A pre-docking role for microtubules in insulin-stimulated glucose transporter 4 translocation

Insulin stimulates glucose uptake by inducing translocation of glucose transporter 4 (GLUT4) from intracellular resides to the plasma membrane. How GLUT4 storage vesicles are translocated from the cellular interior to the plasma membrane remains to be elucidated. In the present study, intracellular transport of GLUT4 storage vesicles and the kinetics of their docking at the plasma membrane were comprehensively investigated at single vesicle level in control and microtubule-disrupted 3T3-L1 adipocytes by time-lapse total internal reflection fluorescence microscopy. It is demonstrated that microtubule disruption substantially inhibited insulin-stimulated GLUT4 translocation. Detailed analysis reveals that microtubule disruption blocked the recruitment of GLUT4 storage vesicles to underneath the plasma membrane and abolished the docking of them at the plasma membrane. These data suggest that transport of GLUT4 storage vesicles to the plasma membrane takes place along microtubules and that this transport is obligatory for insulin-stimulated GLUT4 translocation.     

GLUT4 is retained by an intracellular cycle of vesicle formation and fusion with endosomes

The intracellularly stored GLUT4 glucose transporter is rapidly translocated to the cell surface upon insulin stimulation. Regulation of GLUT4 distribution is key for the maintenance of whole body glucose homeostasis. We find that GLUT4 is excluded from the plasma membrane of adipocytes by a dynamic retention/retrieval mechanism. Our kinetic studies indicate that GLUT4-containing vesicles continually bud and fuse with endosomes in the absence of insulin and that these GLUT4 vesicles are 5 times as likely to fuse with an endosome as with the plasma membrane. We hypothesize that this intracellular cycle of vesicle budding and fusion is an element of the active mechanism by which GLUT4 is retained. The GLUT4 trafficking pathway does not extensively overlap with that of furin, indicating that the trans-Golgi network, a compartment in which furin accumulates, is not a significant storage reservoir of GLUT4. An intact microtubule cytoskeleton is required for insulin-stimulated recruitment to the cell surface, although it is not required for the basal budding/fusion cycle. Nocodazole disruption of the microtubule cytoskeleton reduces the insulin-stimulated exocytosis of GLUT4, accounting for the reduced insulin-stimulated translocation of GLUT4 to the cell surface.     

Insulin action on GLUT4 traffic visualized in single 3T3-l1 adipocytes by using ultra-fast microscopy

A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20-30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5-7 min at 37 degrees C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.     

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.     

Insulin-mediated GLUT4 translocation is dependent on the microtubule network

The GLUT4 facilitative glucose transporter is recruited to the plasma membrane by insulin. This process depends primarily on the exocytosis of a specialized pool of vesicles containing GLUT4 in their membranes. The mechanism of GLUT4 vesicle exocytosis in response to insulin is not understood. To determine whether GLUT4 exocytosis is dependent on intact microtubule network, we measured insulin-mediated GLUT4 exocytosis in 3T3-L1 adipocytes in which the microtubule network was depolymerized by pretreatment with nocodazole. Insulin-mediated GLUT4 translocation was inhibited by more than 80% in nocodazole-treated cells. Phosphorylation of insulin receptor substrate 1 (IRS-1), activation of IRS-1 associated phosphatidylinositide 3-kinase, and phosphorylation of protein kinase B/Akt-1 were not inhibited by nocodazole treatment indicating that the microtubule network was not required for proximal insulin signaling. An intact microtubule network is specifically required for insulin-mediated GLUT4 translocation since nocodazole treatment did not affect insulin-mediated GLUT1 translocation or adipsin secretion. By using in vitro microtubule binding, we demonstrated that both GLUT4 vesicles and IRS-1 bind specifically to microtubules, implicating microtubules in both insulin signaling and GLUT4 translocation. Vesicle binding to microtubules was not mediated through direct binding of GLUT4 or insulin-responsive aminopeptidase to microtubules. A model microtubule-dependent translocation of GLUT4 is proposed.     

Conventional kinesin KIF5B mediates insulin-stimulated GLUT4 movements on microtubules

Insulin stimulates glucose uptake in muscle and adipose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter proteins to the plasma membrane. Here we show in live cultured adipocytes that intracellular membranes containing GLUT4-yellow fluorescent protein (YFP) move along tubulin-cyan fluorescent protein-labeled microtubules in response to insulin by a mechanism that is insensitive to the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin. Insulin increased by several fold the observed frequencies, but not velocities, of long-range movements of GLUT4-YFP on microtubules, both away from and towards the perinuclear region. Genomics screens show conventional kinesin KIF5B is highly expressed in adipocytes and this kinesin is partially co-localized with perinuclear GLUT4. Dominant-negative mutants of conventional kinesin light chain blocked outward GLUT4 vesicle movements and translocation of exofacial Myc-tagged GLUT4-green fluorescent protein to the plasma membrane in response to insulin. These data reveal that insulin signaling targets the engagement or initiates the movement of GLUT4-containing membranes on microtubules via conventional kinesin through a PI3-kinase-independent mechanism. This insulin signaling pathway regulating KIF5B function appears to be required for GLUT4 translocation to the plasma membrane.     

Bi-directional transport of GLUT4 vesicles near the plasma membrane of primary rat adipocytes

Insulin stimulates glucose uptake into adipocytes by mobilizing intracellular membrane vesicles containing GLUT4 proteins to the plasma membrane. Here we applied time-lapse total internal reflection fluorescence microscopy to study moving parameters and characters of exogenously expressed GLUT4 vesicles in basal, insulin and nocodazole treated primary rat adipocytes. Our results showed that microtubules were essential for long-range transport of GLUT4 vesicles but not obligatory for GLUT4 distribution in rat adipocytes. Insulin reduced the mobility of the vesicles, made them tethered/docked to the PM and finally had constitutive exocytosis. Moreover, long-range bi-directional movements of GLUT4 vesicles were visualized for the first time by TIRFM. It is likely that there are interactions between insulin signaling and microtubules, to regulating GLUT4 translocation in rat adipocytes.     

Insulin stimulates membrane fusion and GLUT4 accumulation in clathrin coats on adipocyte plasma membranes

Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by approximately 4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.     

A role for kinesin in insulin-stimulated GLUT4 glucose transporter translocation in 3T3-L1 adipocytes

Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin.     

Microtubule network is required for insulin signaling through activation of Akt/protein kinase B: evidence that insulin stimulates vesicle docking/fusion but not intracellular mobility

The microtubule network has been shown to be required for insulin-dependent GLUT4 redistribution; however, the precise molecular function has not been elucidated. In this article, we used fluorescence recovery after photobleaching (FRAP) to evaluate the role of microtubules in intracellular GLUT4 vesicle mobility. A comparison of the rate of fluorescence recovery (t((1/2))), and the maximum fluorescence recovered (F(max)) was made between basal and insulin-treated cells with or without nocodazole treatment to disrupt microtubules. We found that intracellular mobility of fluorescently tagged GLUT4 (HA-GLUT4-GFP) was high in basal cells. Mobility was not increased by insulin treatment. Basal mobility was dependent upon an intact microtubule network. Using a constitutively active Akt to signal GLUT4 redistribution, we found that microtubule-based GLUT4 vesicle mobility was not obligatory for GLUT4 plasma membrane insertion. Our findings suggest that microtubules organize the insulin-signaling complex and provide a surface for basal mobility of GLUT4 vesicles. Our data do not support an obligatory requirement for long range microtubule-based movement of GLUT4 vesicles for insulin-mediated GLUT4 redistribution to the cell surface. Taken together, these findings suggest a model in which insulin signaling targets membrane docking and/or fusion rather than GLUT4 trafficking to the cell surface.     

Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.     

Intracellular insulin-responsive glucose transporter (GLUT4) distribution but not insulin-stimulated GLUT4 exocytosis and recycling are microtubule dependent

To investigate the potential role of microtubules in the regulation of insulin-responsive glucose transporter (GLUT4) trafficking in adipocytes, we examined the effects of microtubule depolymerizing and stabilizing agents. In contrast to previous reports, disruption or stabilization of microtubule structures had no significant effect on insulin-stimulated GLUT4 translocation. However, consistent with a more recent study (Molero, J. C., J. P. Whitehead, T. Meerloo, and D. E. James, 2001, J Biol Chem 276:43829-43835) nocodazole did inhibit glucose uptake through a direct interaction with the transporter itself independent of the translocation process. In addition, the initial rate of GLUT4 endocytosis was not significantly affected by microtubule depolymerization. However, these internalized GLUT4 compartments are confined to regions just beneath the plasma membrane and were not exposed to the extracellular space. Furthermore, they were unable to undergo further sorting steps and trafficking to the perinuclear region. Nevertheless, these apparent early endocytic GLUT4 compartments fully responded to a second insulin stimulation with an identical extent of plasma membrane translocation. Together, these data demonstrate that although microtubular organization may play a role in the trafficking of GLUT4 early endocytic vesicles back to the perinuclear region, they do not have a significant role in insulin-stimulated GLUT4 exocytosis, initial endocytosis from the plasma membrane and/or recycling back to the plasma membrane.     

Temporal separation of insulin-stimulated GLUT4/IRAP vesicle plasma membrane docking and fusion in 3T3L1 adipocytes

Examination of the time and temperature dependence of insulin-stimulated GLUT4/IRAP-containing vesicle trafficking demonstrated an approximate 7-fold increase in the half-time for plasma membrane translocation at 23 degrees C (t((1)/(2)) = approximately 30 min) compared with 37 degrees C (t((1)/(2)) = approximately 4 min) without a significant change in the extent of either GLUT4 or IRAP translocation. Localization of the endogenous GLUT4 and expressed GLUT4-enhanced green fluorescent protein fusion protein in intact 3T3L1 adipocytes demonstrated that at 23 degrees C there was a time-dependent accumulation of discrete GLUT4-containing vesicles adjacent to the inner face of the cell surface membrane but that was not contiguous and/or physically incorporated into the plasma membrane. Together, these data demonstrate that the temperature-dependent decrease in the rate of GLUT4 and IRAP translocation results from a reduction in GLUT4/IRAP-containing vesicle fusion and not trafficking or docking to the plasma membrane.     

The t-SNAREs syntaxin4 and SNAP23 but not v-SNARE VAMP2 are indispensable to tether GLUT4 vesicles at the plasma membrane in adipocyte

SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.     

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 novel role for myosin II in insulin-stimulated glucose uptake in 3T3-L1 adipocytes

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles from an intracellular pool to the plasma membrane. The studies presented here show that inhibition of myosin II activity impairs GLUT4-mediated glucose uptake but not GLUT4 translocation to the plasma membrane. We also show that adipocytes express both myosin IIA and IIB isoforms, and that myosin IIA is recruited to the plasma membrane upon insulin stimulation. Taken together, the data presented here represent the first demonstration that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. Based on our findings, we hypothesize that myosin II is activated upon insulin stimulation and recruited to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. The identification of myosin II as a key component of GLUT4-mediated glucose uptake represents an important advance in our understanding of the mechanisms regulating glucose homeostasis.     

Insulin recruits GLUT4 from distinct compartments via distinct traffic pathways with differential microtubule dependence in rat adipocytes

In the present study, we investigated the physiological significance of the microtubules in the subcellular localization and trafficking of GLUT4 in rat primary adipocytes. Morphological and biochemical analyses revealed a dose- and time-dependent disruption of the microtubules by treatment with nocodazole. With nearly complete disruption of the microtubules, the insulin-stimulated glucose transport activity was inhibited by 55%. This inhibition was concomitant with a comparable inhibition of GLUT4 translocation measured by the subcellular fractionation and the cell-surface GLUT4 labeling by trypsin cleavage. In addition, the time-course of insulin stimulation of the glucose transport activity was significantly delayed by microtubule disruption (t(1/2) were 7 and 2.3 min in nocodazole-treated and control cells, respectively), while the rate of GLUT4 endocytosis was little affected. The impaired insulin-stimulated glucose transport activity was not fully restored to the level in control cells by blocking GLUT4 endocytosis, suggesting that the inhibition was due to the existence of a microtubule-dependent subpopulation in the insulin-responsive GLUT4 pool. On the other hand, nocodazole partially inhibited insulin-induced translocation of the insulin-regulated aminopeptidase and the vesicle-associated membrane protein (VAMP)-2 without affecting GLUT1 and VAMP-3. In electrically permeabilized adipocytes, the insulin-stimulated glucose transport was inhibited by 40% by disruption of the microtubules whereas that stimulated with GTP gamma S was not affected. Intriguingly, the two reagents stimulated glucose transport to the comparable level by disruption of the microtubules. These data suggest that insulin recruits GLUT4 to the plasma membrane from at least two distinct intracellular compartments via distinct traffic routes with differential microtubule dependence in rat primary adipocytes.     

Snapin interacts with the Exo70 subunit of the exocyst and modulates GLUT4 trafficking

The exocyst is a multisubunit complex that has been implicated in the transport of vesicles from the Golgi complex to the plasma membrane, possibly acting as a vesicle tether and contributing to the specificity of membrane fusion. Here we characterize a novel interaction between the Exo70 subunit of the exocyst and Snapin, a ubiquitous protein known to associate with at least two t-SNAREs, SNAP23 and SNAP25. The interaction between Exo70 and Snapin is mediated via an N-terminal coil-coil domain in Exo70 and a C-terminal helical region in Snapin. Exo70 competes with SNAP23 for Snapin binding, suggesting that Snapin does not provide a direct link between the exocyst and the SNARE complex but, rather, mediates cross-talk between the two complexes by sequential interactions. The insulin-regulated trafficking of GLUT4 to the plasma membrane serves to facilitate glucose uptake in adipocytes, and both SNAP23 and the exocyst have been implicated in this process. In this study, depletion of Snapin in adipocytes using RNA interference inhibits insulin-stimulated glucose uptake. Thus, Snapin interacts with the exocyst and plays a modulatory role in GLUT4 vesicle trafficking.     

Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells

Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.     

Identification of a Role for CLASP2 in Insulin Action

Insulin stimulates the mobilization of glucose transporter 4 (GLUT4) storage vesicles to the plasma membrane, resulting in an influx of glucose into target tissues such as muscle and fat. We present evidence that CLIP-associating protein 2 (CLASP2), a protein previously unassociated with insulin action, is responsive to insulin stimulation. Using mass spectrometry-based protein identification combined with phosphoantibody immunoprecipitation in L6 myotubes, we detected a 4.8-fold increase of CLASP2 in the anti-phosphoserine immunoprecipitates upon insulin stimulation. Western blotting of CLASP2 immunoprecipitates with the phosphoantibody confirmed the finding that CLASP2 undergoes insulin-stimulated phosphorylation, and a number of novel phosphorylation sites were identified. Confocal imaging of L6 myotubes revealed that CLASP2 colocalizes with GLUT4 at the plasma membrane within areas of insulin-mediated cortical actin remodeling. CLASP2 is responsible for directing the distal end of microtubules to the cell cortex, and it has been shown that GLUT4 travels along microtubule tracks. In support of the concept that CLASP2 plays a role in the trafficking of GLUT4 at the cell periphery, CLASP2 knockdown by siRNA in L6 myotubes interfered with insulin-stimulated GLUT4 localization to the plasma membrane. Furthermore, siRNA mediated knockdown of CLASP2 in 3T3-L1 adipocytes inhibited insulin-stimulated glucose transport. We therefore propose a new model for CLASP2 in insulin action, where CLASP2 directs the delivery of GLUT4 to cell cortex landing zones important for insulin action.