Lipid Raft targeting of the TC10 amino terminal domain is responsible for disruption of adipocyte cortical actin

Overexpression of the Rho family member TC10alpha, disrupts adipocyte cortical actin structure and inhibits insulin-stimulated GLUT4 translocation when targeted to lipid raft microdomains. This appears to be independent of effecter domain function because overexpression of the wild-type (TC10/WT), constitutively GTP-bound (TC10/Q75L), and constitutively GDP bound (TC10/T31N) all inhibit adipocyte cortical actin structure and GLUT4 translocation. To examine the structural determinants responsible for these effects, we generated a series of chimera proteins between TC10 with that of H-Ras and K-Ras. Chimera containing the 79 (TC10-79/H-Ras), 41 (TC10-41/H-Ras), or 16 (TC10-16/H-Ras) amino acids of the TC10 amino terminal extension fused to H-Ras disrupted cortical actin and inhibited insulin-stimulated GLUT4 translocation. In contrast, the same amino terminal TC10 extensions fused to K-Ras had no significant effect on either GLUT4 translocation or cortical actin structure. Similarly, expression of TC10beta was without effect, whereas fusion of the amino terminal 8 amino acid of TC10alpha onto TC10beta resulted in an inhibition of insulin-stimulated GLUT4 translocation. Within the amino terminal extension point mutation analysis demonstrated that both a GAG and GPG sequences when lipid raft targeted was essential for these effects. Furthermore, expression of the amino terminal TC10 deletions DeltaNT-TC10/WT or DeltaNT-TC10/T31N had no detectable effect on cortical actin organization and did not perturb insulin-stimulated GLUT4 translocation. Surprisingly, however, expression of DeltaNT-TC10/Q75L remained fully capable of inhibiting insulin-stimulated GLUT4 translocation without affecting cortical actin. These data demonstrate that inhibitory effect of TC10 overexpression on adipocyte cortical actin organization is due to the specific lipid raft targeting of the unusual TC10 amino terminal extension.     

Insulin-stimulated GLUT4 translocation in adipocytes is dependent upon cortical actin remodeling

Rhodamine-labeled phalloidin staining of morphologically differentiated 3T3L1 adipocytes demonstrated that F-actin predominantly exists juxtaposed to and lining the inner face of the plasma membrane (cortical actin) with a smaller amount of stress fiber and/or ruffling actin confined to the cell bottom in contact with the substratum. The extent of cortical actin disruption with various doses of either latrunculin B or Clostridium difficile toxin B (a Rho family small GTP-binding protein toxin) directly correlated with the inhibition of insulin-stimulated glucose uptake and GLUT4 translocation. The dissolution of the cortical actin network had no significant effect on proximal insulin receptor signaling events including insulin receptor autophosphorylation, tyrosine phosphorylation of insulin receptor substrate and Cbl, or serine/threonine phosphorylation of Akt. Surprisingly, however, stabilization of F-actin with jasplakinolide also resulted in a dose-dependent inhibition of insulin-stimulated glucose uptake and GLUT4 translocation. In vivo time-lapse confocal fluorescent microscopy of actin-yellow fluorescent protein demonstrated that insulin stimulation initially results in cortical actin remodeling followed by an increase in polymerized actin in the peri-nuclear region. Importantly, the insulin stimulation of cortical actin rearrangements was completely blocked by treatment of the cells with latrunculin B, C. difficile toxin B, and jasplakinolide. Furthermore, expression of the dominant-interfering TC10/T31N mutant completely disrupted cortical actin and prevents any insulin-stimulated actin remodeling. Together, these data demonstrate that cortical actin, but not stress fibers, lamellipodia, or filopodia, plays an important regulatory role in insulin-stimulated GLUT4 translocation. In addition, cortical F-actin does not function in a static manner (e.g. barrier or scaffold), but insulin-stimulated dynamic cortical actin remodeling is necessary for the GLUT4 translocation process.     

Cyclin-dependent kinase-5 is a key molecule in tumor necrosis factor-α-induced insulin resistance

The mechanism of TNF-α-induced insulin resistance has remained unresolved with evidence for down-regulation of insulin effector targets effects or blockade of proximal as well as distal insulin signaling events depending upon the dose, time, and cell type examined. To address this issue we examined the acute actions of TNF-α in differentiated 3T3L1 adipocytes. Acute (5-15 min) treatment with 20 ng/ml (~0.8 nm) TNF-α had no significant effect on IRS1-associated phosphatidylinositol 3-kinase. In contrast, TNF-α increased insulin-stimulated cyclin-dependent kinase-5 (CDK5) phosphorylation on tyrosine residue 15 through an Erk-dependent pathway and up-regulated the expression of the CDK5 regulator protein p35. In parallel, TNF-α stimulation also resulted in the phosphorylation and GTP loading of the Rho family GTP-binding protein, TC10α. TNF-α enhanced the depolymerization of cortical F-actin and inhibited insulin-stimulated glucose transporter-4 (GLUT4) translocation. Treatment with the MEK inhibitor, PD98059, blocked the TNF-α-induced increase in CDK5 phosphorylation and the depolymerization of cortical F-actin. Conversely, siRNA-mediated knockdown of CDK5 or treatment with the MEK inhibitor restored the impaired insulin-stimulated GLUT4 translocation induced by TNF-α. Furthermore, siRNA-mediated knockdown of p44/42 Erk also rescued the TNF-α inhibition of insulin-stimulated GLUT4 translocation. Together, these data demonstrate that TNF-α-mediated insulin resistance of glucose uptake can occur through a MEK/Erk-dependent activation of CDK5.     

Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.     

A phosphatidylinositol 3-kinase-independent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling.

Recruitment of intracellular glucose transporter 4 (GLUT4) to the plasma membrane of fat and muscle cells in response to insulin requires phosphatidylinositol (PI) 3-kinase as well as a proposed PI 3-kinase-independent pathway leading to activation of the small GTPase TC10. Here we show that in cultured adipocytes insulin causes acute cortical localization of the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) and actin-related protein-3 (Arp3) as well as cortical F-actin polymerization by a mechanism that is insensitive to the PI 3-kinase inhibitor wortmannin. Expression of the dominant inhibitory N-WASP-DeltaWA protein lacking the Arp and actin binding regions attenuates the cortical F-actin rearrangements by insulin in these cells. Remarkably, the N-WASP-DeltaWA protein also inhibits insulin action on GLUT4 translocation, indicating dependence of GLUT4 recycling on N-WASP-directed cortical F-actin assembly. TC10 exhibits sequence similarity to Cdc42 and has been reported to bind N-WASP. We show the inhibitory TC10 (T31N) mutant, which abrogates insulin-stimulated GLUT4 translocation and glucose transport, also inhibits both cortical localization of N-WASP and F-actin formation in response to insulin. These findings reveal that N-WASP likely functions downstream of TC10 in a PI 3-kinase-independent insulin signaling pathway to mobilize cortical F-actin, which in turn promotes GLUT4 responsiveness to insulin.     

Cortactin, an actin binding protein, regulates GLUT4 translocation via actin filament remodeling

Insulin regulates glucose uptake into fat and skeletal muscle cells by modulating the translocation of GLUT4 between the cell surface and interior. We investigated a role for cortactin, a cortical actin binding protein, in the actin filament organization and translocation of GLUT4 in Chinese hamster ovary (CHO-GLUT4myc) and L6-GLUT4myc myotube cells. Overexpression of wild-type cortactin enhanced insulin-stimulated GLUT4myc translocation but did not alter actin fiber formation. Conversely, cortactin mutants lacking the Src homology 3 (SH3) domain inhibited insulin-stimulated formation of actin stress fibers and GLUT4 translocation similar to the actin depolymerizing agent cytochalasin D. Wortmannin, genistein, and a PP1 analog completely blocked insulin-induced Akt phosphorylation, formation of actin stress fibers, and GLUT4 translocation indicating the involvement of both PI3-K/Akt and the Src family of kinases. The effect of these inhibitors was even more pronounced in the presence of overexpressed cortactin suggesting that the same pathways are involved. Knockdown of cortactin by siRNA did not inhibit insulin-induced Akt phosphorylation but completely inhibited actin stress fiber formation and glucose uptake. These results suggest that the actin binding protein cortactin is required for actin stress fiber formation in muscle cells and that this process is absolutely required for translocation of GLUT4-containing vesicles to the plasma membrane.     

Small GTP-binding protein TC10 differentially regulates two distinct populations of filamentous actin in 3T3L1 adipocytes

TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (DeltaN-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominant-interfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/DeltaVCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization.     

Caveolin-associated filamentous actin (Cav-actin) defines a novel F-actin structure in adipocytes

Dynamic actin remodeling has been implicated in the translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Here we show that fully differentiated 3T3L1 adipocytes have unique cortical filamentous actin structure, designated Cav-actin (caveolae-associated F-actin). During 3T3L1 adipocyte differentiation, rhodamine-phalloidin staining demonstrated the formation of a cortical actin cytoskeleton that is composed of small dot-like F-actin spikes lining the inside of the plasma membrane. Double labeling with a caveolin antibody indicated that these F-actin spikes emanate from organized rosette-like clusters of caveolae/lipid raft microdomains. In contrast, there was no obvious relationship between F-actin and caveolin localization and/or organization in 3T3L1 preadipocytes (fibroblasts). Treatments of differentiated adipocytes with latrunculin B, Clostridium difficile toxin B or a dominant-interfering TC10 mutant (TC10/T31N) disrupted the Cav-actin structure without significantly affecting the organization of clustered caveolae. Similarly, disruption of the clustered caveolae with methyl-beta-cyclodextrin also dispersed the Cav-actin structure. These data demonstrate that this novel Cav-actin structure is organized through clustered caveolae but that the formation of caveolae-rosettes are not dependent upon F-actin.     

Decreased insulin-dependent glucose transport by chronic ethanol feeding is associated with dysregulation of the Cbl/TC10 pathway in rat adipocytes

Heavy alcohol consumption is an independent risk factor for type 2 diabetes. Although the exact mechanism by which alcohol contributes to the increased risk is unknown, impaired glucose disposal is a likely target. Insulin-stimulated glucose disposal in adipocytes is regulated by two separate and independent pathways, the PI3K pathway and the Cbl/TC10 pathway. Previous studies suggest that chronic ethanol feeding impairs insulin-stimulated glucose transport in adipocytes in a PI3K-independent manner. In search of potential targets of ethanol that would affect insulin-stimulated glucose transport, we investigated the effects of 4-wk ethanol feeding to male Wistar rats on the Cbl/TC10 pathway in isolated adipocytes. Chronic ethanol feeding inhibited insulin-stimulated cCbl phosphorylation compared with pair feeding. Insulin receptor and Akt/PKB phosphorylation were not affected by ethanol feeding. Chronic ethanol exposure also impaired cCbl and TC10 recruitment to a lipid raft fraction isolated from adipocytes by detergent extraction. Furthermore, chronic ethanol feeding increased the amount of activated TC10 and filamentous actin in adipocytes at baseline and abrogated the ability of insulin to further activate TC10 or polymerize actin. These results demonstrate that the impairment in insulin-stimulated glucose transport observed in adipocytes after chronic ethanol feeding to rats is associated with a disruption of insulin-mediated Cbl/TC10 signaling and actin polymerization.     

Subcellular compartmentalization and trafficking of the insulin-responsive glucose transporter, GLUT4.

Insulin increases glucose transport into cells of target tissues, primarily striated muscle and adipose. This is accomplished via the insulin-dependent translocation of the facilitative glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane. Insulin binds to the cell-surface insulin receptor and activates its intrinsic tyrosine kinase activity. The subsequent activation of phosphatidylinositol 3-kinase (PI 3-K) is well known to be necessary for the recruitment of GLUT4 to the cell surface. Both protein kinase B (PKB) and the atypical protein kinase C(lambda/zeta) (PKClambda/zeta) appear to function downstream of PI 3-K, but how these effectors influence GLUT4 translocation remains unknown. In addition, emerging evidence suggests that a second signaling cascade that functions independently of the PI 3-K pathway is also required for the insulin-dependent translocation of GLUT4. This second pathway involves the Rho-family GTP binding protein TC10, which functions within the specialized environment of lipid raft microdomains at the plasma membrane. Future work is necessary to identify the downstream effectors that link TC10, PKB, and PKClambda/zeta to GLUT4 translocation. Progress in this area will come from a better understanding of the compartmentalization of GLUT4 within the cell and of the mechanisms responsible for targeting the transporter to specialized insulin-responsive storage compartments. Furthermore, an understanding of how GLUT4 is retained within and released from these compartments will facilitate the identification of downstream signaling molecules that function proximal to the GLUT4 storage sites.     

The roles of Cbl-b and c-Cbl in insulin-stimulated glucose transport

Previous studies suggest that the stimulation of glucose transport by insulin involves the tyrosine phosphorylation of c-Cbl and the translocation of the c-Cbl/CAP complex to lipid raft subdomains of the plasma membrane. We now demonstrate that Cbl-b also undergoes tyrosine phosphorylation and membrane translocation in response to insulin in 3T3-L1 adipocytes. Ectopic expression of APS facilitated insulin-stimulated phosphorylation of tyrosines 665 and 709 in Cbl-b. The phosphorylation of APS produced by insulin drove the translocation of both c-Cbl and Cbl-b to the plasma membrane. Like c-Cbl, Cbl-b associates constitutively with CAP and interacts with Crk upon insulin stimulation. Cbl proteins formed homo- and heterodimers in vivo, which required the participation of a conserved leucine zipper domain. A Cbl mutant incapable of dimerization failed to interact with APS and to undergo tyrosine phosphorylation in response to insulin, indicating an essential role of Cbl dimerization in these processes. Thus, both c-Cbl and Cbl-b can initiate a phosphatidylinositol 3-kinase/protein kinase B-independent signaling pathway critical to insulin-stimulated GLUT4 translocation.     

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.     

Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation

Insulin stimulates the rapid translocation of intracellular glucose transporters of the GLUT4 isotype to the plasma membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this membrane-trafficking process have not been fully defined. Recently, we identified a 160-kDa protein in adipocytes, designated AS160, that is phosphorylated by the insulin-activated kinase Akt. This protein contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins required for membrane trafficking. In the present study we have identified six sites of in vivo phosphorylation on AS160. These sites lie in the motif characteristic of Akt phosphorylation, and insulin treatment increased phosphorylation at five of the sites. Expression of AS160 with two or more of these sites mutated to alanine markedly inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not occur when the GAP function in the phosphorylation site mutant was inactivated by a point mutation. These findings strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and that this phosphorylation signals translocation through inactivation of the Rab GAP function.     

Myosin 5a is an insulin-stimulated Akt2 (protein kinase Bbeta) substrate modulating GLUT4 vesicle translocation

Phosphatidylinositol 3-kinase activation of Akt signaling is critical to insulin-stimulated glucose transport and GLUT4 translocation. However, the downstream signaling events following Akt activation which mediate glucose transport stimulation remain relatively unknown. Here we identify an Akt consensus phosphorylation motif in the actin-based motor protein myosin 5a and show that insulin stimulation leads to phosphorylation of myosin 5a at serine 1650. This Akt-mediated phosphorylation event enhances the ability of myosin 5a to interact with the actin cytoskeleton. Small interfering RNA-induced inhibition of myosin 5a and expression of dominant-negative myosin 5a attenuate insulin-stimulated glucose transport and GLUT4 translocation. Furthermore, knockdown of Akt2 or expression of dominant-negative Akt (DN-Akt) abolished insulin-stimulated phosphorylation of myosin 5a, inhibited myosin 5a binding to actin, and blocked insulin-stimulated glucose transport. Taken together, these data indicate that myosin 5a is a newly identified direct substrate of Akt2 and, upon insulin stimulation, phosphorylated myosin 5a facilitates anterograde movement of GLUT4 vesicles along actin to the cell surface.     

Inhibition of GLUT4 translocation by Tbc1d1, a Rab GTPase-activating protein abundant in skeletal muscle, is partially relieved by AMP-activated protein kinase activation

Insulin increases glucose transport by stimulating the trafficking of intracellular GLUT4 to the cell surface, a process known as GLUT4 translocation. A key protein in signaling this process is AS160, a Rab GTPase-activating protein (GAP) whose activity appears to be suppressed by Akt phosphorylation. Tbc1d1 is a Rab GAP with a sequence highly similar to that of AS160 and with the same Rab specificity as that of AS160. The role of Tbc1d1 in regulating GLUT4 trafficking has been unclear. Our previous study showed that overexpressed Tbc1d1 inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, even though insulin caused phosphorylation on its single canonical Akt motif. In the present study, we show in 3T3-L1 adipocytes that Tbc1d1 is only 1/20 as abundant as AS160, that knockdown of Tbc1d1 has no effect on insulin-stimulated GLUT4 translocation, and that overexpressed Tbc1d1 also inhibits GLUT4 translocation elicited by activated Akt expression. These results indicate that endogenous Tbc1d1 does not participate in insulin-regulated GLUT4 translocation in adipocytes and suggest that the GAP activity of Tbc1d1 is not suppressed by Akt phosphorylation. In addition, we discovered that Tbc1d1 is much more highly expressed in skeletal muscle than fat and that the AMP-activated protein kinase (AMPK) activator 5'-aminoimidazole-4-carboxamide ribonucleoside partially reversed the inhibition of insulin-stimulated GLUT4 translocation by overexpressed Tbc1d1 in 3T3-L1 adipocytes. 5'-Aminoimidazole-4-carboxamide ribonucleoside activation of the kinase AMPK is known to cause GLUT4 translocation in muscle. The above findings strongly suggest that Tbc1d1 is a component in the signal transduction pathway leading to AMPK-stimulated GLUT4 translocation in muscle.     

Synip phosphorylation does not regulate insulin-stimulated GLUT4 translocation

Insulin causes the rapid translocation of the glucose transporter GLUT4 from intracellular sites to the plasma membrane in fat and muscle cells. There is considerable evidence that the signaling to this trafficking process is downstream of the insulin-activated protein kinase Akt. One Akt substrate that connects signaling to trafficking is a 160 kDa GTPase activating protein for Rabs. Another potential connecting substrate is the protein Synip, which associates with the SNARE syntaxin4. A recent study presents evidence that Akt phosphorylates Synip on serine 99, at least in vitro, and proposes that this phosphorylation enables GLUT4 translocation by causing the dissociation of Synip from syntaxin4. In the present study we show that marked overexpression of Synip mutant S99A, which lacks this phosphorylation site, has no effect on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. This finding is strong evidence that phosphorylation of Synip on serine 99 is not required for GLUT4 translocation.     

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.     

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.     

Insulin stimulates actin comet tails on intracellular GLUT4-containing compartments in differentiated 3T3L1 adipocytes

Incubation of isolated GLUT4-containing vesicles with Xenopus oocyte extracts resulted in a guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and sodium orthovanadate stimulation of actin comet tails. The in vitro actin-based GLUT4 vesicle motility was inhibited by both latrunculin B and a dominant-interfering N-WASP mutant, N-WASP/Delta VCA. Preparations of gently sheared (broken) 3T3L1 adipocytes also displayed GTP gamma S and sodium orthovanadate stimulation of actin comet tails on GLUT4 intracellular compartments. Furthermore, insulin pretreatment of intact adipocytes prior to gently shearing also resulted in a marked increase in actin polymerization and actin comet tailing on GLUT4 vesicles. In addition, the insulin stimulation of actin comet tails was completely inhibited by Clostridum difficile toxin B, demonstrating a specific role for a Rho family member small GTP-binding protein. Expression of N-WASP/Delta VCA in intact cells had little effect on adipocyte cortical actin but partially inhibited insulin-stimulated GLUT4 translocation. Taken together, these data demonstrate that insulin can induce GLUT4 vesicle actin comet tails that are necessary for the efficient translocation of GLUT4 from intracellular storage sites to the plasma membrane.