Bridging the GAP Between Insulin Signaling and GLUT4 Translocation

Review TRENDS in Biochemical Sciences Vol.31 No.4 April 2006

Bridging the GAP between insulin signaling and GLUT4 translocation

Robert T. Watson and Jeffrey E. Pessin

Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-8651, USA

Upon binding and activating its cell-surface receptor, in both developing and developed countries, has galva- insulin triggers signaling cascades that regulate many nized the research efforts of scientists worldwide [6]. cellular processes. Regarding glucose homeostasis, At the cellular level, understanding glucose uptake insulin suppresses hepatic glucose production and involves two largely separate fields of inquiry: (i) insulin- increases glucose transport into muscle and adipose receptor signaling and (ii) GLUT4 membrane trafficking. tissues. At the cellular level, glucose uptake results from Not surprisingly, identifying the molecules that link insulin the insulin-stimulated translocation of the glucose signaling to GLUT4 translocation has been a major focus of transporter 4 (GLUT4) from intracellular storage sites research [7]. In addition, insulin-signaling pathways can to the plasma membrane. Although the signaling also be functionally separated into two distinct branches, molecules that function proximal to the activated insulin one defined by the Rho-family GTPase TC10 [8] and the receptor have been well characterized, it is not known other by the phosphatidylinositol 3-kinase (PI3-kinase) (see how the distal insulin-signaling cascade interfaces with Ref. [9] for abbreviations of the inositol phosphates and their and mobilizes GLUT4-containing compartments. kinases) (Figure 2). Although the polyphosphoinositide Recently, several candidate signaling molecules, (PPIn) 3-kinase pathway is well established, the TC10 including AS160, PIKfyve and synip, have been identified branch remains controversial [10,11]. Here, we will that might provide functional links between the insulin therefore focus on the PPIn 3-kinase pathway. In recent signaling cascade and GLUT4 compartments. Future years, several molecules, including AS160, PIKfyve, and work will focus on delineating the precise GLUT4 synip, have extended the reach of the PPIn 3-kinase arm of trafficking steps regulated by these molecules. the insulin-signaling cascade, bringing us closer to understanding the cellular mechanisms for bridging the gap between insulin signaling and GLUT4 membrane trafficking. Introduction Elevated levels of circulating sugars and amino acids, as occur following a meal, signal pancreatic b cells to release Insulin signaling: overview and update insulin into the bloodstream. Insulin stimulates periph- When activated, most receptor tyrosine kinases directly eral tissues, primarily muscle and adipose, to absorb recruit downstream effector molecules to their phosphory- glucose from the vascular system. At the cellular level, lated cytoplasmic-tail domains [12]. By contrast, several glucose crosses the plasma membrane through aqueous cytosolic scaffold proteins serve as substrates for the pores formed by facilitative transporters of the GLUT activated insulin receptor, thereby greatly expanding the (glucose transporter) family [1,2]. One GLUT isoform in repertoire of potential downstream signaling opportu- particular, GLUT4, translocates from intracellular nities (Figure 2). With respect to glucose uptake, the four storage sites to the plasma membrane in response to members of the insulin-receptor-substrate family (IRS1, insulin (Figure 1). At the cell surface, GLUT4 facilitates IRS2, IRS3 and IRS4), in addition to Cbl and APS, the passive transport of glucose into muscle and fat cells. undergo tyrosine phosphorylation in response to insulin By contrast, during periods between meals, the liver stimulation [13]. Identified at the molecular level in 1991 normally provides sufficient glucose output to maintain [14], the IRS family has enjoyed considerable attention. circulating levels in the range of 4–7 mM in humans. Both IRS1 and IRS2 have key roles in insulin-stimulated Deviations from this range can have major health glucose uptake in fat and muscle, and the independent consequences. Low blood glucose levels, for example, can genetic ablation of either isoform leads to peripheral lead to seizures, coma and death. Far more common, insulin resistance [15]. However, only Irs2K/K mice show a however, are prolonged elevated glucose levels, as occurs diabetic phenotype, in part, because IRS2 also has in the diabetic state [3–5]. Frank diabetes (when fasting important roles in b-cell function [16]. Mechanistically, blood glucose levels are 126 mg/dl or higher) has many the insulin-dependent tyrosine phosphorylation of IRS associated health risks, including blindness, renal failure, proteins generates docking sites (Tyr–P-Xaa-Xaa-Met) for neuropathy and cardiovascular disease. Indeed, the many Src homology 2 (SH2)-domain-containing down- seemingly inexorable increase in cases of type-2 diabetes, stream effectors, notably the type-1A PPIn 3-kinase. The

Corresponding author: Pessin, J.E. ([email protected]). PPIn 3-kinase has been implicated in numerous biological responses, including mitogenesis, anti-apoptosis, protein www.sciencedirect.com 0968-0004/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2006.02.007 216 Review TRENDS in Biochemical Sciences Vol.31 No.4 April 2006

insulin-stimulated recruitment of GLUT4 to the cell (a) Myc surface [13]. Conversely, expression of a constitutively active p110 subunit, or introduction of a PtdIns(3,4,5)P3 analog into cells, induces GLUT4 translocation [20–24]. Lumen/extracellular The PPIn 3-kinase-catalyzed increase in PtdIns(3,4,5)P3 in the membrane is thought to form lipid-based platforms Cytoplasm for recruiting and concentrating downstream signaling NH2 molecules containing pleckstrin-homology (PH) domains EGFP [25]. PH domains are 100–120 amino acid modules that bind to PtdIns(3,4,5)P3 and other membrane phosphoinositides [26]. PtdIns(3,4,5)P3 promotes the cell-surface (b) EGFP Myc Merge localization of two PH-domain-containing enzymes (i) (ii) (iii) involved in GLUT4 translocation, the 3-phosphoinositide-dependent protein kinase-1 (PDK1) and Akt. The Basal atypical protein kinase C l and z isoforms (aPKCl/z) might also be regulated by PtdIns(3,4,5)P3, although not through PH-domain interactions [27]. In addition, sub- (iv) (v) (vi) stantial evidence supports a role for aPKCl/z in GLUT4 translocation, and the interested reader is referred to Refs Insulin [27,28] for recent authoritative reviews. Activation of Akt requires that it undergoes phosphorylation at two sites [29]. PDK1 phosphorylates Akt at Thr308, a residue located in its kinase-domain activation loop. In addition, Ti BS Ser473 in the C-terminal hydrophobic motif of Akt has Figure 1. The glucose transporter 4 (GLUT4) redistributes to the plasma membrane long been known to undergo phosphorylation but the in response to insulin. (a) Model of the predicted membrane topology of GLUT4. identity of the kinase responsible has been controversial. The N and C termini are cytoplasmic, and the first extracellular loop can accommodate engineered epitope tags, including Myc (shown) and hemagglutinin. Recently, however, an enzyme complex consisting of In non-permeabilized cells transfected with this type of GLUT4 reporter, fully fused mTOR (mammalian target of rapamycin) and RICTOR transporters can be detected by incubating cells with the appropriate antibody. In (rapamycin insensitive companion of mTOR) has been addition, the internalization and recycling of GLUT4 can also be investigated by incubating live cells with the appropriate antibody at 48C, then washing out the shown to phosphorylate Akt at Ser473 in response to antibody for various times at 378C and following its endocytosis with a fluorescent- insulin [30,31] (Figure 3). By contrast, the rapamycin- secondary antibody. The C terminus of GLUT4 can also be modified, for example, sensitive mTOR/RAPTOR (regulatory associated protein with enhanced green fluorescent protein (EGFP), to facilitate live-cell imaging and other applications requiring intrinsic fluorescence. (b) 3T3L1 adipocytes were of mTOR) complex is a downstream target of Akt [32], transiently transfected with myc–GLUT4–EGFP and treated with insulin. Cells were placing mTOR both upstream and downstream of Akt then fixed (without detergent) and labeled with an anti-myc antibody followed by function depending on its associated interacting partner. Texas Red secondary. In the basal state, GLUT4 is present in the perinuclear region and small vesicular structures dispersed in the cytoplasm, as revealed by the In addition to positive regulators of GLUT4 transloca- intrinsic EGFP fluorescence (i). Little myc labeling, other than background tion, negative regulators have also been studied intensely fluorescence, is detectable in the basal state (ii). The merged images are shown because they might provide opportunities for pharmaco- in (iii). In response to insulin, GLUT4 translocates and fuses with the cell surface, thereby exposing the myc-epitope to the exterior of the cell. A prominent ring of logical interventions. For example, the endogenous EGFP fluorescence (iv) and myc labeling (v) is readily visible at the plasma attenuation of PPIn 3-kinase signaling occurs through membrane. Merged images are shown in (vi). the dephosphorylation of PtdIns(3,4,5)P3, and two phos- synthesis and glycogen synthesis [17]. In addition, apart phoinositide phosphateses, SHIP2 (type-II SH2-domain- containing inositol 5-phosphatase) and PTEN (phospha- from the insulin receptor itself, the PPIn 3-kinase is tase and tensin homolog deleted on chromosome ten), have arguably the most-widely accepted signaling enzyme been implicated in negatively regulating insulin signaling necessary for insulin-stimulated glucose transport. Type- [33] (Figure 3). SHIP2 removes the 50 phosphate from 1A PPIn 3-kinases are heterodimers composed of a PtdIns(3,4,5)P3 to generate PtdIns(3,4)P2 [18]. Although regulatory p85 subunit and a catalytic p110 subunit. The K K the Ship2 / mice die shortly after birth and are severely p85 subunit contains SH2 domains that bind phosphotyr- hypoglycemic, heterozygous animals show improved osine residues in IRS and other proteins, thereby glucose tolerance and enhanced insulin sensitivity [34]. allosterically regulating the activity of the p110 catalytic This seems to be due, at least in part, to increased insulin- subunit [18]. PPIn 3-kinases catalyze phosphorylation of stimulated translocation of GLUT4 to the cell surface. 0 the 3 -OH moiety of membrane myo-inositol lipids, and the However, RNA interference (RNAi)-mediated depletion of type-1A isoforms preferentially phosphorylate SHIP2 in cultured 3T3L1 adipocytes fails to enhance phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] insulin responsiveness [35]. Although it could be argued to form phosphatidylinositol 3,4,5-trisphosphate that SHIP2 was only partially knocked down in these [PtdIns(3,4,5)P3] [19]. experiments, it is noteworthy that the PH domain of Akt The insulin-stimulated production of PtdIns(3,4,5)P3 is binds to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with compar- essential for GLUT4 translocation. Indeed, inhibition of able affinity, at least in vitro [36]. In addition, a new PPIn 3-kinase activity, which can be accomplished by a Ship2-knockout mouse, generated with a different target- variety of experimental manoeuvres, prevents the ing construct that eliminates the translation-initiating www.sciencedirect.com Review TRENDS in Biochemical Sciences Vol.31 No.4 April 2006 217

is not a key regulator of insulin-stimulated glucose uptake Insulin 0 GLUT4 in peripheral tissues. In contrast to SHIP2, PTEN is a 3 - receptor speciﬁc PtdIns(3,4,5)P3 phosphatase that generates PtdIns(4,5)P (Figure 3). Overexpression of PTEN pre- Caveolin PDK PtdIns(3,4,5)P3 2 vents the accumulation of PtdIns(3,4,5)P3 and also Flotillin inhibits insulin-stimulated GLUT4 translocation in APS TC10 PI3K IRS 3T3L1 adipocytes [38]. Moreover, the genetic ablation of CAP Cbl C3G Crk Akt aPKC PTEN in adipose tissue of mice has recently been shown to Actin enhance insulin sensitivity and resistance to pharmaco- logically induced diabetes [39]. In addition, Pten mutant mice show increased GLUT4 translocation to the cell surface under both basal and insulin-stimulated conditions. These results are consistent with the RNAi- GLUT4 vesicle mediated depletion of PTEN in cultured 3T3L1 adipocytes, which increases PtdIns(3,4,5)P3 levels, Ti BS enhances insulin-stimulated Akt phosphorylation and

Figure 2. Schematic of two independent insulin-signaling cascades that cooperate increases glucose uptake [35]. Thus, results from both to induce the redistribution of GLUT4 to the plasma membrane. The TC10 pathway intact animals and cell culture studies support a key role (left; yellow) is initiated when insulin phosphorylates Cbl, and seems to require APS for PTEN as a negative regulator of insulin signaling and (adapter protein with Pleckstrin homology and Src homology 2 domains). Cbl- associated protein (CAP) also associates with Cbl and is targeted to lipid-raft GLUT4 translocation. microdomains by interactions with the caveolar protein flotillin. Cbl then recruits Of the three Akt isoforms, Akt2 has been strongly the complex of CT10 regulator of kinase II (CrkII) and the guanylnucleotide linked to GLUT4 translocation. Indeed, knockout of exchange factor C3G, which regulates the lipid-raft-associated Rho GTP-binding protein TC10. In the PPIn 3-kinase pathway (right; cyan), insulin stimulation results Akt2 results in impaired glucose uptake in skeletal in the tyrosine phosphorylation of IRS (insulin receptor substrate) scaffold proteins; muscle and hepatic insulin resistance [40]. Similarly, this event, in turn, promotes the association and activation of the PPIn 3-kinase depletion of Akt2 by RNAi inhibits insulin responsive- (PI3K). The subsequent production of phosphatidylinositol 3,4,5-trisphosphate ness in 3T3L1 adipocytes [41]. Moreover, a mutation in [PtdIns(3,4,5)P3] results in the recruitment and activation of phosphoinositide- dependent kinase 1 (PDK1), which phosphorylates and activates both l and z Akt2 was recently identified in a human family with isoforms of atypical protein kinase C (aPKCl/z) and Akt. Both these signaling severe insulin resistance and diabetes [42]. Initially cascades are thought to converge in a concerted manner to mobilize GLUT4- storage compartments. implicated in GLUT4 translocation in 1996 [43], Akt has since emerged as a crucial transducer of the insulin- ATG, was found to be normal with respect to glucose signaling cascade leading to GLUT4 translocation and homeostasis and insulin sensitivity [37]. These mice were, glucose uptake. however, resistant to diet-induced obesity, which could have practical therapeutic value but suggests that SHIP2 Regulated membrane trafficking of GLUT4 In the basal state, GLUT4 cycles slowly to and from the cell surface, with the vast majority of the transporter I R Insulin located in intracellular compartments. Activation of the insulin receptor initiates signaling cascades that sub- PtdIns(4,5)P2 IRS PI3K stantially increase the exocytic rate of GLUT4 while, at

PtdIns(3,4)P3 the same time, attenuating endocytosis of the transpor- PtdIns(3,4,5)P 3 PTEN ter [13,44,45]. The net effect is to cause a dramatic SHIP2 redistribution of GLUT4 to the plasma membrane. Once PDK1 Akt mTOR/RICTOR glucose is cleared from the bloodstream, circulating Translocation insulin returns to basal levels and GLUT4 is inter- mTOR/RAPTOR nalized through clathrin-coated pits and recycled back aPKC AS160 AS160 to intracellular compartments. Substantial evidence ? GLUT4 p70S6 kinase indicates that GLUT4 resides in a specialized vesicle Rab- Rab- storage compartment that is highly responsive to GTP GDP insulin [44–46]. However, GLUT4 traffics through Protein synthesis several intracellular compartments during its normal Ti BS cycling to and from the cell surface, including endo- Figure 3. Schematic of the PPIn 3-kinase arm of the insulin-signaling cascade. somes and the trans-Golgi network [47–49]. Identifying Negative regulators of insulin signaling leading to GLUT4-vesicle translocation are and biochemically characterizing the specific compart- depicted as stop signs (red octagons); positive regulators are shown as green ment that is insulin-responsive has therefore proven boxes. PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a 0 3 -phosphoinositol phosphatase that converts PtdIns(3,4,5)P3 to PtdIns(4,5)P2. challenging. In addition, there could be multiple SHIP2 (type-II SH2-domain-containing inositol 5-phosphatase) converts insulin-responsive GLUT4-storage compartments that PtdIns(3,4,5)P3 to PtdIns(3,4,)P2. The mTOR/RICTOR complex participates, together might consist of tubulovesicular structures and/or small with PDK1 and PtdIns(3,4,5)P3, in activating the serine/threonine kinase Akt. mTOR, when complexed with RAPTOR, also functions downstream of Akt in the pathway transport vesicles [47,50–53]. leading to protein synthesis. Akt seems to phosphorylate and inactivate the Rab Despite these difficulties, the laboratories of Timothy GTPase-activating protein AS160. This permits the conversion of an as yet unidentified Rab protein to the active GTP-bound form, which is proposed to McGraw (http://www.med.cornell.edu) and David James regulate one or more steps of GLUT4 vesicle translocation to the cell surface. (http://www.garvan.org.au) have independently proposed www.sciencedirect.com 218 Review TRENDS in Biochemical Sciences Vol.31 No.4 April 2006 the presence of two GLUT4 recycling pathways in Candidate Akt substrates involved in GLUT4 adipocytes [54–56]. One pathway corresponds to that of translocation the transferrin receptor and other constitutively AS160 recycling proteins that are continuously trafficking Over the past several years, the Leinhard group (http:// between the plasma membrane and endosome compart- dms.dartmouth.edu) has used the phospho-Akt substrate ments. This pathway is probably present in the (PAS) antibody to immunoprecipitate several candidate majority of cell types and accounts for the observation molecules that might function as endogenous Akt sub- that GLUT4 compartments can be co-immuno- strates, including pp47, pp105, pp250 and AS160 [58,59]. precipitated with constitutively recycling proteins and It is interesting, as has been noted elsewhere [59], that that insulin induces a small (approximately twofold) none of the PAS-immunoreactive molecules identified to translocation of GLUT4 in fibroblasts and other non- date are among the many previously established Akt classical insulin responsive cells. However, early in substrates. This could simply reflect the accessibility or adipogenesis, another series of recycling compartments affinity of PAS epitopes among various proteins or the are formed that efficiently sequester the majority of abundance of target substrate molecules. Regardless, one GLUT4 within the cell [57]. The recycling of GLUT4 candidate substrate in particular, AS160, has quickly within this pathway is fast, whereas exit from this gained experimental support (Figure 3). AS160 contains pathway into the constitutively recycling pathway is six consensus Akt phosphorylation sites in addition to a slow in the basal state. Thus, GLUT4 is excluded from Rab GTPase-activating protein (GAP) domain [60]. The the cell surface by a mechanism of dynamic retention presence of a Rab GAP domain is intriguing because Rab within internal compartments [54]. According to this proteins regulate several steps of membrane transport, model, insulin promotes the entry of GLUT4 into the including vesicle budding, motility, tethering and fusion constitutively recycling pathway, which provides an [61–63] (Box 1). As a working hypothesis, AS160 could, in efficient pathway for GLUT4 to access the cell surface. principle, participate in translating the information However, despite marked advances in our understand- carried by the insulin-signaling cascade into molecular ing of GLUT4 translocation and the development of events associated with recruiting GLUT4 to the cell sophisticated trafficking models, a key question surface. Clearly, the intersection of insulin signaling remains: how does Akt communicate with GLUT4- with GLUT4 trafficking is a crucial step in the overall containing compartments? regulation of GLUT4 translocation and glucose uptake [7].

Box 1. Rab proteins

Rab proteins represent the largest branch of the Ras superfamily of membrane compartments and, in some cases, even to separate small GTP-binding proteins, and O60 members have been identified subdomains of a given compartment [53]. Rabs are targeted to [52]. Like other small GTPases, Rabs function as molecular switches by membranes by lipid modifications at the C terminus. Unlike other Ras- oscillating between a GDP-bound inactive form and a GTP-bound family members, however, most Rabs have two geranylgeranyl active form (Figure I). In the GTP-bound state, Rabs interact with groups. Although the targeting elements responsible for specific downstream effectors that regulate several key steps of membrane membrane-compartment localization remain unclear, the geranylger- trafficking, including transport-vesicle formation at donor mem- anyl groups, together with other sequence information, might have a branes, vesicle motility and vesicle docking or fusion with acceptor role [54]. Targeting individual Rabs to specific compartments is compartments. Initially described as Ras-like proteins in the brain, important because this will determine the membrane localization of many Rabs are ubiquitously expressed, although some show tissue recruited downstream effector molecules. Indeed, because they specificity. At the subcellular level, Rab proteins localize to distinct localize to specific membrane compartments and regulate key molecular events associated with vesicle trafficking, Rabs are thought to participate in maintaining the overall fidelity of membrane transport processes. GEF In addition to the GTPase cycle, Rab proteins are also subject to spatial regulation through reversible associations with their specific target membrane. Rab–GDP dissociation inhibitor (RabGDI) binds to Rab-GDP Rab-GTP and extracts Rab–GDP from membranes, thereby generating a cytosolic pool of inactive Rab–GDP. When complexed with RabGDI, the hydrophobic dual-geranylgeranyl moieties of the Rab protein GDI are shielded by a hydrophobic binding pocket within RabGDI. GAP Additional proteins (e.g. prenylated Rab acceptor 1) seem to be required to displace the RabGDI and promote the re-association of Effector molecules Rabs to their respective target membranes. Rab proteins and other small GTPases usually have low intrinsic GTP hydrolysis and GDP–GTP exchange activities. Thus, these activities are mainly Transport vesicle Docking/fusion of regulated by additional factors: the guanine nucleotide exchange formation transport vesicles factors (GEFs) and GTPase-activating proteins (GAPs). Rab GEFs Vesicle motility promote formation of Rab–GTP, whereas Rab GAPs promote formation of Rab–GDP [53]. By orchestrating the spatial and Ti BS temporal activities of Rab proteins, these complicated control Figure I. Rab proteins function as molecular switches that oscillate between a mechanisms are thought to ensure the precision of membrane GTP-loaded (active) form and a GDP-loaded (inactive) form. GTP loading trafficking processes [52]. AS160 contains a Rab GAP domain and, induces conformational changes that enable Rab proteins to interact with therefore, might inactivate a Rab protein as part of the mechanism downstream effectors. for controlling the trafficking dynamics of GLUT4. www.sciencedirect.com Review TRENDS in Biochemical Sciences Vol.31 No.4 April 2006 219

The identification of molecules that function specifically at [68,69], however the function of AS160 in contraction- this juncture is therefore of tremendous interest, but what induced glucose uptake is less clear. Contraction-induced experimental evidence suggests that AS160 connects glucose transport occurs by a distinct, PPIn 3-kinase- insulin signaling with the mobilization of GLUT4-contain- independent signaling mechanism that seems to involve ing compartments? AMP-activated kinase (AMPK) [69]. Indeed AICAR Of the six consensus Akt phosphorylation sites present (5-aminoimidazole-4-carboxamide ribonucleoside), a phar- in AS160, five are phosphorylated in response to insulin macological AMPK activator, stimulates glucose transport stimulation: Ser318, Ser570, Ser588, Thr642 and Thr571 in intact animals and in isolated muscle tissue in the [60]. In addition, when four of these five sites are mutated absence of insulin [69,71]. This is thought to occur through to alanine, the resulting construct (designated AS160–4P) an AMP-binding conformation change that induces AMPK behaves in a dominant-interfering manner and inhibits phosphorylation by upstream kinases, particularly LKB1 GLUT4 translocation when overexpressed in 3T3L1 (a serine/threonine kinase that phosphorylates the cata- adipocytes. These results are consistent with the hypoth- lytic subunit of AMPK) [69]. Recently, Bruss et al. [70] esis that insulin regulates endogenous AS160 by phos- found that AICAR treatment increases AS160 phosphoryl- phorylation, perhaps by altering its subcellular location or ation without increasing Akt phosphorylation, suggesting by inducing conformational changes that inactivate the that AS160 might be a convergence target for both GAP domain. A myristoylated form of Akt that is contraction and insulin-stimulated glucose uptake. In constitutively targeted to the cell surface and intracellular this study, however, stimulation of isolated epitrochlearis membranes induces GLUT4 translocation in the absence muscles by insulin or contraction enhanced both Akt and of insulin [43,64,65]. This provides a method to directly AS160 phosphorylation, and these effects were blocked by test whether AS160 functions downstream of Akt; wortmannin. Thus, although AS160 might function down- Zeigerer et al. [22] recently demonstrated that expression stream of Akt in skeletal muscle, its precise role – of AS160–4P blocks the ability of myristoylated Akt to particularly with regard to contraction induced GLUT4 induce GLUT4 translocation. In addition, RNAi has translocation – will require experimental clarification. recently been used to reduce the intracellular levels of Indeed, the recent finding that insulin-stimulated AS160 endogenous AS160 in 3T3L1 adipocytes [66,67]. Depletion phosphorylation is decreased in skeletal muscle biopsies of AS160 results in an approximate twofold increase in the taken from type-2 diabetic human subjects will no doubt basal cell-surface localization of a hemagglutinin (HA)– encourage additional research into the role of AS160 in GLUT4–enhanced green fluorescent protein (EGFP) contraction- and insulin-stimulated glucose uptake by reporter construct (Figure 1), and also increases basal muscle tissue [72]. glucose transport activity by approximately twofold. The results described support the hypothesis that Importantly, this effect is rescued by the re-expression of AS160 functions downstream of Akt, but its precise role human AS160. However, mutation of an essential arginine remains to be elucidated. Indeed, it is not yet known which residue to lysine that disables the GAP domain of the Rab isoforms are regulated by the GAP domain of AS160. AS160 rescue construct prevents the rescue of basal However, these results suggest that GLUT4 translocation GLUT4 retention in AS160-knockdown adipocytes. These results strongly indicate that a functional GAP might require an active, GTP-bound Rab. Muscle and domain within AS160 is necessary to maintain the adipose tissues express several Rab isoforms and, to date, intracellular localization of GLUT4 under basal con- Rabs 4, 5 and 11 have been implicated in GLUT4- ditions. However, if AS160 were necessary and sufficient trafficking processes [73]. In addition, Rabs 10, 11 and for intracellular GLUT4 sequestration, then the complete 14 were recently found in association with GLUT4- knockdown of AS160 would be predicted to cause GLUT4 containing membranes in a proteomics screen [67], but translocation to an extent comparable to that of insulin. In the functional significance of this observation has not been AS160-knockdown cells, however, insulin can still experimentally determined. Nevertheless, it seems that increase GLUT4 exocytosis by approximately fourfold enough data have accumulated that we can reasonably [66]. This could be due to incomplete knockdown of ask: how can AS160 regulate GLUT4 translocation? In the AS160 or, alternatively, there could also be AS160- basal state, the GAP activity of AS160 could maintain an independent mechanisms for maintaining the intracellu- as yet unidentified Rab protein in the inactive GDP-bound lar localization of GLUT4. state. Insulin stimulation could then result in the Following a meal, the majority of circulating glucose is phosphorylation of AS160 and the inhibition of its GAP absorbed by skeletal muscle tissue [2,68,69]. Therefore, if domain, thus enabling the conversion of the putative Rab AS160 is a crucial regulator of GLUT4 trafficking, it is protein to the active GTP-bound form. Alternatively, important to determine if it also functions during the insulin could alter the subcellular localization of AS160, insulin and/or contraction-stimulated translocation of for example, by causing its dissociation from GLUT4- GLUT4 in muscle. Using isolated rat epitrochlearis compartments, which would also enable conversion of the muscles, Bruss et al. [70] recently showed that insulin Rab protein to the active form [67]. Regardless of the treatment induces Akt and AS160 phosphorylation. As precise mechanism, once in the active GTP-bound state, expected based on earlier results in adipocytes, this effect the Rab protein could then participate in one or more steps is blocked by pre-exposure to the PPIn 3-kinase inhibitor of GLUT4 translocation. In this model, AS160 is essen- wortmannin. Like insulin, contraction stimulates GLUT4 tially functioning as a brake on GLUT4-vesicle exocytosis translocation and glucose uptake in skeletal muscle and insulin stimulation relieves the inhibitory effect of www.sciencedirect.com 220 Review TRENDS in Biochemical Sciences Vol.31 No.4 April 2006

AS160, thereby permitting GLUT4 translocation translocation of GLUT4 by interfering with the SNARE- (Figure 3). dependent fusion mechanism [83–86]. However, insulin Although many questions remain unanswered regard- has been observed to induce the dissociation of synip from ing the function of AS160, the identification of an insulin- syntaxin 4, thus enabling productive SNARE pairing stimulated Akt substrate with the potential to regulate between syntaxin 4 and VAMP2 [81]. But how does insulin one or more Rab-family proteins provides a tantalizing regulate the interactions between synip and syntaxin 4? link between insulin-signaling processes and GLUT4 Recent work has identified an unusual potential Akt2 membrane-trafficking events. phosphorylation site within synip (Arg-Xaa-Lys-Xaa-Arg- Ser97-Xaa-Ser99) [80]. Interestingly, Ser99 seems to be a PIKfyve specific substrate of Akt2, but not Akt1 or Akt3. Insulin Although many Akt substrates are known [29], identifying stimulation results in the phosphorylation of synip at the essential downstream molecule(s) necessary for Ser99, and this leads to the dissociation of the synip– glucose uptake has been challenging. Using antibodies syntaxin 4 complex. Moreover, mutation of Ser99 to directed against the PAS motif (Arg-Xaa-Arg-Xaa-Xaa- phenylalanine prevents the dissociation of synip from [Ser–P/Thr–P]), several candidate substrates have been syntaxin 4 and also inhibits GLUT4 translocation in a identified that might function downstream of Akt in the dominant interfering manner. Thus, the insulin-stimu- pathway leading to GLUT4 translocation. For example, lated phosphorylation of synip by Akt2 might provide a Berwick et al. [74] fractionated primary adipocytes from mechanism for insulin to regulate the docking or fusion of rat epididymal fat pads and performed western blot GLUT4 vesicles with the cell surface. Other results, analysis using the PAS antibody and identified the however, have suggested that phosphorylation of synip phosphoinositide 5-kinase PIKfyve (phosphoinositide at Ser99 is not necessary for GLUT4 translocation [87].In kinase for five position containing a Fyve finger) as a these latter experiments, Ser99 was changed to alanine, potential Akt substrate. The authors report that PIKfyve and the resulting mutant construct failed to interfere with is phosphorylated at Ser318 by insulin, an effect blocked insulin-stimulated GLUT4 translocation when overex- by wortmannin. Moreover, Berwick et al. [74] showed that pressed in 3T3L1 adipocytes. Clearly, future work is overexpression of a mutant in which Ser318 was changed warranted to resolve these apparent discrepancies. to alanine [PIKfyve(S318A)] enhanced insulin-stimulated However, although the role of synip in GLUT4 transloca- translocation of GLUT4-containing compartments in tion remains controversial, several lines of evidence 3T3L1 adipocytes. indicate that insulin regulates the docking and/or fusion PIKfyve was originally identified in a screen for of GLUT4-containing compartments with the plasma transcripts enriched in fat and muscle [75,76]. In addition, membrane [53,88]. expression of a kinase-dead mutant (PIKfyveK1831E) was previously shown to inhibit insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes [77]. PIKfyve has also Concluding remarks recently been implicated in insulin-stimulated F-actin Although much progress has been made in the intervening rearrangements [78], although the relationship between actin dynamics and GLUT4 translocation is complicated decades since the discovery of insulin in 1922, it remains [10,13,75,79]. Clearly, future work is necessary to delin- unknown how the insulin-signaling cascade interfaces eate the precise steps of GLUT4 translocation that might with GLUT4-storage compartments. It is now widely require PIKfyve activity. accepted that activation of the insulin receptor tyrosine kinase leads to the translocation of the facilitative GLUT4 Synip to the plasma membrane in adipose and striated muscle Another recently identified Akt substrate involved in tissue, and that this effect contributes to the postprandial GLUT4 translocation is the SNARE (soluble N-ethylma- maintenance of the normal glycemic state. Molecularly, leimide sensitive factor-attachment receptor)-associated GLUT4 translocation occurs through a signal-transduc- protein synip [80]. Synip was originally identified in a tion cascade that converts the initial tyrosine kinase yeast two-hybrid screen using the cytosolic domain of signal into a phosphoinositide intermediate that, in turn, syntaxin 4 as bait [81]. Synip has an N-terminal PDZ activates a serine/threonine kinase cascade. Current data domain, central EF and coiled-coiled domains, and a suggest that three potential targets – PIKfyve (another C-terminal WW motif. Syntaxin 4 is a t-SNARE localized phosphoinositide kinase), AS160 (a Rab GAP) and synip to the cell surface, whereas another SNARE molecule, (a SNARE interacting protein) – might link the serine/ VAMP2, is localized to GLUT4 vesicles [82]. Interactions threonine kinase Akt to GLUT4 translocation. Whether between syntaxin 4 and VAMP2, together with other one of these, a combination or additional unknown fusogenic molecules, are thought to drive the fusion of effectors are required remains to be determined. More- GLUT4 vesicle with the plasma membrane [13].Asa over, the exciting observation that muscle contraction working model, synip binds to syntaxin 4 in the basal state might converge at the level of AS160 to induce glucose and blocks the ability of VAMP2 to interact productively uptake in an insulin-independent manner provides a with syntaxin 4 [81]. This might prevent promiscuous possible route to develop novel therapies that could fusions between GLUT4 vesicles and the cell surface. potentially bypass the peripheral insulin-resistance Several other syntaxin-binding proteins including tomo- characteristic of type-2 diabetes, and, equally important, syn and Munc18c have also been reported to prevent the the insulin secretion defect in type-1 diabetes. www.sciencedirect.com Review TRENDS in Biochemical Sciences Vol.31 No.4 April 2006 221

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