REGULATED TRANSPORT OF THE GLUCOSE TRANSPORTER …

Glucose is a fundamental source of energy for alleukaryotic cells. In humans, although all cells use glu-cose for their energy needs, the main consumer underbasal conditions is the brain, which accounts for asmuch as 80% of whole-body consumption. The energyis provided by the breakdown of endogenous glycogenstores that are primarily in the liver. These whole-bodyenergy stores are replenished from glucose in the diet,which, after being digested and absorbed across the gutwall, is distributed among the various tissues of the body(reviewed in REF. 1). This distribution process involves afamily of transport proteins — called GLUTs — whichact as shuttles to move sugar across the cell surface.These polytopic membrane proteins (FIG. 1) form anaqueous pore across the membrane through whichglucose can move. A large family of FACILITATIVE SUGAR

TRANSPORTERS exists in mammals, the individual mem-bers of which differ in their tissue distribution andkinetic properties, as well as in their intracellular local-ization. The latter property is of particular interest forglucose transport in specialized cell types, and providesthe basis for polarized glucose transport in epithelialcells, such as those in the gut, or for acute regulation ofglucose transport, as is observed in muscle and fat cellsafter a meal. Many mammalian tissues, such as thebrain, have a constitutively high glucose requirementand have been endowed with transporters that are con-stitutively targeted to the cell surface (for example,GLUTs 1–3). By contrast, certain tissues, such as muscleand adipose tissue, have acquired a highly specialized

glucose-transport system, the activity of which can berapidly upregulated to allow these tissues to increasetheir rate of glucose transport by 10–40-fold withinminutes of exposure to a particular stimulus (reviewedin REF. 1). This system is crucial during exercise, whenthe metabolic demands of skeletal muscle can increasemore than 100-fold, and during the absorptive period(after a meal), to facilitate the rapid insulin-dependentstorage of glucose in muscle and adipose tissue, so pre-venting large fluctuations in blood glucose levels.Dysfunctional glucose uptake into muscle and fat cellscontributes to the onset of TYPE II DIABETES (BOX 1).

In 1980, it was reported that, in rat adipocytes,insulin triggers the movement of the sugar transporterthat is found in these cells from an intracellular store tothe plasma membrane2,3. This translocation hypothesiswas later confirmed when GLUT4 was identified as themain glucose transporter in these cells. GLUT4, whichis expressed primarily in muscle and fat cells, is found ina complex intracellular tubulo–vesicular network that isconnected to the endosomal–trans-Golgi network(TGN) system. In the absence of stimulation, GLUT4 isalmost completely excluded from the plasma mem-brane (FIG. 2). The addition of insulin, or exercise in thecase of muscle cells, causes GLUT4 to shift from itsintracellular location to the plasma membrane (FIG. 2).Several observations indicate that GLUT4 has a crucialrole in whole-body glucose homeostasis. First, insulin-stimulated glucose transport is an important rate-limit-ing step for glucose metabolism in both muscle and fat

REGULATED TRANSPORT OF THEGLUCOSE TRANSPORTER GLUT4Nia J. Bryant, Roland Govers and David E. James

In muscle and fat cells, insulin stimulates the delivery of the glucose transporter GLUT4 from anintracellular location to the cell surface, where it facilitates the reduction of plasma glucose levels.Understanding the molecular mechanisms that mediate this translocation event involves integratingour knowledge of two fundamental processes — the signal transduction pathways that aretriggered when insulin binds to its receptor and the membrane transport events that need to bemodified to divert GLUT4 from intracellular storage to an active plasma membrane shuttle service.

FACILITATIVE SUGAR

TRANSPORTER

A polytopic membrane proteinthat transports sugars down aconcentration gradient in anenergy-independent manner.

TYPE II DIABETES

Also known as non-insulin-dependent diabetes or maturityonset diabetes.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 3 | APRIL 2002 | 267

Garvan Institute of MedicalResearch, 384 Victoria Road,Darlinghurst, New SouthWales 2010, Australia.Correspondence to D.E.J.e-mail:[email protected]: 10.1038/nrm782

R E V I E W S

268 | APRIL 2002 | VOLUME 3 www.nature.com/reviews/molcellbio

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an insulin-regulated step(s). Although many importantsignalling molecules that are integral to the insulin reg-ulation of GLUT4 translocation have been identified(BOX 2), any convergence between these two approachesremains to be achieved. In this review, we focus on ourcell-biological understanding of GLUT4 transport,and highlight potential regulatory sites of the insulin-signalling cascade.

GLUT4 transportGLUT4 is found in many organelles, including theplasma membrane, sorting endosomes, recycling endo-somes, the TGN and vesicles that mediate the transportof GLUT4 between these compartments (FIG. 3).Presumably this localization represents a complex anddynamic transport itinerary, and it raises several impor-tant questions. How does GLUT4 transport from oneorganelle to another, what is the relationship betweenthese pathways and the intracellular sequestration ofGLUT4 in basal cells, and which of these steps doesinsulin influence to affect GLUT4 exocytosis?

In non-stimulated adipocytes, the rate of GLUT4 exo-cytosis is 10-fold slower than that of the transferrin recep-tor (TfR) — one of the most well-studied constitutiverecycling proteins in mammalian cells6,7. To account forthis, GLUT4 must be selectively retained in one of itsintracellular locations, packaged into a specialized com-partment that remains static in the absence of insulin, orinvolved in a dynamic intracellular transport loop thatexcludes it from recycling endosomal vesicles. Currentevidence favours a role for all three mechanisms, whichemphasizes the complexity of this process.Another pro-tein, the insulin-responsive aminopeptidase (IRAP),which was recently described as a receptor for angiotensinIV (REF. 8), colocalizes with GLUT4 and is transported in avery similar manner9. Below, we discuss studies concern-ing the transport of either GLUT4 or IRAP, with a partic-ular focus on adipocytes and muscle cells. We also com-pare this with transport in cells such as fibroblasts that arenot, or only mildly, responsive to insulin, as key differ-ences have been identified that provide new insights intoour understanding of insulin action.

Endosomal sorting of GLUT4Morphological studies in both muscle and fat cellsindicate that, although there is some overlap of GLUT4with markers of the endocytic system such as the TfR, a

tissue,and is severely disrupted in type II diabetes1 (BOX 1);second, disruption of GLUT4 expression in mice resultsin insulin resistance4; and overexpression of GLUT4ameliorates diabetes in the DB/DB MOUSE model5.Analysing the molecular and cellular regulation ofGLUT4 transport not only promises to provide newinsights into protein sorting, but could also yield newtargets for therapeutic intervention in what could wellbe one of the most prevalent diseases that we will haveto confront in the future.

Understanding the regulation of GLUT4 and glucosetransport has proved to be extremely challenging, prin-cipally because it involves several signal-transductionpathways that are superimposed on a complex series ofvesicle transport processes. Insulin binds to a surfacereceptor on muscle and fat cells and triggers a cascade ofsignalling events (BOX 2) that culminates in GLUT4translocation. Studies of this process have been carriedout using two approaches.‘Outside–in’ approaches havelargely focused on mapping insulin-specific signallingpathways in muscle and fat cells with the view to identi-fying downstream targets that directly control GLUT4translocation. Conversely,‘inside–out’ approaches haveused cell-biological studies to map the intracellulartransport itinerary of GLUT4 with the aim of identifying

Figure 1 | Schematic representation of the GLUT family of proteins. The GLUT family ofproteins comprises 13 members at present, which are predicted to span the membrane 12 timeswith both amino- and carboxyl-termini located in the cytosol. On the basis of sequence homologyand structural similarity, three subclasses of sugar transporters have been defined: Class I (GLUTs1–4) are glucose transporters; Class II (GLUTs 5, 7, 9 and 11) are fructose transporters; and ClassIII (GLUTs 6, 8, 10,12 and HMIT1) are structurally atypical members of the GLUT family, which arepoorly defined at present. The diagram shows a homology plot between GLUT1 and GLUT4.Residues that are unique to GLUT4 are shown in red.

Sugar moiety

NH2

HCOOH

Plasmamembrane

Cytoplasm

Box 1 | Type II diabetes

The prevalence of type II diabetes is increasing at an alarming rate. In 1998, 143 million people worldwide suffered fromthis disease, and it is likely that this number will double over the next 20–30 years71. The incidence of type II diabetesincreases sharply with age, and it is highly prevalent in certain ethnic groups. For example, 10–30% of Australianaborigines are currently thought to have type II diabetes, and this number is predicted to increase to more than 50% inthe next ten years. The disease is characterized by defective insulin action, a condition that is referred to as insulinresistance. Insulin resistance is characterized by dysfunctional glucose uptake into muscle and adipose tissue, inconjunction with an oversupply of glucose from the liver, which results in high circulating plasma glucose levels. Thiscauses many of the complications of type II diabetes, including eye, nerve and kidney disease. The highest contributorto morbidity and mortality in type II diabetes is heart disease and, strikingly, type II diabetes is one of the main causesof heart disease in the Western world.

DB/DB MOUSE

A genetic mouse model of typeII diabetes and obesity. Thedefect has been mapped to thegene for the leptin receptor.


R E V I E W S

GLUT4 from the cell surface of adipocytes, it has beenshown that the transporter is transported through endo-somes into a perinuclear compartment that is distinctfrom recycling endosomes10. Using a similar approach,we have recently shown that this perinuclear compart-ment represents a subdomain of the TGN that alsocontains the SNARE proteins Syntaxin 6 and Syntaxin 16(A. Shewan, S. Martin, D. E. J., unpublished observa-tions). The transport of GLUT4 between endosomesand the TGN is regulated by a unique acidic targetingmotif in the carboxyl terminus of GLUT4 (REF. 19).Intriguingly, the transport of other proteins, such as thepro-protein CONVERTASES furin and PC6B, between endo-somes and the TGN is also regulated by acidic targetingmotifs20,21. The COAT-ASSOCIATED PROTEIN phosphofurinacidic cluster sorting protein 1 (PACS1) has been foundto bind to the acidic motif in the furin tail22. So far, wehave been unable to detect an interaction betweenPACS1 and GLUT4 (S. Rea, D. E. J., unpublished obser-vations), which indicates that other, related coat proteinsmight function in this specific region of the cell.

The recycling of membrane proteins through theTGN is unusual in that most endosomal proteins donot take this route. However, several molecules, includ-ing certain bacterial toxins23, mannose-6-phosphatereceptors, TGN38 and pro-protein convertases(reviewed in REF. 20), have been shown to follow thispathway. Once in the TGN, these molecules are sortedto one of many different destinations — this is animportant function of this organelle. For example,Shiga toxin is transported to the endoplasmic reticu-lum (ER), the mannose-6-phosphate receptors aretransported back to endosomes, and the pro-proteinconvertases enter the secretory pathway. Once GLUT4arrives at the TGN, its fate is uncertain. Despite the fact

significant pool of GLUT4 is not localized toendosomes10. Endosomes can be chemically ablated onuptake of horseradish peroxidase (HRP)-conjugatedtransferrin11. This procedure can be used to determinethe proportion of a protein that is localized to the endo-somal system, and has shown that only 30–40% ofGLUT4 is found in endosomes under basal conditions11.Furthermore, chemical ablation of endosomes does notblock insulin-stimulated GLUT4 translocation inadipocytes12. So, although insulin has a modest effect ongeneral recycling through the endosomal system, whichresults in the translocation of many molecules — includ-ing the TfR — to the plasma membrane, endosomes donot seem to be the main insulin-sensitive GLUT4 storagecompartment. Intriguingly, in fibroblasts, most GLUT4and IRAP colocalizes with the TfR in endosomes13. Thismight explain the relatively small insulin effect that isobserved in these cells (twofold increase), and also indi-cates that the transport of GLUT4 is more specialized inbona fide insulin-responsive cell types. Nevertheless,there is evidence for a selective retention mechanism ofGLUT4 and IRAP in fibroblasts, but this is clearly insuffi-cient to generate the robust insulin effect that is observedin adipocytes. Intriguingly, studies that use EVANESCENCE

WAVE MICROSCOPY in 3T3-L1 fibroblasts13, and an in vitroassay that reconstitutes budding of transport vesiclesfrom endosomes in Chinese hamster ovary (CHO)cells14, have shown that GLUT4 is packaged into endoso-mal transport vesicles that are distinct from those thatcontain the TfR. However, in fibroblasts, the GLUT4transport vesicles that bud from endosomes are veryshort-lived, presumably because they fuse rapidly withthe plasma membrane, even in the absence of insulin.This is clearly not the case in adipocytes, though, as thereis little exocytosis of GLUT4 under basal conditions6.Collectively, these studies implicate an important role forthe segregation of GLUT4 from the endosomal system inthe insulin-responsive transport of GLUT4.

The role of the trans-Golgi networkWhat is the fate of GLUT4-containing endosomal trans-port vesicles in adipocytes? One possibility is that theyare somehow retained in the cytosol in the absence ofinsulin, and are discharged in response to insulin. Thissimple model, however, overlooks the fact that a propor-tion of GLUT4, which does not represent newly synthe-sized protein, is present in the TGN of both muscle andfat cells15,16. Interestingly, recent evidence indicates thatGLUT4 recycles rapidly between the TGN and endo-somes. First, morphological studies in ATRIAL CARDIOMY-

OCYTES indicate that 60% of all the GLUT4 expressed inthis cell type is localized to secretory granules that con-tain atrial natriuretic factor (ANF)17. GLUT4 seems toenter these granules at the TGN, and as the localizationof GLUT4 here is not blocked by protein-synthesisinhibitors, these studies indicate that a GLUT4 recyclingpathway, possibly from endosomes, merges with thesecretory pathway at the TGN. Second, GLUT4 has beenshown to localize to adaptor-protein-1 (AP-1)positive,clathrin-coated, vesicles in the vicinity of the TGN inadipocytes18. Third, by following the internalization of

EVANESCENCE WAVE

MICROSCOPY

A technique in which onlyfluorophores within a100–220 nm field above a glasscoverslip are excited, whichallows localization of moleculesvery close to the cell surface.

ATRIAL CARDIOMYOCYTE

A heart muscle cell.

AP-1

(Adaptor protein complex 1).Adaptor proteins link cargomolecules on membranes withcoat proteins such as clathrin.Several classes of adaptorproteins have been identifiedand shown to be involved indifferent transport steps. AP-1 isthought to regulate transportfrom the trans-Golgi network toendosomes.

SNARE

(soluble N-ethylmaleimidesensitive factor attachmentprotein receptor). A family ofmembrane-tethered coiled-coilproteins that regulate fusionreactions and target specificity inthe vacuolar system. They can bedivided into v-SNAREs (vesicle)and t-SNAREs (target) on thebasis of their localization, or intoQ-SNAREs and R-SNAREs onthe basis of a highly conservedamino acid.

CONVERTASE

An enzyme that is responsiblefor protein activation throughproteolytic activity.

COAT-ASSOCIATED PROTEIN

A protein that links cargomolecules to vesicle coats.

Figure 2 | Insulin triggers the translocation of GLUT4 froman intracellular location to the plasma membrane ofadipocytes. The figure shows a confocal image of 3T3-L1adipocytes incubated either with (right panel), or without (leftpanel) 100 nM insulin for 15 mins. The location of GLUT4 inthese cells is shown using an antibody that specificallyrecognizes GLUT4 and a secondary antibody conjugated toAlexa-488 (shown in green). Confocal-laser-scanned sectionswere obtained from the base of the cells to the perinuclearregion, which were then stacked to create a three-dimensionalreconstruction. Images courtesy of Timo Meerloo, GarvanInstitute of Medical Research, Darlinghurst, Australia.

– Insulin + Insulin


R E V I E W S

The TGN is clearly a complex and central sorting sta-tion in which key sorting decisions are made. Many coat-protein complexes, including AP-1,AP-3,AP-4, as well asthe Golgi-localized, γ-EAR-CONTAINING,ARF-binding (GGA)family of coat proteins28 have been localized to the TGNand might regulate transport into or out of this organelle.Moreover, these coats are multisubunit protein com-plexes, and it has been shown that unique isoforms of justone component of a particular coat is sufficient to gener-ate cell-type specific sorting.At least a portion of GLUT4must be delivered back to endosomes to account for therelatively large pool (~30–40%) that is found in this com-partment. AP-1 coated vesicles have been proposed to

that GLUT4 re-enters the secretory pathway in atrialcardiomyocytes, it is subsequently retrieved from thesegranules before they are delivered to the cell surface,possibly by an AP-1-mediated pathway17. Consistentwith this, GLUT4 does not colocalize with other secre-tory proteins, such as the 30 kDa adipocyte comple-ment-related protein (ACRP30), leptin or adipsin, inadipocytes24–26. Nevertheless, it seems evident that tran-sit through the TGN probably precedes the packagingof GLUT4 into its insulin-responsive compartmentbecause prolonged incubation of adipocytes at 19°C —a temperature that blocks exit from the TGN —inhibits insulin action27.

ARNO

(ARF nucleotide binding-siteopener). This activates ADP-ribosylating factors (ARFs),which are known to have a rolein protein sorting and vesiclebudding.

γ-EAR-CONTAINING

This represents a proteindomain within the γ-subunit ofcoat adaptor proteins.

Box 2 | Insulin signalling pathways that control glucose transport in muscle and fat cells

At least two discrete signalling pathways have been implicatedin insulin-regulated GLUT4 translocation. The first involvesthe lipid kinase phosphatidylinositol 3-kinase (PI3K), and thesecond involves the proto-oncoprotein c-Cbl. Insulin binds toits receptor — a heterotetramer that is comprised of two α- and two β-subunits — on the surface of target cells. Thisbinding induces a conformational change in the receptor, andleads to activation of its tyrosine-kinase domain, which islocated within the intracellular portion of its β-subunits. Onactivation, the receptor phosphorylates several proximalsubstrates, including members of the insulin-receptor-substrate family (IRS-1 and IRS-2 being the most importantin muscle and fat cells) and c-Cbl. Tyrosine-phosphorylatedIRS proteins, which are thought to be held in close proximityto the plasma membrane through association with theunderlying cytoskeleton, recruit more effector molecules,such as PI3K, to this location. Substantial evidence indicatesthat the Class 1a PI3K might have an important role ininsulin-stimulated GLUT4 translocation, although a role forother PI3K isoforms cannot be excluded. Two importanttargets of PI3K in muscle and fat cells that have been shown tohave a role in insulin-stimulated GLUT4 translocation are theserine/threonine kinase Akt/protein kinase B (PKB) and theatypical protein kinase C (PKC) isoform, PKCζ. PI3Kactivates Akt by generating polyphosphoinositides in theinner leaflet of the plasma membrane. This acts as a dockingsite for Akt through its pleckstrin homology domain, therebybringing it in close proximity to its upstream regulatorykinase, phosphatidylinositol-dependent kinase-1 (PDK-1).The mechanism of activation of PKCζ, although not clear,might involve its recruitment to intracellular membranes, andindeed it has been shown to be present in intracellularGLUT4-containing vesicles. Although Akt and PKCζ haveboth been implicated in insulin action, there are numerousdownstream targets of PI3K — including proteins such asARNO that have a role in membrane transport — that mightalso be involved in the insulin regulation of GLUT4translocation. The second, putative signalling pathway thathas been shown to have a role in insulin-stimulated GLUT4translocation operates independently of PI3K and involves a dimeric complex that comprises c-Cbl and the c-Cbl-associated protein CAP. Intriguingly,whereas many growth factors trigger the activation of PI3K, Akt and PKCζ in many cell types, aspects of the c-Cbl–CAP pathway, including the tyrosinephosphorylation and the expression of CAP, seem to be unique to muscle and fat cells. Insulin triggers the movement of this dimeric c-Cbl–CAP complexinto cell-surface lipid rafts through association with the raft protein flotillin. Inhibition of this process inhibits insulin-stimulated GLUT4 translocation inadipocytes72. Tyrosine-phosphorylated c-Cbl then recruits a complex of CrkII, an adaptor protein, and C3G into lipid rafts. C3G is a guanine-nucleotide-exchange factor for the Rho-like GTPase TC10. Because TC10 is constitutively localized to lipid rafts, this catalyses GTP loading and, consequently,activation of TC10.

PKCζPI3K

AKT

PDK

TC10

CAP

Cbl

IRS-1

PKCζ

PKCζ

TC10

IRS-1

PDK

AKT

AKT

PI3K CAP

CblC3G

CrkII

Insulin receptor

Insulin

Polyphosphoinositides

GLUT4 translocation

– Insulin

+ Insulin

GDP

GTP

Flotillin

Lipid raft


R E V I E W S

These include retention mechanisms, dynamic sortingevents and the packaging of GLUT4 into a more sta-tionary population of secretory-type vesicles. It nowseems likely that these different models are not mutuallyexclusive, and indeed facets of each of them must beincorporated into a working model. We propose such amodel in FIG. 4. This model accommodates many of theapparently contradictory observations, and proposesthat GLUT4 transport is controlled by retention mecha-nisms and dynamic sorting, as well as by being pack-aged into a more stationary population of secretory-type vesicles. The main feature of this model is thatGLUT4 is selectively targeted to an intracellular trans-port loop between the TGN and the endosomes (cycle 2in FIG. 4). The entry of GLUT4 into this intracellular,seemingly futile, cycle probably excludes it from the cell-surface recycling pathway (cycle 1 in FIG. 4). This wouldaccount for the very low levels of GLUT4 at the cell sur-face in basal adipocytes compared with other proteinssuch as the TfR that do not enter this cycle. An essentialfeature of this model is that there is an intracellular storeof GLUT4 that represents a slowly exchanging pool thatmoves between the TGN and endosomes.

This intracellular store can presumably fuse witheither endosomes (in the absence of insulin), or withthe cell surface (in the presence of insulin). Severallines of evidence support the existence of this uniquepool of GLUT4 vesicles and the idea that it can fusedirectly with the cell surface in response to insulin. Inparticular, studies of the SNARE proteins that areinvolved in the docking and fusion of GLUT4 storagevesicles (GSVs) with the cell surface (BOX 3) have beenmost enlightening. Disrupting the function of theSyntaxin 4–SNAP23–VAMP2 SNARE complex selec-tively inhibits the insulin-stimulated translocation ofGLUT4 to the cell surface, but not other recycling pro-teins such as GLUT1. These data argue strongly infavour of a model in which a population of vesicles isready to move directly to the cell surface. Once formed,it is highly unlikely that these vesicles remain static inthe absence of an insulin signal, as the endosomal andTGN pools of GLUT4 would become depleted and allthe GLUT4 would be present in GSVs if this were thecase. It therefore seems likely that the GSVs slowly fusewith endosomes, and allow GLUT4 to re-enter theendosomal system. This TGN–endosomal recyclingpathway is not unique to insulin-responsive cells, butprobably exists in all cell types. Numerous examples ofproteins that are transported to the cell surface from anintracellular pool in response to external stimuli havebeen identified (TABLE 1). For example, aquaporin-2 — awater channel that is normally found in the TGN regionof renal epithelial cells — translocates to the plasmamembrane in response to the peptide hormone vaso-pressin. Intriguingly, the SNARE complex that controlsGLUT4 translocation is also responsible for the translo-cation of aquaporin-2.

A similar TGN–endosomal transport pathway hasbeen described in the budding yeast Saccharomyces cere-visiae to account for the upregulation of amino-acidpermeases on the cell surface that occurs in response to

have a role in sorting GLUT4 to endosomes. This wouldexplain the fact that GLUT4 colocalizes with molecules,such as the cation-dependent mannose-6-phosphatereceptor, (CD-MPR)18 that also follow this route.

GLUT4 storage vesiclesDespite the fact that GLUT4 is obviously engaged in arecycling loop between endosomes and the TGN, there isclear evidence for the existence of a more static secretorypool of GLUT4 that can move directly to the cell surfacein response to insulin. Using both morphological andbiochemical methods, a discrete population of small(50 nm diameter) vesicles have been identified29–31. Thesevesicles exclude other recycling proteins, such as the TfRand the CD-MPR, and are highly responsive to insulin.Importantly, these vesicles contain the v-SNARE, vesi-cle associated membrane protein (VAMP2) — thesame v-SNARE that is found in synaptic vesicles andaquaporin-2-containing vesicles — which indicates ageneric role for this molecule in regulated exocytosis inmany cell types. This v-SNARE has been shown to forma complex with the t-SNAREs Syntaxin 4 and SNAP23(BOX 3), which are highly enriched in the plasma mem-brane of fat and muscle cells. The identification of theseSNAREs has provided important clues about the mech-anism of GLUT4 translocation.

A model for GLUT4 transportSeveral models, based on the studies described above,have been proposed to explain the transport of GLUT4.

CD-MPR

(Cation-dependent mannose-6-phosphate receptor). Thisprotein shuttles between thetrans-Golgi network andendosomes.

Figure 3 | Relative GLUT4 distribution throughout organelles of cells from non-stimulatedand insulin-stimulated brown adipose tissue. Cryosections of brown adipose tissue wereimmunolabelled with anti-GLUT4 antibody and gold-conjugated Protein A. Gold particles werecounted and assigned to the following organelles: (1) trans-Golgi network (TGN); (2) tubulo–vesicular(T–V) elements located underneath the plasma membrane; (3) clusters of T–V elements; (4) T–Velements distributed throughout the cytoplasm; (5) T–V elements connected or close to lateendosomal vacuoles (6); (7) T–V elements connected or close to early endosomal vacuoles (8); (9)non-coated invaginations of the plasma membrane; (10) coated pits and vesicles; (11) plasmamembrane; (12) cytoplasm. The graph (right) shows the relative distribution of GLUT4 throughoutthese organelles. Reproduced with permission from REF. 16 ©1991 The Rockefeller University Press.

0 5 10 15 20 25 30 35

GLUT4 distribution (%)

Basal+ Insulin

1

2

34

5

67

8

9

10

11 12


R E V I E W S

adipocytes36. It remains to be seen if this is linked to itsubiquitylation or if there is a role for this process ininsulin resistance.

The futile cycle that is depicted in FIG. 4 might explainseveral observations that are related to GLUT4 trans-port. First, such a cycle might provide the basis for theconsiderable increase in the rate of GLUT4 exocytosis— compared with that of other proteins — in responseto insulin. This would explain the very large increase incell-surface levels of GLUT4. Second, it might explainhow different stimuli mobilize discrete intracellularpools of GLUT4. Most notably, in skeletal muscle exer-cise causes a large increase in GLUT4 translocation tothe plasma membrane, mainly from the endosomalpool rather than the GSVs (REF. 37). Similar observa-tions have been made using other agonists such as GTPγS

(REF. 38). Intriguingly, the regulation of GLUT4 move-ment from these different compartments seems to bequite unique. Whereas wortmannin, which inhibitsphosphatidylinositol 3-kinase (PI3K) activity, completelyinhibits insulin-stimulated GLUT4 translocation, it hasno effect on the translocation of GLUT4 that occurs in

external growth conditions. On rich nitrogen sources,the general amino-acid permease Gap1 is transported tothe vacuole, where it is degraded. By contrast, when cellsare grown on low nitrogen sources, Gap1 is transportedfrom an intracellular storage pool to the cell surface32.Intriguingly, a di-leucine-containing motif in the car-boxyl-terminus of Gap1, which is required for its regu-lated transport, resembles a motif that is required forthe insulin-sensitive transport of GLUT4 (REF. 33). Theregulated transport of Gap1 is controlled by the addi-tion of ubiquitin to the amino terminus of Gap1, whichseems to occur in the TGN. This is intriguing, as it hasbeen reported that GLUT4 is modified by the additionof the ubiquitin-like molecule sentrin, also known asSUMO1, in muscle cells34. So, it is tempting to speculatethat there might be a role for sumoylation and/or ubiq-uitylation in regulating the transport of GLUT4between the TGN and endosomes. Ubiquitylation hasrecently been shown to regulate the entry of membraneproteins into multivesicular bodies, which targets themfor degradation35. Intriguingly, chronic insulin treat-ment markedly reduces the stability of GLUT4 in

GTPγS

A non-hydrolysable analogue ofGTP.

Box 3 | The SNARE hypothesis

The multitude of membrane transport events that occurs in eukaryotic cells are controlled by families of proteins knownas SNAREs and SNARE-associated proteins. v-SNAREs (membrane proteins that are found in transport vesicles) bind ina highly specific manner to t-SNAREs (membrane proteins that are found on the relevant target membrane). Theformation of a stable, ternary complex between the correct set of SNARE proteins brings transport vesicles and targetmembranes into close proximity, and ultimately leads to their fusion. Although the precise role of SNAREs in membranedocking and fusion is still debated, these molecules and their associated proteins clearly have an important role inmembrane fusion. Membrane fusion can be broken down into the three distinct stages, as outlined in the figure.

Vesicle tethering. The small GTPase Rab family of proteins is responsible for tethering the transport vesicles to theappropriate target membrane. Rab proteins bind to specific transport vesicles and seem to function — through theirGTPase activity — as molecular switches, to recruit cytosolic effector molecules that are required for vesicle tethering todocking sites on the appropriate target membrane (reviewed in REF. 74).

Vesicle docking. After a transport vesicle is tethered to its target membrane, the formation of a stable ternary SNAREcomplex docks the transport vesicle onto the target membrane. The Sec1-like/Munc18 (SM) family adds a further level ofregulation to membrane fusion at this stage. SM proteins seem to have both a positive and negative role in SNARE-complex assembly. These proteins bind tightly to t-SNARE molecules and prevent ternary-complex formation. However,their binding also seems to be required to activate the t-SNARE for entry into the ternary complex. The formation of astable SNARE complex completes the docking stage of vesicular transport.

Membrane fusion. The docked vesicle fuses with the target membrane, where it delivers its contents. Every SNARE-dependent fusion event that has so far been identified to date requires the NEM (N-ethylmaleimide) sensitive factor(NSF) and its binding partner α-SNAP, but their precise role remains unclear.

The SNARE, Rab and SM protein families are all highly conserved throughout evolution, as well as throughout the cell. Asituation is emerging in many cellular systems, in which different members of these families mark different transportvesicles and target (or acceptor) membranes. The coordination of the various families of proteins that are involved inmembrane fusion results in a highly regulated process.

Targetmembrane

Transport vesicle

Rab TetherTethering FusionDocking

SNAP23v-SNARE

t-SNARES


R E V I E W S

referred to as ‘intrinsic activation’. First, kinetic studies inL6 myotubes have indicated that the insulin-dependentarrival of GLUT4 at the cell surface precedes the increasein glucose uptake by several minutes41. Intriguingly, asimilar difference is not observed in adipocytes42,43,which raises the possibility that transporters that translo-cate more slowly might account for the increase in glu-cose uptake in L6 cells. Second, a discrepancy in thedose-response effects of wortmannin on insulin-stimu-lated glucose transport compared with GLUT4 translo-cation have been observed in both 3T3-L1 adipocytes44

and L6 cells45. In both of these studies, glucose uptakewas inhibited at a much lower dose of wortmannin thanGLUT4 translocation, which indicates that these twoprocesses are clearly dissociated. Finally, an inhibitor ofthe mitogen-activated protein kinase (MAPK) isoformp38 inhibits insulin-stimulated glucose uptake withoutany apparent effect on GLUT4 translocation46. In addi-tion to these studies, several agents such as leptin47, iso-proterenol48 and dibutyryl cyclic AMP49 decrease glucoseuptake, whereas cycloheximide50 and adenosine48

increase glucose uptake without affecting the amount ofGLUT4 at the plasma membrane. An important limita-tion of the intrinsic-activation hypothesis is that a plau-sible biochemical mechanism for intrinsic activation ofGLUT4 is yet to be described. It is most likely that intrin-sic activation involves some type of covalent or struc-tural change in GLUT4. Several possible mechanisms,such as phosphorylation51, nucleotide binding52 and (atleast in the case of GLUT1) the formation of homo-oligomers53 have been proposed. Moreover, it has beenreported that GLUT4 can be detected in both clathrin-coated pits54 and caveolae55 at the cell surface inadipocytes, and it is possible that within these subdo-mains, the structure of GLUT4, and consequently itsactivity, is constrained in some way.

Integrating the transport with the signalsAs discussed above, there are several steps that areinvolved in maintaining the intracellular pool ofGLUT4 in the absence of insulin, any one of whichcould be a target of insulin action. GLUT4 is trans-ported between several intracellular compartmentseven in the absence of insulin, and this alone involvesselective retention mechanisms, vesicle-budding reac-tions that involve the binding of coat proteins such asAP-1 to GLUT4, the movement of vesicles alongcytoskeletal elements and the docking and fusion oftransport vesicles with their relevant target membrane.Organelles that are potential targets of insulin actioninclude the plasma membrane, endosomes and theTGN, which shows that a vast amount of transportmachinery is involved. An important question is: whichstep does insulin modulate to increase GLUT4 translo-cation to the cell surface? The lack of in vitro assays thatrecapitulate some of these stages of GLUT4 transporthas been a major limitation in answering this and otherquestions. Recently, an assay for the in vitro fusion ofintracellular vesicles that contain GLUT4 with plasmamembranes has been described56.Using this system, it wasshown that insulin modulates targets in both the vesicle

response to exercise or GTPγS (REF. 39). Furthermore, inadipocytes, overexpression of constitutively activemutants of a downstream target of PI3K, Akt, stimulatethe exocytosis of GSVs but not the endosomal pool40.These studies indicate that the exocytic cues that regu-late the movement of GLUT4 from different locationsare quite distinct, and so specificity is probably achievedby the use of a combination of discrete pools of intracel-lular GLUT4, each of which are coupled to unique regu-latory mechanisms. These studies also indicate that, atleast as far as insulin action is concerned, the regulationmight be quite similar in both muscle and fat cells.

Intrinsic activationCan translocation of GLUT4 to the plasma membraneaccount for the stimulatory effects of insulin on glucosetransport in muscle and fat cells, or is its ability to trans-port glucose also subject to regulation? Several recentstudies have shown that insulin-stimulated transportand GLUT4 translocation can be dissociated from eachother under certain conditions, which indicates thatthere might be further means of regulating the transportproperties of GLUT4 — a phenomenon previously

Figure 4 | A model that depicts the transport of GLUT4 in insulin-responsive cells. Themodel depicts two main intracellular-recycling pathways: cycle 1, between the cell surface andendosomes; and cycle 2, between the trans-Golgi network (TGN) and endosomes. GLUT4transport is intricately controlled at several points along these cycles. On entry into the endosomalsystem, GLUT4 is selectively retained at the expense of other recycling transport, such as thetransferrin receptor that constitutively moves through cycle 1. This retention mechanism mightpredispose GLUT4 for sorting into transport vesicles that bud slowly from the endosome and thatare targeted to the TGN. GLUT4 is sorted into a secretory pathway in the TGN. This sorting stepprobably involves a specialized population of secretory vesicles that excludes other secretorycargo, and that does not fuse constitutively with the plasma membrane. Vesicles that emerge fromthis sorting step, which we have previously referred to as GLUT4 storage vesicles or GSVs, mightconstitute most of the GLUT4 that is excluded from the endosomal system. In the absence ofinsulin, GSVs might slowly fuse with endosomes, thereby accounting for the presence of asignificant but small pool of GLUT4 in endosomes, even in the absence of insulin. Insulin wouldthen shift GLUT4 from this TGN–endosome cycle to a pathway that takes GLUT4 directly to thecell surface. The inset shows the SNARE proteins that are thought to regulate docking and fusionof GSVs with the cell surface (reviewed in REF. 73). The t-SNAREs Syntaxin 4 and SNAP23 in theplasma membrane of fat and muscle cells form a ternary complex with the v-SNARE VAMP2,which is present on GSVs. Munc18c has been identified as the SM (Sec1-like/Munc18 family)protein (BOX 3) that controls the formation of this ternary complex.

Plasma membrane

Earlyendosome

Recyclingendosome

Cycle 1

Cycle 2

+ Insulin

Trans-Golgi network

Transportvesicle

GLUT4storagevesicles

SNAP23

VAMP2

Munc18cSyntaxin4

Cytoplasm


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Synip with Syntaxin 4 is reduced on stimulation withinsulin58, but how this dissociation is achieved remainsunknown. Similarly, the VAMP2-binding proteinspantophysin59 and vesicle-associated protein 33(VAP33)60 have been proposed to prevent the entry ofthe v-SNARE into the ternary complex in the absence ofinsulin, but again, the signal that transduces this is notknown. Intriguingly, insulin stimulates the GTP-loadingof Rab4, and GTP–Rab4 is known to bind Syntaxin 4(REFS 61,62). Furthermore, insulin or overexpression ofPKCζ induces serine phosphorylation of VAMP2 in pri-mary cultures of rat skeletal muscle63. So, we can imag-ine that GLUT4 vesicles that are formed from eitherendosomes or the TGN constantly sample the cell sur-face, but that their fusion is limited by the availability oftethering and/or docking sites. Insulin might overcome

and the plasma membranes. So, these data support thenotion that there are several signalling pathways thatconverge on different aspects of GLUT4 transport. Insupport of this, whereas Akt is activated at the plasmamembrane, another downstream target of PI3K, proteinkinase Cζ (PKCζ), is selectively activated inendosomes57. So, it might be of interest to look for spe-cific substrates of each of these kinases at these discretecellular locations.

The most likely targets of insulin action at the cellsurface are the SNARE proteins (BOX 3). Several proteinshave been implicated in regulating the formation of theternary complex — which consists of Syntaxin 4,SNAP23 and VAMP2 — in response to insulin. Forexample, Synip binds to Syntaxin 4 and preventsVAMP2, but not SNAP23, binding. The association of

Table 1 | Proteins that translocate after stimulus (in addition to GLUT4 and IRAP)

Name Cell type Stimulus Intracellular Remarks Referenceslocalization

General amino acid S. cerevisiae Poor nitrogen source Golgi Sec13 dependent 32permease Gap1 (ammonia/urea)

Aquaporin-1 Rat peritoneal Hyperosmotic stimulus Endosomal 75mesothelial cells

Cholangiocytes Secretin Unknown Inhibited at 20ºC and by colchicine 76

Aquaporin-2 Renal epithelium Arginine vasopressin/ trans-Golgi Translocation blocked at 20ºC and by bafilomycin 77–84(renal inner medullary forskolin network A1; VAMP2, Syntaxin-4 and SNAP23 probablycollecting-duct cells) (TGN) involved; cyclic AMP and PKA involved (PKA-mediated

phosphorylation of AQP-2 is probably required fortranslocation); mutation of phosphorylation siteSer256 blocks translocation; might involveheterotrimeric G proteins (Gαi); okadaic acid inducestranslocation independent of AQP-2 phosphorylation;AQP-2 recycles in absence and presence of stimulus

Epithelial Na Renal epithelium cAMP agonists Unknown Process inhibited at 15ºC; PPPXY 85,86channel (ENaC) sequence is involved

Na+-K+-ATPase Kidney epithelium Insulin/arginine Unknown Translocation accompanied by subunit 87–91vasopressin dephosphorylation (insulin + AVP); inhibited by

wortmannin (insulin)Skeletal muscle Exercise/insulin Unknown

Na+/H+ exchanger Renal and intestinal bFGF Recycling Blocked by PI3K inhibitors 92,93NHE3 epithelial cells endosomes

Calcium channel Neuronal cells IGF-1, PDGF, head Unknown Translocation is wortmannin sensitive 94,95GRC activator (neuropeptide)

N-type calcium channel Neuronal cell KCl, ionomycin, Unknown Translocation is BFA-insensitive 96PKC activation

8 pS chloride channel Renal epithelium PKA activation Unknown Translocation is BFA-sensitive 97

H+/K+-ATPase Gastric parietal cells Histamine Vesicles 98

Menkes protein Ubiquitously Copper TGN 99,100MNK expressed

GABA transporter GAT1 Neuronal cells PKC activation (PMA) Unknown 101

Glutamate Neuronal cells PDGF Unknown Translocation inhibited by wortmannin and 102transporter EAAC1 LY 294002, not by PKC inhibitor BisII

Flt3 ligand (growth T lymphocytes Bone-marrow failure Perinuclear Translocation not due to de novo protein 103,104factor for (chemotherapy); synthesis haematopoietic cells) IL-2, -4, -7, -15

κ opioid Magnocellular Salt loading AVP-containing Occurs during neuropeptide release; removed from 105receptor KOR1 neurosecretory secretory plasma membrane within 1 hr of stimulation

neurons vesicles

bFGF, basic fibroblast growth factor; IGF, insulin growth factor; IL, interleukin; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinaseA; PKC, protein kinase C; BFA, brefeldin A.


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tactic to avoid appearing at the cell surface. Elements ofthis system are absent from ‘non-insulin-responsive’ celltypes. So, the adaptations that occur during muscle andfat differentiation to allow the entry of GLUT4 into thisintracellular loop are clearly an important area forfuture study.

We imagine that the complex transport itinerary ofGLUT4 is governed by the protein encountering differ-ent coat complexes throughout the cell. Some of thesewe know, including AP-2 at the cell surface, and AP-1 atthe TGN. But the coats that regulate transport betweenendosomes and the TGN are not yet known. These areprobably somewhat specialized, perhaps by beingexpressed uniquely in insulin-responsive cells and/or bybeing resistant to the effects of BREFELDIN A (BFA).

In addition to GLUT4, GSVs also contain IRAP andVAMP2. The identification of the latter has allowedhuge inroads to our understanding of the docking andfusion of GSVs with the plasma membrane. TheSNAREs and some of their associated proteins are nowknown. Such discoveries will provide a template for thediscovery of new molecules that might be unique to theinsulin-stimulated transport of GLUT4 in muscle andfat cells. A large gap in our current knowledge of theexocytosis of GLUT4 is the identity of the Rab proteinthat is involved in delivery of the transport to the cellsurface. Although Rab4 has been implicated in thisprocess, it might be involved in less specialized aspectsof the transport itinerary of GLUT4, in which case theRab that is responsible for the delivery of GSVs to theplasma membrane remains to be identified.

Perhaps the ultimate question is, what does insulindo? Without more complete answers to the above twoquestions, this question will probably remain unan-swered. Although our knowledge of signalling hasadvanced tremendously over the past few years, we havenot, as yet, identified the intersection point of theinsulin-signalling pathway with the GLUT4 transportpathway. This intersection point might be governed bycoat proteins, cytoskeletal elements, the SNARE pro-teins or, more probably, a combination of all three.Identification of this intersection point will require aconvergence of different approaches. New approachessuch as DNA microarrays will provide knowledge of thegenes that are uniquely expressed in muscle and fat cells,and will offer new insights into the dynamic and regu-lated characteristics of GLUT4 transport. Finally, thedevelopment of in vitro assays that reconstitute variousaspects of insulin-stimulated GLUT4 translocation willbe required for the discovery and characterization ofkey molecules that are involved in this process. All ofthis knowledge will contribute to our understanding ofboth cell biology and type II diabetes.

this barrier by modulating auxiliary regulatory proteinssuch as the Rab proteins or Synip.

If each of the signalling pathways that are implicatedin insulin action (BOX 2) were assigned discrete functionsin the GLUT4-recruitment process, we would predictthat activation of each signalling intermediate on itsown would have little or no effect compared with that ofinsulin. However, in the case of the TC10 pathway thisdoes not seem to be the case. Overexpression of consti-tutively active forms of PI3K (REF. 64), Akt65 or PKC63, butnot TC10 (REF. 66), has a robust stimulatory effect onGLUT4 translocation that is similar to that observedwith insulin. One interpretation of these data is that theTC10 pathway regulates a factor, or process, that is per-missive for GLUT4 translocation to the cell surface. Onesuch process that has recently been proposed is the reg-ulation of the actin cytoskeleton67, which is consistentwith the generalized role of Rho family members inactin rearrangement. Considerable evidence supports arole for the actin cytoskeleton in insulin-stimulated glu-cose transport. Agents that depolymerize actin inhibitGLUT4 translocation68 and, although controversial, ithas been suggested that insulin might modulate the cor-tical actin cytoskeleton in adipocytes69. This leads to amodel in which actin might be involved in tethering theGLUT4 vesicles at the cell surface, and this might pre-cede the docking/fusion step. More recently, it has beenshown that insulin stimulates the formation of actintails that are associated with GLUT4-containing mem-branes70, which raises the possibility that actin might beinvolved in propelling the vesicles towards the cell sur-face. In either case, we can imagine that this step, whichis regulated by the TC10 pathway, might not be suffi-cient to activate GLUT4 translocation, and this mightalso explain why activation of either the Akt or PKCpathways on their own might overcome the need forthis pathway. So, until the function of TC10 has beenmore clearly defined, with particular attention to theidentification of its downstream targets in muscle andfat cells, it is difficult to assign an important role for thispathway in insulin action.

Conclusions and perspectivesSo, to return to the central questions that we posed atthe beginning of this review: how is GLUT4 transportedfrom one organelle to another, and what is the rela-tionship between these pathways and the intracellularsequestration of GLUT4 in the absence of insulin? Theintracellular movement of GLUT4 is complex, andinvolves many organelles and perhaps also a uniquestorage compartment — GSVs. In the absence ofinsulin, GLUT4 is trapped in an intracellular circuitbetween endosomes and the TGN as a diversionary

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Online links

DATABASESThe following terms in this article are linked online to:Interpro: http://www.ebi.ac.uk/interpro/pleckstrin homology domainLocusLink: http://www.ncbi.nlm.nih.gov/LocusLinkAkt | TfROMIM: http://www.ncbi.nlm.nih.gov/OmimType II diabetesSwiss-Prot: http://www.expasy.ch/ACRP30 | adipsin | ANF | c-Cbl | Gap1 | GLUT1 | GLUT4 | GLUT5 |GLUT8 | GLUT9 | GLUT10 | GLUT11 | IRS-1 | IRS-2 | p38 | PKCζ |Rab4 | sentrin | SNAP23 | Syntaxin 4 | Syntaxin 6 | Syntaxin 16 |Synip | TC10 | TGN38 | VAMP2 | VAP33Access to this interactive links box is free online.

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