BCM_Sem_12_Traffic control of GLUT4.pdf

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Vesicle exocytosis has a crucial role in the delivery ofcellular material to the plasma membrane and extra-cellular space1. Whereas some Golgi-derived vesicles areconstantly delivered to the plasma membrane throughconstitutive exocytosis, other populations of vesicles thatare enriched in particular lipids and/or proteins undergoaccelerated exocytosis in response to extracellular stim-uli. This regulated exocytosis controls the release ofsoluble factors (for example, thrombin-stimulated secre-tion of von Willebrand factor from endothelial cells) 2,the integration of membrane proteins into the plasmamembrane (for example, insertion of aquaporin 2 intothe apical membrane of polarized kidney duct cells inresponse to Ca 2+)3 and the delivery of intracellular mem-branes for cell surface expansion (for example, insulin-like growth factor 1 (IGF1)-stimulated vesicle exocytosisduring neurite extension) 4. The controlled exocytosis ofthese specialized vesicles depends on the coordinated

regulation of the vesicle trafficking machinery by signal-ling pathways. Uncovering how signalling pathwayscommunicate with proteins that are involved in vesicleformation, movement, targeting and fusion is crucial forunderstanding a variety of physiological processes.

One well-studied example of regulated exocytosi s isthat of the hexose transporter GLUT4 (glucose trans-porter type 4), which mediates insulin-stimulatedglucose transport in fat and muscle 5. Following a meal,increased nutrients in the blood lead to secretion ofinsulin. This hormone in turn prevents gluconeogenesis in the liver and promotes glucose uptake into muscle andadipose tissue through regulated trafficking of GLUT4

from intracellular stores to the plasma membrane 6.Glucose uptake is the rate limiting step in glucose uti-lization and/ or storage and as such has a key role in themaintenanc e of glucose homeostasis (BOX 1) .

GLUT4 is one of 14 members of the GLUT family offacilitative transmembrane hexose transporters, eachof which has a distinct affinity and specificity for par-ticular hexoses, as well as unique tissue distribution,subcellular localization and physiological function 7.GLUT4 is a high-affinity glucose transporter that ispredominantly expressed in muscle cells and adipocytes.In the absence of insulin, the majority of GLUT4 isdistributed between endosomes, the trans -Golgi network (TGN) and heterogeneous tubulovesicular structuresthat consist of endosomal sorting intermediates and spe-cialized GLUT4 storage vesicles (GSVs). In the absenceof insulin, only ~5% of the total transporter pool isfound on the cell surface 8,9. Exclusion of GLUT4 from

the cell surface depends on efficient sorting and seques-tration into GSVs that do not readily cycle to the plasmamembrane in the absence of stimulation 10 but translocatethere in response to insulin or exercise, which resultsin a tenfold increase in glucose uptake 11. The failureof GLUT4 to translocate to the plasma membrane inresponse to insulin is an early step in the developmentof insulin resistance and type 2 diabetes mellitus 6.

About 90% of insulin-stimulated glucose uptakeoccurs in skeletal muscle 12. Although adipose tissueaccounts for only 10% of insulin-stimulated glucoseuptake, this process is important for controlling whole-body energy homeostasis, as adipocytes serve as a

1Life Sciences Institute,University of Michigan,210 Washtenaw Avenue,

Ann Arbor, Michigan 48105,USA.2Departments of Molecularand Integrative Physiologyand Internal Medicine,Life Sciences Institute,

Ann Arbor, Michigan 48105,USA.e-mails: [email protected] ; [email protected]:10.1038/nrm3351

GluconeogenesisDe novo synthesis of glucosefrom non-carbohydrate carbonsources.

Trans -Golgi network(TGN). The terminal Golgi stackwhere proteins are sorted andpackaged into vesicles fordelivery to their cellulardestination.

Insulin resistancePhysiological condition that isdefined by a failure of tissuesand organs to respond tonormal concentrationsof insulin.

Regulation of glucose transport byinsulin: traffic control of GLUT4Dara Leto 1 and Alan R. Saltiel 2

Abstract | Despite daily fasting and feeding, plasma glucose levels are normally maintainedwithin a narrow range owing to the hormones insulin and glucagon. Insulin increases glucoseuptake into fat and muscle cells through the regulated trafficking of vesicles that containglucose transporter type 4 (GLUT4). New insights into insulin signalling reveal thatphosphorylation events initiated by the insulin receptor regulate key GLUT4 traffickingproteins, including small GTPases, tethering complexes and the vesicle fusion machinery.These proteins, in turn, control GLUT4 movement through the endosomal system, formationand retention of specialized GLUT4 storage vesicles and targeted exocytosis of thesevesicles. Understanding these processes may help to explain the development of insulinresistance in type 2 diabetes and provide new potential therapeutic targets.

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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 13 | JUNE 2012 | 383

2012 Macmillan Publishers Limited. All rights reserved
mailto:[email protected]:[email protected]:[email protected]:[email protected]


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Brain

Liver

Adipokines,FFAs

Nutrients

Muscle

Glucose

Glycogen

Adipose tissue

Glucose

Triglycerides

Digestive system

Glucose

Insulin

Pancreas

cellular rheostat that senses the energy status, andresponds by secreting numerous hormones, such ascytokines and chemokines that regulate metabolism inmuscle, the liver and the brain 13,14. Thus, understandingglucose transport mechanisms in both skeletal muscleand adipocytes is crucial for elucidating the mechanismsthat underlie the physiology and pathophysiology ofenergy metabolism.

In this Review, we discuss the current understand-ing of how GLUT4 is compartmentalized, the roles ofspecific membrane trafficking proteins in maintainingthis compartmentalization and the emerging points ofintersection between insulin signalling and trafficking,with particular attention to the role of small GTPases .

Insulin signalling and GLUT4 trafficInsulin regulates energy metabolism by initiating severalsignalling cascades that control cell growth and survival,as well as protein, glycogen and lipid uptake, synthesisand hydrolysis 6. Some of these pathways, including theRAS extracellular signal-regulated kinase (ERK) and

mammalian target of rapamycin complex 1 (mTORC1)pathways, are not important for controlling glucosetransport 15,16. Instead, glucose transport in adipose tis-sue requires a phosphoinositide 3-kinase (PI3K) and anAPS (adaptor protein with pleckstrin homology (PH)and Src homology 2 (SH2) domains; also known asSH2B2) signalling cascade. Although the importance ofthe PI3K signalling pathway for glucose uptake in muscleis well established, the requirement for the APS signal-ling pathway remains untested in this tissue. Together,these pathway s ensure the efficient delivery of GLUT4 tothe cell surface by assembling signalling platforms at theplasma membrane that are comprised of lipids, proteinkinases, lipid kinases, small GTPases and adaptor pro-teins. These signalling pathways engage the traffickingmachinery to regulate GLUT4 cycling (FIG. 1).

The targets of insulin signalling. GLUT4 delivery tothe cell surface requires its mobilization from intra-cellular membrane compartments, recognition ofGLUT4-containing vesicles at the plasma membraneand finally fusion of these two membranes. Insulin sig-nalling coordinates these steps by engaging a series ofsmall GTPases that cycle between an active GTP-boundconformation, in which they mediate their biologicaleffects, and an inactive GDP-bound state. Insulin altersthe activation state of small GTPases by regulatingguanine nucleotid e exchange factors (GEFs) and GTPase-activating proteins (GAPs), both of which control thisnucleotide cycling. Active GTPases interact with multiplecomponents of the trafficking machinery to confer direc-tionality and specificity in membrane flow 17. In additionto small GTPases, insulin signalling directly targetsmotor proteins, membrane tethers and fusion-regulatin gproteins, which suggests that insulin acts at multiplesteps in the GLUT4 trafficking itinerary to increase theconcentratio n of the transporter on the surface of the cell.

The insulin signalling pathways acutely upregulateGLUT4 surface levels largely by increasing exocytosisof GSVs. However, there is evidence that insulin might

Box 1 | Muscle and adipose tissue in energy homeostasis

Maintenance of energy homeostasis during changing energy demands and availabilitydepends on the concerted action of multiple organs and tissues, including thedigestive system, pancreas, brain, liver, muscle and adipose tissue. Together, thesetissues sense energy and communicate fuel availability to other organs through therelease of metabolites and hormones (see the figure). Defects in the sensing ofthe energy status, and the ability to respond appropriately, result in metabolic

diseases such as type 2 diabetes mellitus.The anabolic hormone insulin is released from pancreatic -cells when dietary

carbohydrates or amino acids are abundant. Insulin stimulates the conversion ofsimple energy units such as monosaccharides (including glucose) and amino acids intocomplex macromolecules such as proteins, lipids and glycogen. This is accomplished,in part, by increasing glucose uptake in muscle and adipose tissue. The majority ofinsulin-stimulated glucose uptake occurs in skeletal muscle 12, where glucose is storedas glycogen, which is mobilized when fuel demands are high or glucose is notabundant. About 10% of insulin-stimulated glucose uptake occurs in adipose tissue,where energy is stored as triglycerides. Triglycerides are released from adipocytes asfree fatty acids (FFAs) and utilized as an energy source by other tissues when fuelavailability is low.

Adipose tissue serves an additional endocrine function by secreting adipokines andlipids 167 . These factors communicate the energy status to other tissues in the body,including the liver, muscle and brain, to alter fuel usage and feeding patterns.

Adipocyte-specific Glut4 (glucose transporter type 4; also known as Slc2a4 )-knockout(AG4KO) mice display several markers of insulin resistance, including an increase incirculating insulin levels, a failure of insulin to suppress hepatic glucose productionthrough gluconeogenesis and a reduction in insulin-stimulated glucose uptake inmuscle 14 , which demonstrates that glucose uptake supports the endocrine functionof adipose tissue. How adipocytes translate changes in glucose uptake or storage intosignals that affect global energy balance is not completely understood but is at leastpartially explained by the actions of adipokines such as leptin, adiponectin, resistinand retinol-binding protein 4 (RBP4) 167,168 . Finally, the brain has an additional role inrelaying information about the energy status back to other organs including the liverand adipose tissue.

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384 | JUNE 2012 | VOLUME 13 www.nature.com/reviews/molcellbio



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CIP4

APSCAP

Vesicletargeting

Glucose

Insulina b

Insulinreceptor

c-CBL CRK

C3GTC10 TC10

GAPEX5

IRSPI3K PDK1

AKT

AS160

RGC

mTORC2

Vesicle translocation

Vesicletargeting

PIP3PIP2

Glucose

Vesicle retention

GDP GTP

P P P

PP

P

P

P

Exocyst

GSV

GLUT4-mediatedglucose uptake

GSV

GSV

GSV

GLUT4-mediatedglucose uptake

Type 2 diabetes mellitusA chronic metabolic disorder

that is characterized byincreased plasma glucoselevels that result from aninability of tissues torespond to insulin.

Anabolic hormoneSecreted peptide that signalsto cells to upregulate metabolicprocesses that convert simpleenergy sources intomacromolecules.

AdipokinesHormones and cytokines thatare released by adipocytesand signal to other tissues toalter feeding behaviour andmetabolism.

Small GTPases20 35 kDa guaninenucleotide-binding proteinsthat switch between aninactive GDP-boundconformation and an activeGTP-bound conformation.

Guanine nucleotideexchange factors(GEFs). A family of enzymesthat activate GTPases bycatalysing GDP release,

thus allowing cytoplasmicGTP to bind to the GTPase.

GTPase-activating proteins(GAPs). A family of enzymesthat inactive GTPases bycatalysing GTP hydrolysis.

SNARE regulatory proteins(Soluble N -ethylmaleimide-sensitive factor attachmentprotein receptor regulatoryproteins). A family of smallhelical proteins that bridgetwo membranes and drivemembrane fusion events.

also affect other stages of GLUT4 trafficking, includingendocytosis, sorting in the endosomal system and theformation of GSVs. The development of new tools forhigh-resolution analysis of the different intra cellularmembrane compartments that contain GLUT4 hasaided in pinpointing some of the steps that regulateGLUT4 cycling.

PI3K-dependent insulin signalling. The PI3K-dependentsignalling pathway is initiated following insulin bind-ing to its cognate Tyr kinase receptor on the cell surface,which leads to the recruitment and Tyr phosphoryla-tion of the insulin receptor substrate (IRS) family ofadaptor proteins 6,1820. Tyr-phosphorylated IRS proteinsserve as docking sites for the SH2 domain of the p85

regulatory subunit of class I PI3K, and the interactionof IRS proteins and PI3K results in PI3K activation andthe subsequent synthesis of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P 3) from PtdIns(4,5)P 2 at theplasma membrane. PtdIns(3,4,5)P 3 in turn serves as adocking site for several PH domain-containing Ser/Thrkinases that are implicated in glucose uptake, includin gphosphoinositide-dependent kinase 1 (PDK1) andAKT (also known as PKB)21. PDK1 and mTORC2activate AKT through dual Ser/ Thr phosphorylation.Knockout and knockdown studies have demonstratedthat IRS adaptor proteins and AKT are absolutely nec-essary for insulin-stimulated glucose uptake 2224, and

overexpression of constitutively active AKT can largely,but not completely, mimic the effect of insulin 25,26.

AKT is a central hub connecting insulin signallingwith downstream regulators of GLUT4 t rafficking.Microscopy studies that examined GLUT4 trafficking inthe presence of AKT inhibitors or dominant-negativ eAKT constructs indicate that the kinase affects the exo-cytic arm of the GLUT4 trafficking itinerary, probablyby regulating the translocation, targeting and fusion ofGLUT4-containing vesicles 2729. The most extensivelystudied targets of AKT are the RAB GAP AS160 (alsoknown as TBC1D4, which targets RAB8, RAB10 andRAB14) and the RALGAP complex (RGC, whichis comprised of a regulatory subunit (RGC1) and acatalytic subunit (RGC2) and targets RALA). These

small GTPases are involved in GLUT4 vesicle trans-location and targeting to the plasma membrane 28,30,31.Some SNARE regulatory proteins , including Synip (alsoknown as STXBP4) and CDP138 (138 kDa C2 domain-containing phosphoprotein), are also direct substratesfor AKT and regulate GLUT4 vesicle fusion with theplasma membrane 3234. Whether AKT regulates othersteps in GLUT4 trafficking, including endocytosis, sort-ing and GSV formation, is currently unclear. The searchfor additional AKT targets in muscle and adipose cellscontinues and will probably give further insight into thespecific steps of GLUT4 trafficking that are regulatedby this kinase.

Figure 1 | Insulin signalling regulates GLUT4 exocytosis by engaging the trafficking machinery. The APS (adaptorprotein with pleckstrin homology (PH) and Src homology 2 (SH2) domains)insulin signalling cascade is initiated when theactivated insulin receptor recruits the adaptor proteins APS, c-CBL and c-CBL-associated protein (CAP) ( a). Insulinreceptor-catalysed Tyr phosphorylation of c-CBL in turn triggers the recruitment of the adaptor protein CRK and theguanine nucleotide exchange factor (GEF) C3G to the plasma membrane, where C3G activates the small GTPase TC10.GTP-bound TC10 interacts with the exocyst complex, thereby creating targeting sites for the glucose transporter type 4(GLUT4) storage vesicle (GSV) at the plasma membrane. TC10 also interacts with CDC42interacting protein 4 (CIP4), whichis associated with the RAB5 and RAB31 GEF GAPEX5. Translocation of GAPEX5 to the cell surface modulates the activationstate of its target small GTPases, which are involved in GSV retention and translocation. The insulin receptor simultaneouslyinitiates the phosphoinositide 3-kinase (PI3K)-dependent signalling cascade by phosphorylating insulin receptorsubstrate (IRS) proteins, thus producing docking sites for the recruitment and activation of PI3K ( b). This kinase convertsphosphatidylinosito l-4,5-diphosp hate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3), which serves as a platformfor the recruitment of phosphoinositide-dependent kinase 1 (PDK1) and AKT. When at the plasma membrane, AKT isphosphorylated by PDK1 and mammalian target of rapamycin comple x 2 (mTORC2), wh ich results in AKT activation.AKT promotes GSV exocytosis by phosphorylating and inactivating two GTPaseactivating proteins (GAPs), AS160 and theRALGAP complex (RGC, consisting of a regulatory subunit (RGC1) a nd a catalytic subunit (RGC2)), which regulate smallGTPases that are involved in GSV retention and targeting, respectively.

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Clathrin-mediatedendocytosis

Dynamin

GLUT4

CaveolinClathrin

AP2

Microtubule

RAB5

RAB5

RAB5

Adipocytes Adipocytes and muscle

Cholesterol

Dynein

Sorting endosome

Cholesterol-dependent endocytosis

RAB5

RAB5

Lipid raftsRigid regions of the plasma

membrane that areenriched in cholesteroland glycosphingolipids.

EffectorA protein that preferentiallybinds to an activated smallGTPase.

ExocystAn evolutionarily conservedprotein complex that consistsof eight subunits and targetsexocytic vesicles to sites ofdocking and fusion at theplasma membrane.

The APSinsulin signalling pathway. Insulin alsoinitiate s a PI3K-independent signalling pathwayby recruiting the adaptor protein APS, which bindswith high affinity to the activated insulin receptor 35.Following insulin receptor phosphorylation, APS recruits

a complex that comprises the proto-oncogene c-CBLand c-CBL-associated protein (CAP) 36,37. This triggersinsulin receptor-catalysed Tyr phosphorylation of c-CBL.Phosphorylated c-CBL then interacts with the adaptorprotein CRK, which is in complex with the GEF C3G 38.C3G in turn catalyses the activation of TC10, a memberof the RHO-family of small GTPases 39,40 that is localizedin lipid rafts in the plasma membrane.

Active TC10 interacts with effector proteins that regu-late GLUT4 vesicle exocytosis. The TC10-binding pro-tein CIP4 (CDC42-interacting protein 4) forms a stablecomplex with the RAB GEF GAPEX5, which regulatesthe activity of RAB5 family GTPases that are involved in

GLUT4 vesicle retention and translocation. The TC10effector EXO70, which is a subunit of the exocyst teth-ering complex, has been implicated in GLUT4 vesicletargeting. Together, these molecular targets support arole for APS signalling in the release of intracellular GSVretention mechanisms and in GLUT4 vesicle targeting tothe plasma membrane.

Disrupting individual components of thePI3K-dependent or the APS signalling pathways inadipo cytes, either with pharmacological inhibitors or bysmall interfering RNA (siRNA)-mediated knockdown,inhibits GLUT4 exocytosis and glucose uptake 22,24,4144,which suggests that these pathways converge to con-trol the GLUT4 trafficking machinery. In muscle cells,the PI3K-dependent signalling pathway is absolutelyrequired for GLUT4 exocytosis; however, the necessityof the APS signalling pathway is unknown. In vivo thesituation is less clear; knockout studies have shown thatAKT isoforms seem to have different effects on cellularmetabolism and growth 21,45. Although knockout micefor the majority of components in the APS signalling

pathway have not yet been analysed, CAP-deficientmice are paradoxically more sensitive to insulin whenplaced on a high-fat diet than control mice 46. The basis ofthis is unclear, but CAP is also expressed in many othertissues, including macrophages, and seems to have acrucial role in these cells 46. Moreover, CAP is a memberof a larger family of sorbin homology (SoHo) proteins,and its function may be partially redundant with othermember s of this family 47.

Despite extensive cell biology data supporting therole of insulin in increasing surface GLUT4 levels, manyquestions still remain regarding the mechanisms con-trolling GLUT4 localization, both in the absence andpresence of the hormone. Below, we focus on the steps inGLUT4 cycling, our current understanding of the cell-ular machineries regulating these steps, and finally if andhow insulin signalling intervenes at different stages of theGLUT4 trafficking itinerary.

GLUT4 endocytosisThe amount of cell surface GLUT4 is determined by the netrate of GLUT4 endocytosis and exocytosis. In the absenceof insulin, hypoglycaemia is avoided by rapid endocytosisand slow exocytosis of GLUT4 in adipose and muscletissues48,49. After stimulation with insulin, surface GLUT4levels increase, which may be a result of an increasedexocytosis rate, a decreased endo cytosis rate or the mod-

ulation of both. Although it is widely accepted that mostof the effects of insulin occur through increased GLUT4exocytosis48,49, whether and how GLUT4 endocytosi s isaltered by insulin remains unclear.

GLUT4 is endocytosed through at least two separatepathways: clathrin-mediated endocytosis and cholesterol-dependent endocytosis 5052, both of which internalizeGLUT4 into a sorting endosome compartment (FIG. 2) . Inmuscle cells, clathrin-mediated endocytosis is mainlyresponsible for GLUT4 internalization; however, adipo-cytes use both the clathrin-mediated and the cholesterol-dependent pathways 53. Internalization of GLUT4 viaclathrin-mediated endocytosis probably requires the

Figure 2 | Molecular mechanisms of GLUT4 internalization. Glucose transportertype 4 ( GLUT4) is internalized via clathrin-mediated and cholesterol-dependentendocytosis. Internalization of GLUT4 through clathrin-mediated endocytosis requiresthe adaptor protein AP2. AP2 coordinates packaging of this glucose transporter intoendocytic vesicles by recruiting clathrin to the plasma membrane and by binding to anamino-terminal F 5QQI sequence in GLUT4. Vesicle scission from the plasma membranerequires the GTPase dynamin, which assembles at the neck of the invaginating vesicleand, following GTP hydrolysis, constricts and breaks the membrane. Cholesterol-dependent endocytosis requires the membrane-deforming protein caveolin, whicholigomerizes in lipid rafts and forms large cave-like structures, or caveolae. Cholesterol-dependent endocytosis is likely to also require dynamin to liberate caveolae from thecell surface. Vesicles carrying newly endocytosed cargo move towards the cell interior

on dynein motors, which attach to the vesicles via RAB5. Endocytosed vesicles fusewith endosomes, where their cargo is sorted for degradation or recycling. Whereasclathrin-mediated endocytosis is the predominant mode of GLUT4 endocytosis in musclecells, both clathrin-mediated and caveolin-dependent endocytosis have been implicatedin GLUT4 endocytosis in adipocytes. In adipocytes, an insulin-stimulated switch fromcholesterol-dependent to clathrin-mediated endocytosis may alter the rate ofGLUT4 internalization.

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HypoglycaemiaPhysiological condition that isdefined by abnormally lowblood glucose levels.

Clathrin-mediatedendocytosisA mechanism for internalizingextracellular molecules andportions of the plasmamembrane. This pathway isdependent on the membranecurvature-inducing coatprotein clathrin.

Cholesterol-dependentendocytosisA clathrin-independentmechanism for internalizingmolecules. This mechanism isblocked by drugs that depletecellular cholesterol and often

requires the lipid raft proteincaveolin.

Sorting endosomeA membrane compartmentthat is localized close to thecell surface where recentlyendocytosed proteins aredelivered and sorted fordegradation or recycling.

Recycling endosomesMembrane compartmentsthat many recycling proteinspass through before returningto the cell surface.

adaptor protein AP2, which recognizes and inter-acts with an F 5QQI sequence at the amino ter minusof GLUT4 (REFS 51,54,55) . By contrast, cholesterol-dependen t internalization of GLUT4 seems to requirecaveolin, a protein that localizes in lipid rafts 50,52; how-ever, it should be noted that several studies have foundno enrichment of GLUT4 in caveolae in adipocytesor muscle cells56,57. Abscission of GLUT4-containingclathrin-coated pits, and probably also caveolae, requiresthe GTPase dynamin 58,59. Once formed, movement ofendocytosed vesicles towards the cell interior is mediatedby microtubule-associated dynein motors, which attachto endocytic vesicles through the small GTPase RAB5 inadipocytes60,61. The relative importance of these two inde-pendent endocytic pathways is unclear. Although somestudies have suggested that the two routes may endo-cytose GLUT4 at different rates, it is also possible thatthese pathways sort GLUT4 into distinct vesicle pools inthe endosomal system.

Reconciling the role of insulin. Whereas insulin does

not affect GLUT4 internalization rates in muscle cells 62,the effect of the hormone in adipocytes is less clear.Several studies have reported that insulin augments sur-face GLUT4 levels in adipocytes not only by increasing therate of exocytosis but also by decreasing the rate of GLUT4endocytosis by ~two- to threefold 48,51,63,64. Treatment withthe cholesterol-depleting drug nystatin blocks GLUT4internalization in basal adipocytes, whereas knockdown of Ap2 blocks GLUT4 internalization in insulin-stimulate dadipocytes, which suggests that stimulation with thehormone results in a switch from cholesterol-dependentinternalization to clathrin-mediated internalization 51.A difference in the efficiency of GLUT4 internalization via caveolin and clathrin could result in a slower rate ofGLUT4 internalization in the presence of insulin.

Although decreased internalization of GLUT4 inresponse to insulin in adipocytes would seem to supportthe effect of the hormone in increasing glucose transport,the importance of a small reduction in the endocytic ratein modulating overall surface levels of GLUT4 is con-troversial. Furthermore, it was recently suggested thatthe methods used previously to measure the endocyticrate in adipocytes did not properly account for the rateof GLUT4 exocytosis, and that this led to a perceiveddecrease in endocytic rates 65. After correcting for thesefactors, no change was observed in the rate of endocytosisafter the addition of insulin. Other studies in adipocytes

have found that insulin increases GLUT4 traffickingthrough the recycling system 49,66, suggesting that endo-cytosis of the transporter may be important for maintain-ing a pool of endosomes that quickly return to the plasmamembrane. Although the jury is still out on this point,it seems reasonable that GLUT4 endocytosis might beunchanged or even increased by insulin to creat e a readilyreleasable pool of vesicles.

GLUT4 sorting and retentionAfter endocytosis, recycled membrane proteins canreturn quickly to the plasma membrane from sortin gendosomes or they can sort through intracellular

compartments, including recycling endosomes , lateendosomes and the TGN, before returning to the plasmamembrane 67. This diversity of recycling pathways allowscells to regulate the protein and lipid composition at dis-tinct locations within the cell. In addition to the recyclingmembrane system, some specialized cell types containnon-secretory storage compartments that retain specificmembrane proteins in the cell until a stimulus signals fortheir exocytosis to the plasma membrane 1.

A single GLUT4 molecule undergoes multiplecycles of exocytosis and endocytosis during its lifetime.Studies of GLUT4 trafficking indicate that in unstimu-lated adipo cytes, a pool of GLUT4 constantly trafficsbetween the cell surface and the cell interior throughendosomal pathways; however, at least 50% of the trans-porter pool is sequestered away from this recyclingsystem in specialized post-endosomal GSVs that undergoexocytosis in response to insulin 49,66,68 (FIG. 3).

Dynamic regulation of the GSV. The existence of aGLUT4 storage compartment was proposed on the

basis of ablation studies that depleted adipocytes ofendosomes that contain transferrin receptor (TfR), aprotein that traffics through the recycling endosomalsystem and only exhibits a twofold change in trans-location in response to insulin 6870. These studies showedthat, in the basal state, at least half of the GLUT4 pop-ulation is found in a vesicle compartment that largelylacks TfR and other recycling proteins, includingmannose-6-phosphate receptor (M6PR) and GLUT1.Stimulation with insulin depletes a proportion of theseGLUT4-enriched vesicles, and this led to the hypo thesisthat adipocytes and muscle cells sort GLUT4 into aspecialized pool of insulin-sensitive vesicles 71.

The isolation and biochemical characterization ofthese GSVs has been challenging, mainly because GLUT4and other proteins that are enriched in these vesiclesalso traffic through the endosomal system, resulting ina lack of GSV-specific markers. For example, electronmicroscopy studies showed that GLUT4 is found inall endosomal compartments 8,72. Efforts to purify andanalyse GSVs have used a combination of techniques,including compartment ablation, cell fractionation,immuno-isolatio n using GLUT4-specific antibodiesand immunodepletion of other vesicle populationsusing antibodies against non-GSV proteins, followedby immunoanalysis or mass spectrometry analysis.However, it is important to note that, so far, the purity

of the GLUT4-containing vesicles used in mass spec-trometry analysis studies has been hard to ascertain.Nevertheless, these studies have provided valuableinsight into the protein composition of GSVs and haveidentified GLUT4, insulin-regulated aminopeptidase(IRAP), sortilin, vesicle-associated membrane protein 2(VAMP2) and low-density lipoprotein receptor-relatedprotein 1 (LRP1) as major components of these vesi-cles69,7375. Although the functions of these proteins inGSVs are not completely understood, sortilin, IRAP andLRP1 all positively affect GLUT4 sorting into GSVs75,76,whereas VAMP2 is the v-SNARE required for fusion ofthese vesicles with the plasma membrane 77,78.

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Trans -Golgi network

TC10

IRAPARF6

ClathrinAdaptorprotein

GLUT4

GLUT1

TransferrinreceptorLRP1

SortilinVAMP2

GSVs

ARF6

RALA

RAB5 RAB4

RAB11

Fastrecycling

Slowrecycling

Sortingendosome

Recyclingendosome

Exocyst

Plasma membrane

RAB31 InsulinInsulinRAB8,

RAB10,RAB14

Donor compartment

GSVs

a b

Formation of GSV multimericcomplexes

Adaptor-drivenprotein sorting

GSVs do not cycle to the cell surface in the absence ofinsulin, or do so only very slowly, suggesting that adipo-cytes and muscle cells contain a specialized machinerythat retains this vesicle population within the cell 49,66,79.Although insulin causes rapid translocation of GSVs tothe plasma membrane, prolonged stimulation with thehormone (~15 minutes) triggers GLUT4 movement tothe plasma membrane in endosomes, suggesting thatwhen preformed GSVs have been depleted, GLUT4

recycling from endosomes is favoured rather thanreforming of new GSVs 49,80. Thus, insulin may increasesurface GLUT4 levels by acting on at least two processesin GLUT4 trafficking: exocytosis of GSVs and recycling via endosomes (FIG. 3).

Small GTPases regulate GLUT4 sorting. UnderstandingGLUT4 sorting and retention has proven difficult forseveral technical reasons. In addition to the difficultyin isolating a pure population of GSVs for characteriza-tion, the organization of the perinuclear endomembranesystem from which GSVs arise is not well defined, andso trafficking of GLUT4 through this system is poorly

understood. Furthermore, because GLUT4 traffickingbetween the interior of the cell and the cell surface is acircular process, disruption of one trafficking step neces-sarily affects all other steps. Thus, the identification ofthe key players for any particular step is difficult.

After endocytosis into sorting endosomes, GLUT4is sorted into recycling endosomes and/or the TGN,and one or both of these compartments probably serveas the donor membrane for GSVs 81,82. GLUT4 sorting

and delivery into GSVs relies on the actions of smallGTPases of the RAB and ARF families, which assembleeffectors that mediate vesicle budding, transport andfusion. In adipocytes and muscle cells, RAB4, RAB5,RAB8, RAB10, RAB11, RAB14 and RAB31 have beenimplicated in regulating different steps in GLUT4sorting, although additional small GTPases (includ-ing ARF6 and RALA) have been found to associatewith GLUT4-containing vesicles, and some of theseGTPases also affect GSV formation 83. RAB5, whichdrives homo- and heterotypic early endosomal fusion,is activated at the plasma membrane by insulin throughTC10, which recruits the RAB5 GEF GAPEX5 to the

Figure 3 | The GLUT4 trafficking itinerary. a | Glucose transporter type 4 (GLUT4) is constantly cycled between theplasma membrane and intracellular membrane-bound compartments. After internalization, GLUT4 can take severaldifferent paths back to the plasma membrane. GLUT4 can recycle back to the plasma membrane from sorting endosomes(termed fast recycling) or recycling endosomes (termed slow recycling), or this glucose transporter can be packaged intoGLUT4 storage vesicles (GSVs) that bud from recycling endosomes and/or the trans- Golgi network (TGN). GSVs do notcycle to the plasma membrane in the absence of insulin owing to intracellular retention mechanisms that may involvefutile cycling with recycling endosomes or the TGN, or physical anchoring to an intracellular structure. Insulin increasessurface GLUT4 levels 10- to 40-fold by increasing GLUT4 exocytosis from GSVs and endosomes. GLUT4 traffickingthrough the endosomal system and the formation of GSVs require the actions of multiple small GTPases, including RAB5,RAB4, RAB11, RAB31, ARF6 and RALA.b | Hypothetical model for GSV formation. The GSV resident proteins GLUT4,insulin-regulated aminopeptidase (IRAP), sortilin and low-density lipoprotein receptor-related protein 1 (LRP1) interactwith each other through their lumenal domains to form oligomeric protein complexes in the GSV donor compartments.The small GTPase ARF6 drives vesicle formation on a subdomain of donor membranes by recruiting adaptor proteins,

which interact with clathrin and GSV-resident proteins. In the absence of insulin, GSVs may be prevented from cyclingto the plasma membrane through a TGNGSV futile cycle that is maintained by active RAB31. Insulin may increaseGSV release by inactivating RAB31 and by activating RAB10 in adipocytes and RAB8 and RAB14 in muscle cells.

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cell surface. RAB5 may regulate mobility and sortingof GLUT4-containing vesicles after endocytosis 61,84,85.The generation of PtdIns(3)P by RAB5 has also beenshown to regulate maturation of endosomes 86. The roleof RAB4 in GLUT4 trafficking is poorly understood;however, given its known function in protein sortingin endosomes in other systems 85, and the observationthat manipulating RAB4 function in adipocytes altersGLUT4 levels at the plasma membrane 87,88, RAB4 maypromote fast recycling of the transporter from sortingendosomes.

GSV formation. Disrupting RAB11 function in adipo-cytes by overexpressing the binding domain of itseffector protein RAB11-binding protein (RAB11BP)increases the amount of GLUT4 that colocalizes with TfR,inhibits basal TfR and GLUT4 recycling and decreasesinsulin-stimulated GLUT4 translocation 79. Together,these data suggest a role for RAB11 in GSV formationand traffickin g in the endosomal system.

The AKT substrate AS160 is a RAB GAP that targets

RAB8 and RAB14 in muscle cells89 and RAB10 in adipo-cytes83,90. These RABs have a positive role in GLUT4translocation, suggesting that they may regulate GSVformation and/or intracellular retention 8992. As insulinstimulates AKT-catalysed phosphorylation of AS160,blocking the function of the protein, this should relievethe inhibitory effect of AS160 on its target RABs 93,94 (FIG. 1) . Whereas the activation of RAB8 and RAB14 byinsulin has been demonstrated in muscle cells 89, so farthe activation of RAB10 in adipocytes has not beendetected 92. Nevertheless, RAB10 is necessary for maxi-mal GLUT4 exocytosis in response to insulin, and sev-eral lines of evidence indicate that cycling of this smallGTPase may increase glucose uptake 90,95. Together, theactivation of RAB4, RAB5, RAB8, RAB11 and RAB14seems to be a key means by which insulin signallingpathways alter GLUT4 trafficking 96,97.

GSV budding from recycling endosomes and/or theTGN depends on vesicle coats that assemble on sub-domains of the donor compartments (FIG. 4) . Althoughthe molecular forces that drive the packaging of GSVcomponents into budding vesicles are incompletelyunderstood, the observation that GLUT4, IRAP, sortili nand LRP1 interact with one another through theirlumena l domains has led to a mass action hypothesisof GSV formation. This hypothesis posits that GSVcomponents, which are highly expressed in mature

adipocytes and muscle cells compared with other recy-cling proteins, find and interact with one another due totheir high concentration in donor compartments. Theformation of large multimeric complexes that containGSV components would then allow for efficient sort-ing of these proteins into newly forming vesicles 75,76,98.Adaptor proteins, which are recruited to GSV donormembranes by the small GTPase ARF6, interact withGSV components and clathrin to facilitate protein sort-ing and vesicle buddin g. The adaptor proteins GGA(Golgi-localized -ear-containing ARF-binding protein),ACAP1 (ARF-GAP with coiled-coil ankyrin repeat andPH domain-containing protein 1), AP1 and AP3 have

all been implicated in GSV formation 99102. Althoughthe specific roles of these different adaptor proteins arenot currently understood, it is possible that they act ina coordinated fashion to drive vesicle budding fromone donor membrane. Alternatively, they may assem-ble on different membrane-bound compartments andact at different steps in GLUT4 trafficking (for example,sorting at early endosomes and recycling endosomes) toultimately drive GSV formation.

GSV retention. GLUT4 is sequestered away fromthe recycling system in the basal state through intra-cellular retention of GSVs, which occurs through twomechanisms that may require a common machinery:futile cycling and retention by anchoring proteins.A futile cycle in which GSVs, the TGN and/or recyclingendosomes undergo repeated cycles of fusion and fis-sion is thought to disfavour GLUT4 translocation to theplasma membrane in the absence of insulin 82. Little isknown about the proteins that are necessary for main-taining a futile cycle, but the opposing actions of at

least two RAB proteins are probably involved. RAB31 ishypothesized to participate in futile cycling in the basalstate; in support of this, RAB31 negatively regulatesGLUT4 translocation in adipocytes, and it participatesin TGN-to-endosome trafficking in other cell types 103,104.Insulin may promote the release of GLUT4 from futilecycling by recruiting the RAB31 GEF GAPEX5 to theplasma membrane, thereby decreasing RAB31 activityon intracellular membranes 103.

Anchoring proteins are hypothesized to promoteGSV retention by either physically tethering GSVs to anunidentified intracellular site or by constraining GSVcycling to minimize exchange with the plasma mem-brane 105,106. For example, TUG (tether containing UBXdomain for GLUT4) interacts with GLUT4 and mayanchor GSVs within the cell 107. Overexpression of TUGsequesters GLUT4 away from TfR-positive structures,whereas the disruption of TUG function enhances basalglucose uptake and GLUT4 levels at the cell surface andincreases dispersion of GLUT4 throughout the endo-somal system 106,107. Although these data may indicate arole for TUG as a scaffold that physically tethers GSVs,these results could also be consistent with a role for TUGin intracellular sorting of GLUT4. Insulin abrogates theinteraction between TUG and GLUT4, suggesting thatTUG is an important target of insulin action 107.

Exocytosis of GSVsInsulin increases the amount of GLUT4 at the cell surfacemainly by promoting exocytosis of GSVs 48,108 and to alesser extent by increasing exocytosis from the recyclingsystem 70. GSV exocytosis can be separated into threedistinct processes: translocation to the cell periphery;targeting; and fusion (FIG. 4) . The application of totalinternal reflection fluorescence micro scopy (TIRFM)(BOX 2) to study GSV exocytosis has shown that insu-lin probably regulates each of these steps by recruitingGLUT4-containing vesicles to the cell periphery andthen modulates the rates of targeting and fusion 27,28,109,110.These imaging studies are consistent with earlier

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Exocyst

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P

P

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c Fusionb Targeting and disengagementa Translocation

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EXO70TC10 CDP138

Synip

RALA

MUNC18C

AKT

Insulin

Insulinreceptor

RALA

TUG

Insulin

TUG

RAB14RAB10

RAB8

AS160P

AKT

Kinesin

MYO1C

GSV

PAKT

PKC

VAMP2

GLUT4

Plasma membrane

DOC2

Actin lament

RGC

SNAP23

SAP97

evidence from biochemical and cell biological studiesthat identified molecular motors 18,96,111, tethers 112 andfusion proteins 33,113115 as important components of thecell machinery that are necessary for insulin-stimulatedGLUT4 exocytosis.

Translocation. Insulin stimulates the accumulationof GLUT4-containing vesicles at the periphery of thecell within 5 minutes of exposure to the hormone 116,117.High-resolution imaging of GLUT4 shows thatGLUT4-containing vesicles move along the cytoskeletaltracks in a linear fashion both in the presence and absenceof insulin 110,116119. Treatment with drugs that depoly-merize either microtubules or actin partially blocksinsulin-stimulated accumulation of GLUT4 at the plasmamembrane and glucose uptake 117,120122, and disruptionof both microtubules and actin completely inhibit sthe effects of insulin on GLUT4 relocalization 116,123.

Whereas microtubules participate in long-range move-ment of GSVs to the periphery of the cell, cortical actin isinvolved in short movements that are coupled to dockingand/or fusion near the plasma membrane 123126.

Whether insulin regulates GSV movement on

cytoskeletal tracks is still debated. Some studies havereported an increase in the speed of movement or anincrease in the recognition of GSVs by the cyto skeleton following insulin stimulation 29,96,127. By contrast, othergroups report that GLUT4-containing vesicles are con-stantly moving along cytoskeletal tracks in the basalstate, suggesting that they continually sample theplasma membrane. In this model, insulin would regulatethe recognition of GSVs at the plasma membrane, ratherthan the mobilization of GSVs themselves 27,29,110,119.

Several microtubule-based kinesin and actin-basedmyosin motors have been implicated in GSV trans-port along cytoskeletal tracks, including the kinesins

Figure 4 | Insulin targets several steps in GLUT4 storage vesicle exocytosis. a | Insulin stimulates glucosetransporter type 4 (GLUT4) storage vesicle (GSV) translocation by inhibiting the interaction between TUG (tethercontaining UBX domain for GLUT4) and GLUT4 and by stimulating AKTcatalysed phosphorylation of AS160, whichinhibits the GTPaseactivating protein (GAP) activity of AS160 towards RAB8, RAB10 and RAB14. Microtubulebasedkinesin motors drive GSV movement from the perinuclear region to the periphery of the cell. Close to the cell surface,GSVs move along a meshwork of cortical actin to targeting sites on the plasma membrane. The actin-based myosin motorMYO1C, which drives these movements, recognizes GSVs by interacting with the vesiclelocalized small GTPase RALA.Insulin may regulate the movement of GSVs along cytoskeletal tracks by increasing the ATPase activity of motor proteinsor by modulating recognition of vesicles by molecular motors via factors such as calmodulin. b | Insulin assemblesGSV targeting sites at the plasma membrane by activating the small GTPase TC10. GTPloaded TC10 and lipidraftlocalized synapse-associated protein 97 (SAP97) recruit and stabilize the exocyst complex at the plasma membrane.Simultaneously, AKT activates RALA by phosphorylating and inhibiting the RALGAP complex (RGC, consisting of aregulatory subunit (RGC1) and a catalytic subunit (RGC2)). GTPloaded RALA guides GSVs to targeting sites on the plasmamembrane by interacting with the exocyst subunits SEC5 and EXO84. After the arrival of GSVs at the plasma membrane,protein kinase C (PKC)-catalysed phosphorylation of SEC5 inhibits the interaction between the exocyst and RALA, thusallowing GSVs to disengage from the targeting machinery before vesicle fusion. c | GSV fusion with the plasma membraneis driven by the assembly of SNARE complexes comprised of vesiclelocalized vesicleassociated membrane protein 2(VAMP2), plasma membranelocalized syntaxin 4 (STX4) and synaptosomalassociated protein 23 (SNAP23). The activatedinsulin receptor phosphorylates the fusion regulatory protein MUNC18C, which then stimulates GSV fusion, perhaps byrecruiting DOC2 (double C2like domaincontaining protein ) to SNARE complexes. AKT catalyses phosphorylation ofthe syntaxin 4 binding protein Synip, which may release its inhibition of GSV fusion. AKT also phosphorylates and recruitsCDP138 to the cell surface, where CDP138 acts as a positive regulator of GSV fusion.

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VAMP2:

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Glut4

VAMP2

GSV

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Plasma membraneTIRF zone

STX4

FusionTargetingTranslocation

SNAP23

KIF3 (REFS 96,123) and KIF5B119, myosin 5A (MYO5A)127,MYO5B91 and MYO1C18,111,123,128 . In yeast and mamma-lian cells, small GTPases couple motor proteins to vesi-cles61,96,128132. In adipocytes, KIF3 (REF. 96) interacts withRAB4, and MYO1C interacts with RALA128. Inhibiting anyof these motor proteins with blocking peptides, by siRNA-mediated knockdown, expression of dominant-negative

constructs or by disruption of RAB proteins that theyinteract with, inhibits insulin-stimulate d GLUT4translocation 96,111,119,127.

So far, the role of the motor protein MYO1C in GSVmobility has been most extensively investigated. MYO1Cmoves from the cytoplasm to the plasma membrane inresponse to insulin 111 and targets GSVs to sites of fusion atthe plasma membrane. MYO1C does not recognize GSVsdirectly; instead, the motor protein is coupled to these vesicles through its interaction with RALA on GSVs 128.Calmodulin is proposed to modulate MYO1C functionin adipocytes, but its precise role is not completely under-stood. The interaction between MYO1C and RALA hasbeen shown to be calmodulin-dependent, suggestingthat this protein might potentiate GSV recognition bythe actin cytoskeleton 128. However, insulin has also beenseen to decrease the association between MYO1C andcalmodulin, suggesting that calmodulin release mightbe necessary for the motor activity of MYO1C 18. Thus,the role of calmodulin in GSV translocatio n remains tobe elucidated.

Targeting. The delivery of vesicles to the correct cellula rcompartment requires a targeting step in which the vesicles are init ially recognized by the dest inationmembrane. In adipocytes, GSV targeting to lipids raftson the cell surface is important for efficient insertionof GLUT4 into the plasma membrane 133. After stimula-tion of cells with insulin, the level of immunodetectableGLUT4 increases in the plasma membrane fraction sev-eral minutes before GLUT4 is exposed extracellularly,raising the possibility that insulin regulates GSV target-ing and/or fusion at the plasma membrane 48. TIRFMstudies carried out in adipocytes also indicate thatinsulin increases the rate and decreases the duration oftethering 27,110. However, these studies are limited by theability to clearly distinguish tethering from fusion. Inthis regard, the application of pH-sensitive fluorophoresthat gain their fluorescence signal only after the fusionpore opens should more clearly define the initiation offusion and thus provide insights into distinct events thatoccur at the plasma membrane (BOX 2) .

A crucial component of the GLUT4 targetingmachinery is the exocyst, which is an evolutionarilyconserved octameric complex that assembles at sites ofexocytosis and tethers exocytic vesicles to the plasmamembrane 134. The exocyst is thought to mediate the ini-tial flexible contact between exocytic vesicles and the

plasma membrane from a relatively long distance andcan thus concentrate GSVs before the final membranefusion step. Inhibition of exocyst assembly in adipocytesdisrupts fusion of GSVs without affecting their translo-cation, demonstratin g that this complex is necessary for vesicle targeting at the plasma membrane 112.

Insulin regulates exocyst-mediated targetingthrough three steps: exocyst assembly; engagement ofthe exocyst by GSVs; and GSV disengagement from theexocyst to enable fusion 112,128,133,135. First, insulin directsthe assembly of the exocyst at the plasma membraneby promoting an interaction between active TC10and the exocyst scaffolding subunit EXO70 (REF. 112) .

Box 2 | Study of GLUT4 vesicle exocytosis by TIRFM

Over the past 10 years, the understanding of how insulin affects glucose transportertype 4 (GLUT4) trafficking to the plasma membrane has been greatly aided by totalinternal reflection fluorescence microscopy (TIRFM). This technique allows thevisualization of events near the cell surface by illuminating a region of the cytoplasmthat is within ~100200 nm of the plasma membrane. The first TIRFM experimentstracked the movements of hundreds of single GLUT4-containing vesicles in live cells

and showed that insulin increases the amount of GLUT4 within the visualized region,thereby confirming previous biochemical and cell biological evidence that suggestedthat insulin predominantly targets GLUT4 exocytic trafficking 28.

Advances in TIRFM technology in the past few years have enabled researchers tostudy individual steps in GLUT4 vesicle exocytosis by using automated high-resolutioncapture of exocytic events 27,109 and dual-colour TIRFM 33,80. pHsensitive fluorophores,which are quenched in the acidic vesicle lumen but fluoresce after opening of the fusionpore at the plasma membrane, have also been used to functionally separate targetingof GLUT4 vesicles versus fusion. For example, TIRFM imaging of the vesicle-localizedSNARE VAMP2 (vesicleassociated membrane protein 2) and GLUT4 showed thatGLUT4 fluorescence can be observed during both targeting and fusion, whereas VAMP2fluorescence is observed specifically during fusion (see the figure). Together, thesestudies have shown that insulin increases the number of vesicles in the TIRF zone,decreases the duration of vesicle targeting and increases the frequency of fusionevents 27,109 . Advances in TIRFM technology have also allowed researchers to investigate

the roles of specific proteins in GLUT4 exocytosis, including TUG (tether containingUBX domain for GLUT4) and CDP138 (138 kDa C2 domaincontaining phosphoprotein),which affect vesicle retention and fusion, respectively 80,33.

However, there are still limitations of this technique; in particular, the study ofGLUT4 exocytosis is restricted by the inability to easily distinguish different typesof GLUT4containing vesicles. As a consequence, TIRFM studies examine a mixedpopulation of vesicles that include both GLUT4 storage vesicles (GSVs) and endosomes.These vesicles are likely to approach and fuse with the plasma membrane with differentkinetics, different dependencies on insulin action and different molecular machinery.The development and use of techniques that can reliably identify GSVs, including a newapproach that separates vesicle carriers on the basis of size 80, will be important forelucidating the functions of specific signalling and trafficking proteins in GSVexocytosis. Immunofluorescence images are reproduced, with permission, fromREF. 80 (2011) The Rockefeller University Press.

SNAP23, synaptosomalassociated protein 23; STX4, syntaxin 4.

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EXO70 is constitutively associated with other exo-cyst subunits and thereby assembles the complex atthe plasma membrane 133. Other interactions betweenexocyst subunits and the plasma membrane facilitatecomplex assembly at discrete locations. For example,an inter action between the exocyst subunit SEC8 (alsoknown as EXOC4) and synapse-associated protein 97(SAP97; also known as DLG1) in lipid rafts anchorsthe exocyst complex in this subdomain of the plasmamembrane 133. EXO70 and SEC3 (also known as EXOC1)interact with PtdIns(4,5)P 2, and may thus target the exo-cyst to zones of the plasma membrane that are enrichedin this phospholipid 136138. PtdIns(4,5)P 2 is a known con-stituent of lipid rafts, and disruption of exocyst assemblyin raft subdomains inhibits GLUT4 insertion into theplasma membrane and glucose uptake, further implicat-ing this membrane subdomain in GLUT4 targeting 133,139.Thus, multiple interactions between plasma membraneconstituents and exocyst subunits coordinate exocystassembly in lipid rafts and localized GLUT4 targeting.

The exocyst is recognized by GSVs via the small

GTPase RALA, which is present on GLUT4-containing vesicles. Insulin controls RALA activity by inhibitinga complex of proteins with RAL-GAP activity. TheRAL-GAP complex is composed of RGC1 and RGC2,which contains a GAP domain with specific activit ytowards RAL-GTPases. AKT-catalysed phosphorylationof RGC2 on at least three residues inhibits the complexand allows for GTP loading on RALA 30. siRNA-mediatedknockdown of RGC1 or RGC2 increases RALA activityand insulin-stimulated glucose uptake, demonstratin gthe regulatory role of this complex 30. Moreover, siRNA-mediated knockdown of Rala or overexpression ofa dominant-negative RALA mutant blocks insulin-stimulate d glucose uptake and GLUT4 insertion intothe plasma membrane. By contrast, constitutively activeRALA mutants increase the effect of insulin, indicatingthat the activation of this small GTPase is required forinsulin-regulated GLUT4 exocytosis 128. Once activated,RALA interacts with exocyst subunits SEC5 (also knownas EXOC2) and EXO84 (REFS 128,140,141) . Although theprecise role of these two RALA-binding proteins remainsuncertain, both are required for insulin-stimulatedglucose uptake 128.

Large tethering complexes such as the exocyst arethought to disengage from vesicles and/or disassemblebefore fusion occurs 142. Disengagement may allow forfusion of opposing membranes and recycling of the teth-

ering machinery for additional rounds of vesicle target-ing. Indeed, RALA dissociates from the exocyst throughinsulin-dependent, protein kinase C (PKC)-catalysedphosphorylation of Ser89 in the RALA-binding domainof SEC5, which triggers SEC5 release135. Mutation ofSer89 to either Ala (to block phosphorylation) or toAsp (to mimic phosphorylation) blocks GLUT4 inser-tion into the plasma membrane, suggesting that bothengagement and disengagement from the targetingmachinery are necessary steps preceding fusion 135. Thisphosphorylatio n-dependent release of GSVs from theexocyst raises the possibility that the exocyst also servesa gatekeeper function in controlling GSV fusion.

Although RAB10 has been implicated in insulin-stimulated GSV translocation to the cell surface in adi-pocytes, it may have an additional role at the plasmamembrane. This function of RAB10 has largely emergedfrom TIRFM experiments performed in adipocytes byoverexpressing AS160-4P (in which the four phospho-rylation sites have been mutated to Ala), a mutant formof the RAB10 GAP that is not regulated by insulin. Thesecells exhibit decreased tethering of GSVs at the plasmamembrane, with little to no effect on fusion 27,28,143. Thereare currently no known RAB10 effectors in adipocytes;however, in MadinDarby canine kidney (MDCK) cells,RAB10 interacts with the exocyst, and GTP loading onRAB10 may enhance this interaction 144. It will be ofinterest to determine whether such an interaction is alsoregulated by insulin in adipocytes and, if so, how thissmall GTPase functions with RALA and TC10 to regu-late exocyst-mediated targeting of GSVs to the plasmamembrane.

SNARE regulation during GSV fusion. Following vesi-

cle tethering, SNARE proteins on both the GSV andthe plasma membrane (the donor and acceptor com-partments, respectively) form tight complexes thatprovide the driving force for membrane mixing andfusion 145. GSV fusion with the plasma membrane isdriven by SNARE ternary complex assembly betweenthe GSV-localized v-SNARE VAMP2, the plasmamembrane-localized t-SNARE syntaxin 4 (STX4) andits plasma membrane-associated accessory proteinsynaptosomal-associated protein 23 (SNAP23) 77,78,146148.TIRFM studies indicate that insulin increases the rateof GSV fusion, probably in an AKT-dependent man-ner 80,143. Proteins that have been implicated as targetsof insulin action include the SNARE regulatory pro-teins MUNC18C (also known as STXBP3), Synip (alsoknown as STXBP4), tomosyn (also known as STXBP5)and CDP138, suggesting that SNARE complex assemblymay be controlled by insulin 149.

The most extensively studied regulator of SNAREcomplex formation in GSV trafficking is the SEC1MUNC18 family member MUNC18C, which binds toSTX4 on the plasma membrane. SEC1MUNC18 pro-teins are thought to switch from negative regulators ofSNARE assembly to indispensible components of thefusogenic machinery during the fusion process. Thisdual function may occur through a change in their modeof binding to syntaxins 150. Studies in adipocytes support

both a negative role 151155 and a required role 156158 forMUNC18C in GSV exocytosis.

MUNC18C regulation by insulin is still not com-pletely understood. Some studies have suggested thatinsulin alters the mode of binding between MUNC18Cand STX4, thus enhancing its fusogenic function 113,159.Alternatively, others have reported that the insulinrecepto r catalyses phosphorylation of MUNC18C atTyr219 and Tyr521, which results in its dissociationfrom STX4 (REF. 115) . Interestingly, in pancreatic -cells,Tyr phosphorylation of MUNC18C increases its bind-ing to double C2-like domain-containing protein-(DOC2) in a manner that precludes STX4 binding 160.

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DOC2 belongs to a class of proteins that increase mem-brane curvature during fusion of synaptic vesicles withthe plasma membrane 161 and has been implicated as apositive regulator of GLUT4 exocytosis in adipocytes 114.MUNC18C may promote fusion by recruiting DOC2to sites of SNARE assembly at the plasma membrane.

Two other STX4-binding proteins, Synip andtomosy n, have been implicated as negative regulatorsof SNARE complex assembly in adipocytes. Stimulationwith insulin decreases the binding affinity between STX4and Synip in adipocytes, thus presumably freeingSTX4 to interact with VAMP2 on the incoming GSV 32.Insulin-mediated regulation of Synip is proposed tooccur through AKT-catalysed phosphorylation at Ser99(REFS 34,162) , but the importance of this phosphorylationevent has been disputed 163. Although tomosyn can alsocompete with VAMP2 for binding to STX4 and SNAP23in adipocytes, its regulation by insulin has not yet beeninvestigated 164.

CDP138, a previously uncharacterized protein con-taining a Ca2 +-binding C2 domain, has been identi-

fied as a novel AKT substrate that positively regulatesGLUT4 insertion into the plasma membrane in adipo-cytes. Overexpression of a phosphorylation-deficientS197A mutant or a Ca2 +- and lipid binding-deficientmutant of CDP138 blocked GLUT4 vesicle fusion buthad no effect on vesicle accumulation at the plasmamembrane 33. The C2 domain of CDP138 shares homol-ogy with those found in synaptotagmin proteins, whichpromote vesicle fusion by binding to lipids in a Ca2 +-dependent manner and thereby induce membranecurvature 165. Determining the mechanism by whichCDP138 acts and elucidating the role of AKT-mediatedphosphorylation in promoting its positive function inGLUT4 vesicle fusion are important areas for futureinvestigations.

Conclusions and future directionsNearly 25 years after the discovery that insulin stimu-lates glucose uptake by altering the trafficking itineraryof GLUT4 in muscle and adipose cells, we are begin-ning to understand the varied paths that this hexose

transporter can follow through the complex cellularmembrane network. New insights into how insulin sig-nalling intersects with these trafficking intermediateshave revealed that phosphorylation events initiated bythe insulin receptor can lead to the regulation of smallGTPases by modulating the activity or localization ofGEFs and GAPs. These small GTPases in turn mediat ecrucial steps and define: when and where GLUT4 cycles;the regulation of endocytosis; the entry into and theexit out of the recycling endosome system; the forma-tion of specialized storage vesicles and the retentionof these GSVs within the cell; and finally the release ofthe vesicles and the transport to discrete regions in theplasma membrane for tethering and fusion. Moreover,the generation of different phosphoinositide speciesdownstream of these phosphorylation events seems toprovide localization signals for these signalling events,thereby ensuring their efficiency.

Despite this progress, many questions remain. Howdoes GLUT4 move through the endomembrane sys-tem? Although it has been possible to study GLUT4

vesicle trafficking close to the plasma membrane withTIRFM, new microscopy techniques may permit thestudy of trafficking that occurs deeper within the cell.This should help to provide insights into how GSVsare formed and then retained in cells. For example, itis not yet clear whether GSVs are dynamically cycledback into the endosome network, or whether a physica ltether retains these vesicles in cells. It will also be impor-tant to understand how individual components of theexocyst complex contribute to GLUT4 targeting, andhow they control accessibility of GSVs to the fusionmachinery. All eight members of the exocyst complexare conserved from mammals to yeast, and in each spe-cies every component is required for secretion 166. Thesefindings suggest that the exocyst proteins are likely tohave additional uncharacterized regulatory roles. Howand why do these pathways differ between muscle andfat cells? It may take another 25 years to answer these keyquestions, and hopefully new insights will be gained asto how these processes are altered in obesity and otherstates that give rise to insulin resistance.

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