of GLUT4 and other GSV constituents. Its cytoplasmic tail interacts with the insulin-signaling pathway target, AS160 (Akt substrate of 160 kDa). Depletion of LRP1 from 3T3-L1 adipocytes reduces GLUT4 expression and correspondingly results in decreased insulin-stimulated 2-[³H]deoxyglucose uptake. Furthermore, adipose-specific LRP1 knock-out mice also exhibit decreased GLUT4 expression. These findings suggest LRP1 is an important component of GSVs, and its expression is needed for the formation of fully functional GSVs.     

以消散场成像和单微粒跟踪技术揭示GLUT4囊泡和分泌囊泡的不同动态过程

葡萄糖转运子蛋白4(glucose transporter 4,GLUT4)在维持体内葡萄糖动态平衡的过程中起着至关重要的作用。GLUT4贮存囊泡(GLUT4 storage vesicle,GSV)和神经内分泌细胞中的分泌囊泡含有许多相同的蛋白。研究证明这些蛋白调节了分泌囊泡的胞内转运过程,但是GLUT4囊泡和分泌囊泡是否具有相同的胞内动态过程还未阐明。文章以3T3-L1纤维原细胞中的GSV和神经内分泌细胞PC12细胞中的分泌囊泡:致密核心大囊泡(large dense core vesicle,LDCV)为研究对象,使用消散场显微成像技术和单微粒跟踪技术直观观察了活体细胞内单个GSV和LDCV的三维运动轨迹。通过以适当方程拟合单个囊泡的均方位移曲线,发现两种囊泡都具有三种运动模式。定量分析显示作自由扩散运动和方向性扩散运动的GSV数量明显多于LDCV。对比GSV和LDCV的三维扩散系数,发现GSV的扩散系数中值为7.2×10-4μm2/s,而LDCV的扩散系数中值仅为1.94×10-4μm2/s。这一结果说明GSV的活动性远大于LDCV,提示GSV的胞内转运过程涉及不同的分子机制。     

Rab10 and myosin-Va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes

Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.     

Dynamic tracking and mobility analysis of single GLUT4 storage vesicle in live 3T3-L1 cells

Glucose transporter 4 (GLUT4) is responsible for insulin-stimulated glucose transporting into the insulin-sensitive fat and muscle cells. The dynamics of GLUT4 storage vesicles (GSVs) remains to be explored and it is unclear how GSVs are arranged based on their mobility. We examined this issue in 3T3-L1 cells via investigating the three-dimensional mobility of single GSV labeled with EGFP-fused GLUT4. A thin layer of cytosol right adjacent to the plasma membrane was illuminated and successively imaged at 5 Hz under a total internal reflection fluorescence microscope with a penetration depth of 136 nm. Employing single particle tracking, the three-dimensional subpixel displacement of single GSV was tracked at a spatial precision of 22 nm. Both the mean square displacement and the diffusion coefficient were calculated for each vesicle. Tracking results revealed that vesicles moved as if restricted within a cage that has a mean radius of 160 nm, suggesting the presence of some intracellular tethering matrix. By constructing the histogram of the diffusion coefficients of GSVs, we observed a smooth distribution instead of the existence of distinct groups. The result indicates that GSVs are dynamically retained in a continuous and wide range of mobility rather than into separate classes.     

Dual-mode of insulin action controls GLUT4 vesicle exocytosis

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.     

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.     

GLUT4在胰岛素调控葡萄糖转运中作用

机体的血糖平衡调节主要依赖于胰岛素,其中一个重要的机制是胰岛素通过调控GLUT4的囊泡运转来调节脂肪细胞和肌细胞对葡萄糖的摄取。由胰岛素受体介导的一系列磷酸化过程能调节一些关键的GLUT4转运相关蛋白质的活性,这些蛋白质包括小GTP酶、拴系复合体和囊泡融合体。而这些蛋白质又反过来通过内膜系统调节GLUT4储存囊泡的生成、滞留,并调控这些囊泡的靶向出胞方式。了解这些过程有助于解释2型糖尿病中胰岛素耐受的机制,并可能为糖尿病提供新的靶向治疗方法。     

Characterization of GLUT4-containing vesicles in 3T3-L1 adipocytes by total internal reflection fluorescence microscopy

Insulin-responsive GLUT4 (glucose transporter 4) translocation plays a major role in regulating glucose uptake in adipose tissue and muscle. Whether or not there is a specialized secretory GSV (GLUT4 storage vesicle) pool, and more importantly how GSVs are translocated to the PM (plasma membrane) under insulin stimulation is still under debate. In the present study, we systematically analyzed the dynamics of a large number of single GLUT4-containing vesicles in 3T3-L1 adipocytes by TIRFM (total internal reflection fluorescence microscopy). We found that GLUT4-containing vesicles can be classified into three groups according to their mobility, namely vertical, stable, and lateral GLUT4-containing vesicles. Among these groups, vertical GLUT4-containing vesicles exclude transferrin receptors and move towards the PM specifically in response to insulin stimulation, while stable and lateral GLUT4-containing vesicles contain transferrin receptors and show no insulin responsiveness. These data demonstrate that vertical GLUT4-containing vesicles correspond to specialized secretory GSVs, which approach the PM directly and bypass the constitutive recycling pathway. Contributed equally to this work Supported by the National Natural Science Foundation of China (Grant Nos. 30470448 and 30130230), the National key Basic Research Program of China (Grant No. 2004CB720000), the Knowledge Innovative Program of The Chinese Academy of Sciences (Grant Nos. KSCX2-SW-224 and Y2004018), the Li Foundation and the Sinogerman Scientific Center.     

GLUT4 and Transferrin Receptor Are Differentially Sorted Along the Endocytic Pathway in CHO Cells

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750–12754.). In this study, we demonstrate that at 37°C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37°C, cell surface–labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15°C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37°C after accumulating labeled GLUT4 at 15°C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15°C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37°C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15° and 37°C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.     

Identification of P-Rex1 as a novel Rac1-guanine nucleotide exchange factor (GEF) that promotes actin remodeling and GLUT4 protein trafficking in adipocytes

Phosphoinositide 3-kinase (PI3K) signaling promotes the translocation of the glucose transporter, GLUT4, to the plasma membrane in insulin-sensitive tissues to facilitate glucose uptake. In adipocytes, insulin-stimulated reorganization of the actin cytoskeleton has been proposed to play a role in promoting GLUT4 translocation and glucose uptake, in a PI3K-dependent manner. However, the PI3K effectors that promote GLUT4 translocation via regulation of the actin cytoskeleton in adipocytes remain to be fully elucidated. Here we demonstrate that the PI3K-dependent Rac exchange factor, P-Rex1, enhances membrane ruffling in 3T3-L1 adipocytes and promotes GLUT4 trafficking to the plasma membrane at submaximal insulin concentrations. P-Rex1-facilitated GLUT4 trafficking requires a functional actin network and membrane ruffle formation and occurs in a PI3K- and Rac1-dependent manner. In contrast, expression of other Rho GTPases, such as Cdc42 or Rho, did not affect insulin-stimulated P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or expression of a P-Rex1 dominant negative mutant reduced but did not completely inhibit glucose uptake in response to insulin. Collectively, these studies identify a novel RacGEF in adipocytes as P-Rex1 that, at physiological insulin concentrations, functions as an insulin-dependent regulator of the actin cytoskeleton that contributes to GLUT4 trafficking to the plasma membrane.     

How many signals impinge on GLUT4 activation by insulin?

GLUT4 is the main glucose transporter activated by insulin in skeletal muscle cells and adipocytes. GLUT4 storage vesicles (GSVs) traffic in endocytic and exocytic compartments. In the basal state, GLUT4 compartments are preferentially sequestered in perinuclear deposits wherein stimuli including insulin and non-insulin factors can increase GLUT4 vesicle formation, its exocytosis, and fusion to plasma membrane. In addition to well-established effectors of insulin signaling pathway, such as PKCzeta and Akt, the cytoskeletal network is implicated in GLUT4 translocation. This review will discuss the mechanisms and activation of GLUT4 trafficking and incorporating to PM from three aspects: known molecules of the insulin signaling pathway; Rho and Rab family proteins and cytoskeletal molecules.     

脂肪细胞中Munc18c的缺失不影响胰岛素诱导的GLUT4转运(已撤稿2016-01-13)

动物脂肪和肌肉组织中葡萄糖的摄取是通过受胰岛素调控的GLUT4储存囊泡的运输实现的.Sec1p的同源物Munc18c被认为是通过控制SNARE复合物的装配来使GLUT4囊泡锚定到质膜上的重要物质.我们发现Munc18c的缺失没有影响GLUT4的转运上膜,也没有影响Syntaxin4在细胞膜上的定位.在缺少Munc18c和功能性Syntaxin2的时候,GLUT4的转运可能和Munc18b有关.在3T3-L1脂肪细胞中与Syntaxin4具有强烈相互作用的是Munc18c而不是Munc18a和Munc18b.然而,当缺少Munc18c时,Munc18a和Munc18b与Syntaxin4体现出较弱的相互作用.因此,Syntaxin4可能在胰岛素刺激GLUT4转运过程中起到重要的作用,且与SM蛋白的相互作用是有代偿性的.     

Adipsin and the glucose transporter GLUT4 traffic to the cell surface via independent pathways in adipocytes

Insulin increases the exocytosis of many soluble and membrane proteins in adipocytes. This may reflect a general effect of insulin on protein export from the trans Golgi network. To test this hypothesis, we have compared the trafficking of the secreted serine protease adipsin and the integral membrane proteins GLUT4 and transferrin receptors in 3T3-L1 adipocytes. We show that adipsin is secreted from the trans Golgi network to the endosomal system, as ablation of endosomes using transferrin-HRP conjugates strongly inhibited adipsin secretion. Phospholipase D has been implicated in export from the trans Golgi network, and we show that insulin stimulates phospholipase D activity in these cells. Inhibition of phospholipase D action with butan-1-ol blocked adipsin secretion and resulted in accumulation of adipsin in trans Golgi network-derived vesicles. In contrast, butan-1-ol did not affect the insulin-stimulated movement of transferrin receptors to the plasma membrane, whereas this was abrogated following endosome ablation. GLUT4 trafficking to the cell surface does not utilise this pathway, as insulin-stimulated GLUT4 translocation is still observed after endosome ablation or inhibition of phospholipase D activity. Immunolabelling revealed that adipsin and GLUT4 are predominantly localised to distinct intracellular compartments. These data suggest that insulin stimulates the activity of the constitutive secretory pathway in adipocytes possibly by increasing the budding step at the TGN by a phospholipase D-dependent mechanism. This may have relevance for the secretion of other soluble molecules from these cells. This is not the pathway employed to deliver GLUT4 to the plasma membrane, arguing that insulin stimulates multiple pathways to the cell surface in adipocytes.     

Palmitate-induced Down-regulation of Sortilin and Impaired GLUT4 Trafficking in C2C12 Myotubes

Elevated saturated FFAs including palmitate (C16:0) are a primary trigger for peripheral insulin resistance characterized by impaired glucose uptake/disposal in skeletal muscle, resulting from impaired GLUT4 translocation in response to insulin. We herein demonstrate that palmitate induces down-regulation of sortilin, a sorting receptor implicated in the formation of insulin-responsive GLUT4 vesicles, via mechanisms involving PKCθ and TNF-α-converting enzyme, but not p38, JNK, or mitochondrial reactive oxygen species generation, leading to impaired GLUT4 trafficking in C2C12 myotubes. Intriguingly, unsaturated FFAs such as palmitoleate (C16:1) and oleate (C18:1) had no such detrimental effects, appearing instead to effectively reverse palmitate-induced impairment of insulin-responsive GLUT4 recycling along with restoration of sortilin abundance by preventing aberrant PKCθ activation. On the other hand, shRNA-mediated reduction of sortilin in intact C2C12 myotubes inhibited insulin-induced GLUT4 recycling without dampening Akt phosphorylation. We found that the peroxisome proliferator-activated receptor γ agonist troglitazone prevented the palmitate-induced sortilin reduction and also ameliorated insulin-responsive GLUT4 recycling without altering the palmitate-evoked insults on signaling cascades; neither highly phosphorylated PKCθ states nor impaired insulin-responsive Akt phosphorylation was affected. Taken together, our data provide novel insights into the pathogenesis of PKCθ-dependent insulin resistance with respect to insulin-responsive GLUT4 translocation, which could occur not only through defects of insulin signaling but also via a reduction of sortilin, which directly controls trafficking/sorting of GLUT4 in skeletal muscle cells. In addition, our data suggest the insulin-sensitizing action of peroxisome proliferator-activated receptor γ agonists to be at least partially mediated through the restoration of proper GLUT4 trafficking/sorting events governed by sortilin.     

Biogenesis of insulin-responsive GLUT4 vesicles is independent of brefeldin A-sensitive trafficking

Insulin stimulates translocation of GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. As a significant amount of GLUT4 is localised to the TGN, independently of the biosynthetic pathway, one possibility is that trafficking via the TGN is important in either intracellular sequestration or insulin-dependent movement to the cell surface. In this study we have used immuno-electron microscopy to show that GLUT4 is localised to AP-1 vesicles in the TGN region in 3T3-L1 adipocytes. To dissect the role of this trafficking pathway we used brefeldin A (BFA) to disrupt AP-1 association with membranes. Despite a reorganisation of GLUT4 compartments following BFA treatment, the intracellular sequestration of GLUT4, and its insulin-dependent movement to the cell surface, was unaffected. BFA increased the half time of reversal of insulin-stimulated glucose transport from 17 to 30 min but did not prevent complete reversal. Furthermore, following reversal re-stimulation of glucose transport activity by insulin was not compromised. We conclude that under basal conditions GLUT4 cycles between the TGN and endosomes via the AP-1 pathway. However, neither this pathway, nor any other BFA-sensitive pathway, appears to play a major role in insulin-dependent recruitment of GLUT4 to the cell surface.