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.     

Identification of Three Distinct Functional Sites of Insulin-mediated GLUT4 Trafficking in Adipocytes Using Quantitative Single Molecule Imaging

Insulin stimulation of glucose uptake is achieved by redistribution of insulin-responsive glucose transporters, GLUT4, from intracellular storage compartment(s) to the plasma membrane in adipocytes and muscle cells. Although GLUT4 translocation has been investigated using various approaches, GLUT4 trafficking properties within the cell are largely unknown. Our novel method allows direct analysis of intracellular GLUT4 dynamics at the single molecule level by using Quantum dot technology, quantitatively establishing the behavioral nature of GLUT4. Our data demonstrate the predominant mechanism for intracellular GLUT4 sequestration in the basal state to be “static retention” in fully differentiated 3T3L1 adipocytes. We also directly defined three distinct insulin-stimulated GLUT4 trafficking processes: 1) release from the putative GLUT4 anchoring system in storage compartment(s), 2) the speed at which transport GLUT4-containing vesicles move, and 3) the tethering/docking steps at the plasma membrane. Intriguingly, insulin-induced GLUT4 liberation from its static state appeared to be abolished by either pretreatment with an inhibitor of phosphatidylinositol 3-kinase or overexpression of a dominant-interfering AS160 mutant (AS160/T642A). In addition, our novel approach revealed the possibility that, in certain insulin-resistant states, derangements in GLUT4 behavior can impair insulin-responsive GLUT4 translocation.     

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.     

Akt activation is required at a late stage of insulin-induced GLUT4 translocation to the plasma membrane

Insulin stimulates the translocation of glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (PM). This involves multiple steps as well as multiple intracellular compartments. The Ser/Thr kinase Akt has been implicated in this process, but its precise role is ill defined. To begin to dissect the role of Akt in these different steps, we employed a low-temperature block. Upon incubation of 3T3-L1 adipocytes at 19 C, GLUT4 accumulated in small peripheral vesicles with a slight increase in PM labeling concomitant with reduced trans-Golgi network labeling. Although insulin-dependent translocation of GLUT4 to the PM was impaired at 19 C, we still observed movement of vesicles toward the surface. Strikingly, insulin-stimulated Akt activity, but not phosphatidylinositol 3 kinase activity, was blocked at 19 C. Consistent with a multistep process in GLUT4 trafficking, insulin-stimulated GLUT4 translocation could be primed by treating cells with insulin at 19 C, whereas this was not the case for Akt activation. These data implicate two insulin-regulated steps in GLUT4 translocation: 1) redistribution of GLUT4 vesicles toward the cell cortex-this process is Akt-independent and is not blocked at 19 C; and 2) docking and/or fusion of GLUT4 vesicles with the PM-this process may be the major Akt-dependent step in the insulin regulation of glucose transport.     

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.     

Molecular machinery involved in the insulin-regulated fusion of GLUT4-containing vesicles with the plasma membrane (review).

The GLUT4 facilitative glucose transporter protein is primarily expressed in muscle and adipose tissue and accounts for the majority of post-prandial glucose uptake. In the basal or non-stimulated state, GLUT4 is localized to intracellular membrane compartments sequestered away from circulating glucose. However, in response to agonist stimulation, there is a marked redistribution of the GLUT4 protein to the cell surface membrane providing a transport route for the uptake of glucose. This GLUT4 translocation can be divided into four general steps: (i) GLUT4 vesicle trafficking out of the storage pool, (ii) docking just below the cell surface, (iii) priming via the interactions of the SNARE proteins present on the vesicular and plasma membranes, and (iv) fusion of the GLUT4 vesicle with the plasma membrane. This review focuses on recent advances made in identification and characterization of the molecular events and protein interactions involved in these steps of insulin-stimulated GLUT4 translocation.     

Book reviews

The GLUT4 facilitative glucose transporter protein is primarily expressed in muscle and adipose tissue and accounts for the majority of post-prandial glucose uptake. In the basal or non-stimulated state, GLUT4 is localized to intracellular membrane compartments sequestered away from circulating glucose. However, in response to agonist stimulation, there is a marked redistribution of the GLUT4 protein to the cell surface membrane providing a transport route for the uptake of glucose. This GLUT4 translocation can be divided into four general steps: (i) GLUT4 vesicle trafficking outofthe storage pool, (ii) docking just below the cell surface, (iii) priming via the interactions of the SNARE proteins present on the vesicular and plasma membranes, and (iv) fusion of the GLUT4 vesicle with the plasma membrane. This review focuses on recent advances made in identification and characterization of the molecular events and protein interactions involved in these steps of insulin-stimulated GLUT4 translocation.     

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy- methyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA-AM. While the ionophores or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degrees C, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/calmodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.     

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.     

Insulin signaling diverges into Akt-dependent and -independent signals to regulate the recruitment/docking and the fusion of GLUT4 vesicles to the plasma membrane

Insulin modulates glucose disposal in muscle and adipose tissue by regulating the cellular redistribution of the GLUT4 glucose transporter. Protein kinase Akt/PKB is a central mediator of insulin-regulated translocation of GLUT4; however, the GLUT4 trafficking step(s) regulated by Akt is not known. Here, we use acute pharmacological Akt inhibition to show that Akt is required for insulin-stimulated exocytosis of GLUT4 to the plasma membrane. Our data also suggest that the AS160 Rab GAP is not the only Akt target required for insulin-stimulated GLUT4 translocation. Using a total internal reflection microscopy assay, we show that Akt activity is specifically required for an insulin-mediated prefusion step involving the recruitment and/or docking of GLUT4 vesicles to within 250 nm of the plasma membrane. Moreover, the insulin-stimulated fusion of GLUT4 vesicles with the plasma membrane can occur independently of Akt activity, although based on inhibition by wortmannin, it is dependent on phosphatidylinositol 3' kinase activity. Hence, to achieve full redistribution of GLUT4 into the plasma membrane, insulin signaling bifurcates to independently regulate both fusion and a prefusion step(s).     

Role of EHD1 and EHBP1 in perinuclear sorting and insulin-regulated GLUT4 recycling in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipose tissues by recruiting intracellular membrane vesicles containing the glucose transporter GLUT4 to the plasma membrane. The mechanisms involved in the biogenesis of these vesicles and their translocation to the cell surface are poorly understood. Here, we report that an Eps15 homology (EH) domain-containing protein, EHD1, controls the normal perinuclear localization of GLUT4-containing membranes and is required for insulin-stimulated recycling of these membranes in cultured adipocytes. EHD1 is a member of a family of four closely related proteins (EHD1, EHD2, EHD3, and EHD4), which also contain a P-loop near the N terminus and a central coiled-coil domain. Analysis of cultured adipocytes stained with anti-GLUT4, anti-EHD1, and anti-EHD2 antibodies revealed that EHD1, but not EHD2, partially co-localizes with perinuclear GLUT4. Expression of a dominant-negative construct of EHD1 missing the EH domain (DeltaEH-EHD1) markedly enlarged endosomes, dispersed perinuclear GLUT4-containing membranes throughout the cytoplasm, and inhibited GLUT4 translocation to the plasma membranes of 3T3-L1 adipocytes stimulated with insulin. Similarly, small interfering RNA-mediated depletion of endogenous EHD1 protein also markedly dispersed perinuclear GLUT4 in cultured adipocytes. Moreover, EHD1 is shown to interact through its EH domain with the protein EHBP1, which is also required for insulin-stimulated GLUT4 movements and hexose transport. In contrast, disruption of EHD2 function was without effect on GLUT4 localization or translocation to the plasma membrane. Taken together, these results show that EHD1 and EHBP1, but not EHD2, are required for perinuclear localization of GLUT4 and reveal that loss of EHBP1 disrupts insulin-regulated GLUT4 recycling in cultured adipocytes.     

Adrenergic receptor stimulation attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes by inhibiting GLUT4 translocation

Activation of the sympathetic nervous system inhibits insulin-stimulated glucose uptake. However, the underlying mechanisms are incompletely understood. Therefore, we studied the effects of catecholamines on insulin-stimulated glucose uptake and insulin-stimulated translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. We found that epinephrine (1 microM) nearly halved insulin-stimulated 2-deoxyglucose uptake. The beta-adrenoceptor antagonist propranolol (0.3 microM) completely antagonized the inhibitory effect of epinephrine on insulin-stimulated glucose uptake, whereas the alpha-adrenoceptor antagonist phentolamine (10 microM) had no effect. When norepinephrine was used instead of epinephrine, the results were identical. None of the individual selective beta-adrenoceptor antagonists (1 microM, beta(1): metoprolol, beta(2): ICI-118551, beta(3): SR-59230A) could counteract the inhibitory effect of epinephrine. Combination of ICI-118551 and SR-59230A, as well as combination of all three selective beta-adrenoceptor antagonists, abolished the effect of epinephrine on insulin-stimulated glucose uptake. After differential centrifugation, we measured the amount of GLUT1 and GLUT4 in the plasma membrane and in intracellular vesicles by means of Western blotting. Both epinephrine and norepinephrine reduced insulin-stimulated GLUT4 translocation to the plasma membrane. These results show that beta-adrenergic (but not alpha-adrenergic) stimulation inhibits insulin-induced glucose uptake in 3T3-L1 adipocytes, most likely via the beta(2)- and beta(3)-adrenoceptor by interfering with GLUT4 translocation from intracellular vesicles to the plasma membrane.     

C2 domain-containing phosphoprotein CDP138 regulates GLUT4 insertion into the plasma membrane

The protein kinase B(β) (Akt2) pathway is known to?mediate insulin-stimulated glucose transport through increasing glucose transporter GLUT4 translocation from intracellular stores to the plasma membrane (PM). Combining quantitative phosphoproteomics with RNAi-based functional analyses, we show that a previously uncharacterized 138?kDa C2 domain-containing phosphoprotein (CDP138) is a substrate for Akt2, and is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4 vesicles with the PM in live adipocytes. The purified C2 domain is capable of binding Ca(2+) and lipid membranes. CDP138 mutants lacking the Ca(2+)-binding sites in the C2 domain or Akt2 phosphorylation site S197 inhibit insulin-stimulated GLUT4 insertion into the PM, a rate-limiting step of GLUT4 translocation. Interestingly, CDP138 is dynamically associated with the PM and GLUT4-containing vesicles in response to insulin stimulation. Together, these results suggest that CDP138 is a key molecule linking the Akt2 pathway to the regulation of GLUT4 vesicle-PM fusion.     

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.     

A Highlights from MBoC Selection: Insulin-promoted mobilization of GLUT4 from a perinuclear storage site requires RAB10

Insulin controls glucose uptake into muscle and fat cells by inducing a net redistribution of glucose transporter 4 (GLUT4) from intracellular storage to the plasma membrane (PM). The TBC1D4-RAB10 signaling module is required for insulin-stimulated GLUT4 translocation to the PM, although where it intersects GLUT4 traffic was unknown. Here we demonstrate that TBC1D4-RAB10 functions to control GLUT4 mobilization from a trans-Golgi network (TGN) storage compartment, establishing that insulin, in addition to regulating the PM proximal effects of GLUT4-containing vesicles docking to and fusion with the PM, also directly regulates the behavior of GLUT4 deeper within the cell. We also show that GLUT4 is retained in an element/domain of the TGN from which newly synthesized lysosomal proteins are targeted to the late endosomes and the ATP7A copper transporter is translocated to the PM by elevated copper. Insulin does not mobilize ATP7A nor does copper mobilize GLUT4, and RAB10 is not required for copper-elicited ATP7A mobilization. Consequently, GLUT4 intracellular sequestration and mobilization by insulin is achieved, in part, through utilizing a region of the TGN devoted to specialized cargo transport in general rather than being specific for GLUT4. Our results define the GLUT4-containing region of the TGN as a sorting and storage site from which different cargo are mobilized by distinct signals through unique molecular machinery.     

Chromium activates glucose transporter 4 trafficking and enhances insulin-stimulated glucose transport in 3T3-L1 adipocytes via a cholesterol-dependent mechanism

Evidence suggests that chromium supplementation may alleviate symptoms associated with diabetes, such as high blood glucose and lipid abnormalities, yet a molecular mechanism remains unclear. Here, we report that trivalent chromium in the chloride (CrCl3) or picolinate (CrPic) salt forms mobilize the glucose transporter, GLUT4, to the plasma membrane in 3T3-L1 adipocytes. Concomitant with an increase in GLUT4 at the plasma membrane, insulin-stimulated glucose transport was enhanced by chromium treatment. In contrast, the chromium-mobilized pool of transporters was not active in the absence of insulin. Microscopic analysis of an exofacially Myc-tagged enhanced green fluorescent protein-GLUT4 construct revealed that the chromium-induced accumulation of GLUT4-containing vesicles occurred adjacent to the inner cell surface membrane. With insulin these transporters physically incorporated into the plasma membrane. Regulation of GLUT4 translocation by chromium did not involve known insulin signaling proteins such as the insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, and Akt. Consistent with a reported effect of chromium on increasing membrane fluidity, we found that chromium treatment decreased plasma membrane cholesterol. Interestingly, cholesterol add-back to the plasma membrane prevented the beneficial effect of chromium on both GLUT4 mobilization and insulin-stimulated glucose transport. Furthermore, chromium action was absent in methyl-beta-cyclodextrin-pretreated cells already displaying reduced plasma membrane cholesterol and increased GLUT4 translocation. Together, these data reveal a novel mechanism by which chromium may enhance GLUT4 trafficking and insulin-stimulated glucose transport. Moreover, these findings at the level of the cell are consistent with in vivo observations of improved glucose tolerance and decreased circulating cholesterol levels after chromium supplementation.     

TCGAP,a multidomain Rho GTPase-activating protein involved in insulin-stimulated glucose transport

Insulin stimulates glucose uptake in fat and muscle cells via the translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the cell surface. The signaling pathways linking the insulin receptor to GLUT4 translocation in adipocytes involve activation of the Rho family GTPases TC10alpha and beta. We report here the identification of TCGAP, a potential effector for Rho family GTPases. TCGAP consists of N-terminal PX and SH3 domains, a central Rho GAP domain and multiple proline-rich regions in the C-terminus. TCGAP specifically interacts with cdc42 and TC10beta through its GAP domain. Although it has GAP activity in vitro, TCGAP is not active as a GAP in intact cells. TCGAP translocates to the plasma membrane in response to insulin in adipocytes. The N-terminal PX domain interacts specifically with phos phatidylinositol-(4,5)-bisphosphate. Overexpression of the full-length and C-terminal fragments of TCGAP inhibits insulin-stimulated glucose uptake and GLUT4 translocation. Thus, TCGAP may act as a downstream effector of TC10 in the regulation of insulin-stimulated glucose transport.     

MEK inhibitors impair insulin-stimulated glucose uptake in 3T3-L1 adipocytes

In 3T3-L1 adipocytes, insulin activates three major signaling cascades, the phosphoinositide 3-kinase (PI3K) pathway, the Cbl pathway, and the mitogen-activated protein kinase (MAPK) pathway. Although PI3K and Cbl mediate insulin-stimulated glucose uptake by promoting the translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane, the MAPK pathway does not have an established role in insulin-stimulated glucose uptake. We demonstrate in this report that PI3K inhibitors also inhibit the MAPK pathway. To investigate the role of the MAPK pathway separately from that of the PI3K pathway in insulin-stimulated glucose uptake, we used two specific inhibitors of MAPK kinase (MEK) activity, PD-98059 and U-0126, which reduced insulin-stimulated glucose uptake by approximately 33 and 50%, respectively. Neither MEK inhibitor affected the activation of Akt or PKCzeta/lambda, downstream signaling molecules in the PI3K pathway. Inhibition of MEK with U-0126 did not prevent GLUT4 from translocating to the plasma membrane, nor did it inhibit the subsequent docking and fusion of GLUT4-myc with the plasma membrane. MEK inhibitors affected glucose transport mediated by GLUT4 but not GLUT1. Importantly, the presence of MEK inhibitors only at the time of the transport assay markedly impaired both insulin-stimulated glucose uptake and MAPK signaling. Conversely, removal of MEK inhibitors before the transport assay restored glucose uptake and MAPK signaling. Collectively, our studies suggest a possible role for MEK in the activation of GLUT4.     

Insulin-stimulated glucose uptake involves the transition of glucose transporters to a caveolae-rich fraction within the plasma membrane: implications for type II diabetes.

BACKGROUND: Adipose and muscle tissues express an insulin-sensitive glucose transporter (GLUT4). This transporter has been shown to translocate from intracellular stores to the plasma membrane following insulin stimulation. The molecular mechanisms signalling this event and the details of the translocation pathway remain unknown. In type II diabetes, the cellular transport of glucose in response to insulin is impaired, partly explaining why blood-glucose levels in patients are not lowered by insulin as in normal individuals. MATERIALS AND METHODS: Isolated rat epididymal adipocytes were stimulated with insulin and subjected to subcellular fractionation and to measurement of glucose uptake. A caveolae-rich fraction was isolated from the plasma membranes after detergent solubilization and ultracentrifugal floatation in a sucrose gradient. Presence of GLUT4 and caveolin was determined by immunoblotting after SDS-PAGE. RESULTS: In freshly isolated adipocytes, insulin induced a rapid translocation of GLUT4 to the plasma membrane fraction, which was followed by a slower transition of the transporter into a detergent resistant caveolae-rich region of the plasma membrane. The insulin-stimulated appearance of transporters in the caveolae-rich fraction occurred in parallel with enhanced glucose uptake by cells. Treatment with isoproterenol plus adenosine deaminase rapidly inhibited insulin-stimulated glucose transport by 40%, and at the same time GLUT4 disappeared from the caveolae-rich fraction and from plasma membranes as a whole. CONCLUSIONS: Insulin stimulates glucose uptake in adipocytes by rapidly translocating GLUT4 from intracellular stores to the plasma membrane. This is followed by a slower transition of GLUT4 to the caveolae-rich regions of the plasma membrane, where glucose transport appears to take place. These results have implications for an understanding of the defect in glucose transport involved in type II diabetes.     

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.