Rabs 8A and 14 are targets of the insulin-regulated Rab-GAP AS160 regulating GLUT4 traffic in muscle cells

Insulin causes translocation of glucose transporter GLUT4 to the membrane of muscle and fat cells, a process requiring Akt activation. The Rab GTPase-activating protein (Rab-GAP) AS160 is inhibited upon phosphorylation by insulin-activated Akt, thereby allowing GLUT4 translocation. Although several Rab proteins are detected on GLUT4 vesicles, the target Rabs of AS160 involved in the GLUT4 translocation have not been identified. We test whether Rabs 8A, 10, and 14 (in vitro targets of AS160) rescue the inhibition of GLUT4 translocation caused by 'constitutively active' 4P-AS160 in L6 muscle cells. Coexpression of GFP-tagged Rabs 8A or Rab14 with 4P-AS160 prevented the inhibition of GLUT4 translocation imposed by 4P-AS160. GFP-tagged, constitutively active Rab8A also elicited this rescue. In contrast, neither wild-type nor constitutively active GFP-tagged Rab10 restored GLUT4 translocation. These results suggest that Rab8A and possibly Rab14 may be targets of AS160 leading to GLUT4 translocation in L6 muscle cells.     

Muscle cells engage Rab8A and myosin Vb in insulin-dependent GLUT4 translocation

Insulin causes translocation of glucose transporter 4 (GLUT4) to the membrane of muscle and fat cells, a process requiring Akt activation. Two Rab-GTPase-activating proteins (Rab-GAP), AS160 and TBC1D1, were identified as Akt substrates. AS160 phosphorylation is required for insulin-stimulated GLUT4 translocation, but the participation of TBC1D1 on muscle cell GLUT4 is unknown. Moreover, there is controversy as to the AS160/TBC1D1 target Rabs in fat and muscle cells, and Rab effectors are unknown. Here we examined the effect of knockdown of AS160, TBC1D1, and Rabs 8A, 8B, 10, and 14 (in vitro substrates of AS160 and TBC1D1 Rab-GAP activities) on insulin-induced GLUT4 translocation in L6 muscle cells. Silencing AS160 or TBC1D1 increased surface GLUT4 in unstimulated cells but did not prevent insulin-induced GLUT4 translocation. Knockdown of Rab8A and Rab14, but not of Rab8B or Rab10, inhibited insulin-induced GLUT4 translocation. Furthermore, silencing Rab8A or Rab14 but not Rab8B or Rab10 restored the basal-state intracellular retention of GLUT4 impaired by AS160 or TBC1D1 knockdown. Lastly, overexpression of a fragment of myosin Vb, a recently identified Rab8A-interacting protein, inhibited insulin-induced GLUT4 translocation and altered the subcellular distribution of GTP-loaded Rab8A. These results support a model whereby AS160, Rab8A, and myosin Vb are required for insulin-induced GLUT4 translocation in muscle cells, potentially as part of a linear signaling cascade. glucose transporter 4; insulin signaling; Rab guanosine 5'-triphosphatases; Rab-guanosine 5'-triphosphatase-activating protein; myosin Vb     

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.     

Substrate specificity and effect on GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1

Insulin stimulation of the trafficking of the glucose transporter GLUT4 to the plasma membrane is controlled in part by the phosphorylation of the Rab GAP (GTPase-activating protein) AS160 (also known as Tbc1d4). Considerable evidence indicates that the phosphorylation of this protein by Akt (protein kinase B) leads to suppression of its GAP activity and results in the elevation of the GTP form of a critical Rab. The present study examines a similar Rab GAP, Tbc1d1, about which very little is known. We found that the Rab specificity of the Tbc1d1 GAP domain is identical with that of AS160. Ectopic expression of Tbc1d1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect. Insulin treatment led to the phosphorylation of Tbc1d1 on an Akt site that is conserved between Tbc1d1 and AS160. These results show that Tbc1d1 regulates GLUT4 translocation through its GAP activity, and is a likely Akt substrate. An allele of Tbc1d1 in which Arg(125) is replaced by tryptophan has very recently been implicated in susceptibility to obesity by genetic analysis. We found that this form of Tbc1d1 also inhibited GLUT4 translocation and that this effect also required a functional GAP domain.     

Insulin-stimulated Phosphorylation of the Rab GTPase-activating Protein TBC1D1 Regulates GLUT4 Translocation

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular locations to the plasma membrane in adipose and muscle cells. Prior studies have shown that Akt phosphorylation of the Rab GTPase-activating protein, AS160 (160-kDa Akt substrate; also known as TBC1D4), triggers GLUT4 translocation, most likely by suppressing its Rab GTPase-activating protein activity. However, the regulation of a very similar protein, TBC1D1 (TBC domain family, member 1), which is mainly found in muscle, in insulin-stimulated GLUT4 translocation has been unclear. In the present study, we have identified likely Akt sites of insulin-stimulated phosphorylation of TBC1D1 in C2C12 myotubes. We show that a mutant of TBC1D1, in which several Akt sites have been converted to alanine, is considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1. This result thus indicates that similar to AS160, Akt phosphorylation of TBC1D1 enables GLUT4 translocation. We also show that in addition to Akt activation, activation of the AMP-dependent protein kinase partially relieves the inhibition of GLUT4 translocation by TBC1D1. Finally, we show that the R125W variant of TBC1D1, which has been genetically associated with obesity, is equally inhibitory to insulin-stimulated GLUT4 translocation, as is wild-type TBC1D1, and that healthy and type 2 diabetic individuals express approximately the same level of TBC1D1 in biopsies of vastus lateralis muscle. In conclusion, phosphorylation of TBC1D1 is required for GLUT4 translocation. Thus, the regulation of TBC1D1 resembles that of its paralog, AS160.Insulin stimulates glucose transport into adipose and muscle cells by increasing the amount of the GLUT4 glucose transporter at the cell surface by a process termed GLUT4 translocation (1, 2). Unstimulated adipocytes and myotubes sequester GLUT4 in intracellular compartments. Insulin activates signaling cascades that lead to the trafficking of specialized GLUT4 vesicles to the cell membrane and fusion of the vesicles therewith. A key signaling pathway for GLUT4 translocation proceeds from the insulin receptor through the activation of the protein kinase Akt. One Akt substrate that connects signaling to GLUT4 trafficking is the Rab GTPase-activating protein (GAP)³ known as AS160. There is now considerable evidence for the following scheme (2, 3): under basal conditions, AS160 acts as a brake on GLUT4 translocation by maintaining one or more Rab proteins required for translocation in their inactive GDP state; in response to insulin, Akt phosphorylates AS160 and thereby suppresses its GAP activity; as a consequence, the elevation of the GTP form of the Rab proteins occurs, leading to the increased docking and subsequent fusion of the GLUT4 vesicles at the plasma membrane.More recently, we and others have characterized a paralog of AS160 known as TBC1D1 (4–7). Overall, TBC1D1 is 47% identical to AS160, with the GAP domain being 79% identical (4). Its GAP domain has the same Rab specificity as the GAP domain of AS160 (4). TBC1D1 is predominantly expressed in skeletal muscle; its expression in adipocytes is very low (5, 6). Nevertheless, 3T3-L1 adipocytes are a convenient cell type in which to examine the role of proteins in GLUT4 translocation, because insulin causes an ∼10-fold increase in GLUT4 at the cell surface. Previously, we examined the role of TBC1D1 in GLUT4 translocation by overexpressing it in 3T3-L1 adipocytes. Surprisingly, even though insulin led to phosphorylation of TBC1D1 on Akt site(s), ectopic TBC1D1 potently inhibited GLUT4 translocation (4, 5). By contrast, overexpression of AS160 did not inhibit GLUT4 translocation (8). This difference suggested that the regulation of TBC1D1 might be fundamentally different from that of AS160. In the present study, we show that this is not the case. By reducing the level of ectopic TBC1D1, we have obtained evidence that phosphorylation of TBC1D1 on several likely Akt sites relieves the inhibitory effect on GLUT4 translocation. In addition, we have examined the effect of a variant of TBC1D1 genetically associated with obesity on GLUT4 translocation and determined the relative levels of TBC1D1 in muscle biopsies from healthy and type 2 diabetic individuals.     

Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs

Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.     

Thrifty Tbc1d1 and Tbc1d4 proteins link signalling and membrane trafficking pathways

Establishing a complete pathway which links occupancy of the insulin receptor to GLUT4 translocation has been particularly elusive because of the complexities involved in studying both signalling and membrane trafficking processes. However, Lienhard's group has now discovered two related molecules that could function in this linking role. These proteins, Tbc1d4 (also known as AS160) and now Tbc1d1, as reported in this issue of the Biochemical Journal, have been demonstrated to be Rab GAPs (GTPase-activating proteins) that link upstream to Akt (protein kinase B) and phosphoinositide 3-kinase and downstream to Rabs involved in trafficking of GLUT4 vesicles. The data from Leinhard and colleagues suggest that high levels of Rab GAP activity lead to suppression of GLUT4 translocation and this observation has wide significance and is likely to be relevant to the recent discovery that mutations in the Tbc1d1 gene lead to some cases of severe human obesity.     

Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane

GLUT4 trafficking to the plasma membrane of muscle and fat cells is regulated by insulin. An important component of insulin-regulated GLUT4 distribution is the Akt substrate AS160 rab GTPase-activating protein. Here we show that Rab10 functions as a downstream target of AS160 in the insulin-signaling pathway that regulates GLUT4 translocation in adipocytes. Overexpression of a mutant of Rab10 defective for GTP hydrolysis increased GLUT4 on the surface of basal adipocytes. Rab10 knockdown resulted in an attenuation of insulin-induced GLUT4 redistribution to the plasma membrane and a concomitant 2-fold decrease in GLUT4 exocytosis rate. Re-expression of a wild-type Rab10 restored normal GLUT4 translocation. The basal increase in plasma-membrane GLUT4 due to AS160 knockdown was partially blocked by knocking down Rab10 in the same cells, further indicating that Rab10 is a target of AS160 and a positive regulator of GLUT4 trafficking to the cell surface upon insulin stimulation.     

TBC1D13 is a RAB35 Specific GAP that Plays an Important Role in GLUT4 Trafficking in Adipocytes

Insulin stimulates glucose transport in adipocytes by triggering translocation of GLUT4 glucose transporters to the plasma membrane (PM) and several Rabs including Rab10 have been implicated in this process. To delineate the molecular regulation of this pathway, we conducted a TBC/RabGAP overexpression screen in adipocytes. This identified TBC1D13 as a potent inhibitor of insulin-stimulated GLUT4 translocation without affecting other trafficking pathways. To determine the potential Rab substrate for TBC1D13 we conducted a yeast two-hybrid screen and found that the GTP bound forms of Rabs 1 and 10 specifically interacted with TBC1D13 but not with eight other TBC proteins. Surprisingly, a comprehensive in vitro screen for TBC1D13 GAP activity revealed Rab35 but not Rab10 as a specific substrate. TBC1D13 also displayed in vivo GAP activity towards Rab35. Overexpression of constitutively active Rab35 but not constitutively active Rab10 reversed the block in insulin-stimulated GLUT4 translocation observed with TBC1D13 overexpression. These studies implicate an important role for Rab35 in insulin-stimulated GLUT4 translocation in adipocytes.     

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.     

Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulin-regulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.     

Dissecting multiple steps of GLUT4 trafficking and identifying the sites of insulin action

Insulin-stimulated GLUT4 translocation is central to glucose homeostasis. Functional assays to distinguish individual steps in the GLUT4 translocation process are lacking, thus limiting progress toward elucidation of the underlying molecular mechanism. Here we have developed a robust method, which relies on dynamic tracking of single GLUT4 storage vesicles (GSVs) in real time, for dissecting and systematically analyzing the docking, priming, and fusion steps of GSVs with the cell surface in vivo. Using this method, we have shown that the preparation of GSVs for fusion competence after docking at the surface is a key step regulated by insulin, whereas the docking step is regulated by PI3K and its downstream effector, the Rab GAP AS160. These data show that Akt-dependent phosphorylation of AS160 is not the major regulated step in GLUT4 trafficking, implicating alternative Akt substrates or alternative signaling pathways downstream of GSV docking at the cell surface as the major regulatory node.     

The Rab GTPase-Activating Protein TBC1D4/AS160 Contains an Atypical Phosphotyrosine-Binding Domain That Interacts with Plasma Membrane Phospholipids To Facilitate GLUT4 Trafficking in Adipocytes

The Rab GTPase-activating protein TBC1D4/AS160 regulates GLUT4 trafficking in adipocytes. Nonphosphorylated AS160 binds to GLUT4 vesicles and inhibits GLUT4 translocation, and AS160 phosphorylation overcomes this inhibitory effect. In the present study we detected several new functional features of AS160. The second phosphotyrosine-binding domain in AS160 encodes a phospholipid-binding domain that facilitates plasma membrane (PM) targeting of AS160, and this function is conserved in other related RabGAP/Tre-2/Bub2/Cdc16 (TBC) proteins and an AS160 ortholog in Drosophila. This region also contains a nonoverlapping intracellular GLUT4-containing storage vesicle (GSV) cargo-binding site. The interaction of AS160 with GSVs and not with the PM confers the inhibitory effect of AS160 on insulin-dependent GLUT4 translocation. Constitutive targeting of AS160 to the PM increased the surface GLUT4 levels, and this was attributed to both enhanced AS160 phosphorylation and 14-3-3 binding and inhibition of AS160 GAP activity. We propose a model wherein AS160 acts as a regulatory switch in the docking and/or fusion of GSVs with the PM.     

Metformin induces Rab4 through AMPK and modulates GLUT4 translocation in skeletal muscle cells

Metformin is a major oral anti‐diabetic drug and is known as an insulin sensitizer. However, the mechanism by which metformin acts is unclear. In this study, we found that AICAR, an AMPK activator, and metformin increased the expression of Rab4 mRNA and protein levels in skeletal muscle C2C12 cells. The promoter activity of Rab4 was increased by metformin in an AMPK‐dependent manner. Metformin stimulated the phosphorylation of AS160, Akt substrate, and Rab GTPase activating protein (GAP), and also increased the phosphorylation of PKC‐zeta, which is a critical molecule for glucose uptake. Knockdown of AMPK blocked the metformin‐induced phosphorylation of AS160/PKC‐zeta. In addition, a colorimetric absorbance assay showed that insulin‐induced translocation of GLUT4 was suppressed in Rab4 knockdown cells. Moreover, Rab4 interacted with PKC‐zeta but not with GLUT4. The C‐terminal‐deleted Rab4 mutant, Rab4ΔCT, showed diffuse sub‐cellular localization, while wild‐type Rab4 localized exclusively to the perinuclear membrane. Unlike Rab4ΔCT, wild‐type Rab4 co‐localized with PKC‐zeta. Together, these results demonstrate that metformin induces Rab4 expression via AMPK‐AS160‐PKC‐zeta and modulates insulin‐mediated GLUT4 translocation. J. Cell. Physiol. 226: 974–981, 2011. © 2010 Wiley‐Liss, Inc.     

Insulin and AMPK regulate FA translocase/CD36 plasma membrane recruitment in cardiomyocytes via Rab GAP AS160 and Rab8a Rab GTPase

The FA translocase cluster of differentiation 36 (CD36) facilitates FA uptake by the myocardium, and its surface recruitment in cardiomyocytes is induced by insulin, AMP-dependent protein kinase (AMPK), or contraction. Dysfunction of CD36 trafficking contributes to disordered cardiac FA utilization and promotes progression to disease. The Akt substrate 160 (AS160) Rab GTPase-activating protein (GAP) is a key regulator of vesicular trafficking, and its activity is modulated via phosphorylation. Our study documents that AS160 mediates insulin or AMPK-stimulated surface translocation of CD36 in cardiomyocytes. Knock-down of AS160 redistributes CD36 to the surface and abrogates its translocation by insulin or the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). Conversely, overexpression of a phosphorylation-deficient AS160 mutant (AS160 4P) suppresses the stimulated membrane recruitment of CD36. The AS160 substrate Rab8a GTPase is shown via overexpression and knock-down studies to be specifically involved in insulin/AICAR-induced CD36 membrane recruitment. Our findings directly demonstrate AS160 regulation of CD36 trafficking. In myocytes, the AS160 pathway also mediates the effect of insulin, AMPK, or contraction on surface recruitment of the glucose transporter GLUT4. Thus, AS160 constitutes a point of convergence for coordinating physiological regulation of CD36 and GLUT4 membrane recruitment.     

Insulin-stimulated GLUT4 protein translocation in adipocytes requires the Rab10 guanine nucleotide exchange factor Dennd4C

Insulin-stimulated translocation of the glucose transporter GLUT4 to the cell surface in fat and muscle cells is the basis for insulin-stimulated glucose transport. Studies in adipocytes strongly support the following molecular mechanism for this process. Insulin-elicited phosphorylation of the GTPase-activating protein TBC1D4 (AS160) suppresses its activity toward Rab10 and thereby leads to an increase in the GTP-bound form of Rab10, which in turn triggers movement of vesicles containing GLUT4 to the plasma membrane and their fusion with the membrane. This process is expected to require the participation of a guanine nucleotide exchange factor (GEF) to generate the GTP-bound form of Rab10, but this GEF has not hitherto been identified. The present study identifies Dennd4C, a recently described GEF for Rab10, as the primary GEF required for GLUT4 translocation. Knockdown of Dennd4C markedly inhibited GLUT4 translocation, and ectopic expression of Dennd4C slightly stimulated it. Dennd4C was found in isolated GLUT4 vesicles. This study thus identifies another key component in the machinery of GLUT4 translocation. Moreover, it provides a potential explanation for the moderate association of a variant in the Dennd4C gene with type 2 diabetes.     

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).     

AS160 regulates insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle

Insulin and contraction are potent stimulators of GLUT4 translocation and increase skeletal muscle glucose uptake. We recently identified the Rab GTPase-activating protein (GAP) AS160 as a putative point of convergence linking distinct upstream signaling cascades induced by insulin and contraction in mouse skeletal muscle. Here, we studied the functional implications of these AS160 signaling events by using an in vivo electroporation technique to overexpress wild type and three AS160 mutants in mouse tibialis anterior muscles: 1) AS160 mutated to prevent phosphorylation on four regulatory phospho-Akt-substrate sites (4P); 2) AS160 mutated to abolish Rab GTPase activity (R/K); and 3) double mutant AS160 containing both 4P and R/K mutations (2M). One week following gene injection, protein expression for all AS160 isoforms was elevated over 7-fold. To determine the effects of AS160 on insulin- and contraction-stimulated glucose uptake in transfected muscles, we measured [3H]2-deoxyglucose uptake in vivo following intravenous glucose administration and in situ muscle contraction, respectively. Insulin-stimulated glucose uptake was significantly inhibited in muscles overexpressing 4P mutant AS160. However, this inhibition was completely prevented by concomitant disruption of AS160 Rab GAP activity. Transfection with 4P mutant AS160 also significantly impaired contraction-stimulated glucose uptake, as did overexpression of wild type AS160. In contrast, overexpressing mutant AS160 lacking Rab GAP activity resulted in increases in both sham and contraction-stimulated muscles. These data suggest that AS160 regulates both insulin- and contraction-stimulated glucose metabolism in mouse skeletal muscle in vivo and that the effects of mutant AS160 on the actions of insulin and contraction are not identical. Our findings directly implicate AS160 as a critical convergence factor for independent stimulators of skeletal muscle glucose uptake.     

GLUT4 vesicle recruitment and fusion are differentially regulated by Rac, AS160, and Rab8A in muscle cells

Insulin increases glucose uptake into muscle by enhancing the surface recycling of GLUT4 transporters. In myoblasts, insulin signals bifurcate downstream of phosphatidylinositol 3-kinase into separate Akt and Rac/actin arms. Akt-mediated Rab-GAP AS160 phosphorylation and Rac/actin are required for net insulin gain of GLUT4, but the specific steps (vesicle recruitment, docking or fusion) regulated by Rac, actin dynamics, and AS160 target Rab8A are unknown. In L6 myoblasts expressing GLUT4myc, blocking vesicle fusion by tetanus toxin cleavage of VAMP2 impeded GLUT4myc membrane insertion without diminishing its build-up at the cell periphery. Conversely, actin disruption by dominant negative Rac or Latrunculin B abolished insulin-induced surface and submembrane GLUT4myc accumulation. Expression of non-phosphorylatable AS160 (AS160-4P) abrogated membrane insertion of GLUT4myc and partially reduced its cortical build-up, an effect magnified by selective Rab8A knockdown. We propose that insulin-induced actin dynamics participates in GLUT4myc vesicle retention beneath the membrane, whereas AS160 phosphorylation is essential for GLUT4myc vesicle-membrane docking/fusion and also contributes to GLUT4myc cortical availability through Rab8A.     

Full intracellular retention of GLUT4 requires AS160 Rab GTPase activating protein

Insulin controls glucose flux into muscle and fat by regulating the trafficking of GLUT4 between the interior and surface of cells. Here, we show that the AS160 Rab GTPase activating protein (GAP) is a negative regulator of basal GLUT4 exocytosis. AS160 knockdown resulted in a partial redistribution of GLUT4 from intracellular compartments to the plasma membrane, a concomitant increase in basal glucose uptake, and a 3-fold increase in basal GLUT4 exocytosis. Reexpression of wild-type AS160 restored normal GLUT4 behavior to the knockdown adipocytes, whereas reexpression of a GAP domain mutant did not revert the phenotype, providing the first direct evidence that AS160 GAP activity is required for basal GLUT4 retention. AS160 is the first protein identified that is specially required for basal GLUT4 retention. Our findings that AS160 knockdown only partially releases basal GLUT4 retention provides evidence that insulin signals to GLUT4 exocytosis by both AS160-dependent and -independent mechanisms.