REVIEWS

REGULATED TRANSPORT OF THE GLUT4

Nia J. Bryant, Roland Govers and David E. James In muscle and fat cells, insulin stimulates the delivery of the glucose transporter GLUT4 from an intracellular location to the cell surface, where it facilitates the reduction of plasma glucose levels. Understanding the molecular mechanisms that mediate this translocation event involves integrating our knowledge of two fundamental processes — the signal transduction pathways that are triggered when insulin binds to its receptor and the membrane transport events that need to be modified to divert GLUT4 from intracellular storage to an active plasma membrane shuttle service.

FACILITATIVE SUGAR Glucose is a fundamental source of energy for all glucose-transport system, the activity of which can be TRANSPORTER eukaryotic cells. In humans, although all cells use glu- rapidly upregulated to allow these tissues to increase A polytopic cose for their energy needs, the main consumer under their rate of glucose transport by 10–40-fold within that transports sugars down a basal conditions is the , which accounts for as minutes of exposure to a particular stimulus (reviewed concentration gradient in an energy-independent manner. much as 80% of whole-body consumption. The energy in REF.1). This system is crucial during exercise, when is provided by the breakdown of endogenous glycogen the metabolic demands of skeletal muscle can increase TYPE II DIABETES stores that are primarily in the . These whole-body more than 100-fold, and during the absorptive period Also known as non-insulin- energy stores are replenished from glucose in the diet, (after a meal), to facilitate the rapid insulin-dependent dependent diabetes or maturity onset diabetes. which, after being digested and absorbed across the gut storage of glucose in muscle and adipose tissue, so pre- wall, is distributed among the various tissues of the body venting large fluctuations in blood glucose levels. (reviewed in REF.1). This distribution process involves a Dysfunctional glucose uptake into muscle and fat cells family of transport proteins — called GLUTs — which contributes to the onset of TYPE II DIABETES (BOX 1). act as shuttles to move sugar across the cell surface. In 1980, it was reported that, in rat adipocytes, These polytopic membrane proteins (FIG. 1) form an insulin triggers the movement of the sugar transporter aqueous pore across the membrane through which that is found in these cells from an intracellular store to glucose can move. A large family of FACILITATIVE SUGAR the plasma membrane2,3. This translocation hypothesis TRANSPORTERS exists in mammals, the individual mem- was later confirmed when GLUT4 was identified as the bers of which differ in their tissue distribution and main glucose transporter in these cells. GLUT4, which kinetic properties, as well as in their intracellular local- is expressed primarily in muscle and fat cells, is found in ization. The latter property is of particular interest for a complex intracellular tubulo–vesicular network that is glucose transport in specialized cell types, and provides connected to the endosomal–trans-Golgi network the basis for polarized glucose transport in epithelial (TGN) system. In the absence of stimulation, GLUT4 is cells, such as those in the gut, or for acute regulation of almost completely excluded from the plasma mem- Garvan Institute of Medical glucose transport, as is observed in muscle and fat cells brane (FIG. 2). The addition of insulin, or exercise in the Research, 384 Victoria Road, after a meal. Many mammalian tissues, such as the case of muscle cells, causes GLUT4 to shift from its Darlinghurst, New South brain, have a constitutively high glucose requirement intracellular location to the plasma membrane (FIG. 2). Wales 2010,Australia. and have been endowed with transporters that are con- Several observations indicate that GLUT4 has a crucial Correspondence to D.E.J. e-mail: stitutively targeted to the cell surface (for example, role in whole-body glucose homeostasis. First, insulin- [email protected] GLUTs 1–3). By contrast, certain tissues, such as muscle stimulated glucose transport is an important rate-limit- DOI: 10.1038/nrm782 and adipose tissue, have acquired a highly specialized ing step for glucose in both muscle and fat

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Sugar moiety an insulin-regulated step(s). Although many important signalling molecules that are integral to the insulin reg- ulation of GLUT4 translocation have been identified (BOX 2), any convergence between these two approaches remains to be achieved. In this review, we focus on our Plasma membrane cell-biological understanding of GLUT4 transport, and highlight potential regulatory sites of the insulin- NH Cytoplasm 2 signalling cascade. COOHH GLUT4 transport GLUT4 is found in many organelles, including the plasma membrane, sorting endosomes, recycling endo- somes, the TGN and vesicles that mediate the transport Figure 1 | Schematic representation of the GLUT family of proteins. The GLUT family of of GLUT4 between these compartments (FIG. 3). proteins comprises 13 members at present, which are predicted to span the membrane 12 times Presumably this localization represents a complex and with both amino- and carboxyl-termini located in the . On the basis of sequence homology and structural similarity, three subclasses of sugar transporters have been defined: Class I (GLUTs dynamic transport itinerary, and it raises several impor- 1–4) are glucose transporters; Class II (GLUTs 5, 7, 9 and 11) are fructose transporters; and Class tant questions. How does GLUT4 transport from one III (GLUTs 6, 8, 10,12 and HMIT1) are structurally atypical members of the GLUT family, which are organelle to another, what is the relationship between poorly defined at present. The diagram shows a homology plot between GLUT1 and GLUT4. these pathways and the intracellular sequestration of Residues that are unique to GLUT4 are shown in red. GLUT4 in basal cells, and which of these steps does insulin influence to affect GLUT4 exocytosis? In non-stimulated adipocytes, the rate of GLUT4 exo- tissue, and is severely disrupted in type II diabetes1 (BOX 1); cytosis is 10-fold slower than that of the transferrin recep- second, disruption of GLUT4 expression in mice results tor (TfR) — one of the most well-studied constitutive in insulin resistance4; and overexpression of GLUT4 recycling proteins in mammalian cells6,7. To account for ameliorates diabetes in the DB/DB MOUSE model5. this, GLUT4 must be selectively retained in one of its Analysing the molecular and cellular regulation of intracellular locations, packaged into a specialized com- GLUT4 transport not only promises to provide new partment that remains static in the absence of insulin, or insights into protein sorting, but could also yield new involved in a dynamic intracellular transport loop that targets for therapeutic intervention in what could well excludes it from recycling endosomal vesicles. Current be one of the most prevalent diseases that we will have evidence favours a role for all three mechanisms, which to confront in the future. emphasizes the complexity of this process. Another pro- Understanding the regulation of GLUT4 and glucose tein, the insulin-responsive aminopeptidase (IRAP), transport has proved to be extremely challenging, prin- which was recently described as a receptor for angiotensin cipally because it involves several signal-transduction IV (REF.8), colocalizes with GLUT4 and is transported in a pathways that are superimposed on a complex series of very similar manner9. Below, we discuss studies concern- vesicle transport processes. Insulin binds to a surface ing the transport of either GLUT4 or IRAP,with a partic- receptor on muscle and fat cells and triggers a cascade of ular focus on adipocytes and muscle cells. We also com- signalling events (BOX 2) that culminates in GLUT4 pare this with transport in cells such as fibroblasts that are translocation. Studies of this process have been carried not, or only mildly, responsive to insulin, as key differ- out using two approaches.‘Outside–in’ approaches have ences have been identified that provide new insights into largely focused on mapping insulin-specific signalling our understanding of insulin action. pathways in muscle and fat cells with the view to identi- fying downstream targets that directly control GLUT4 Endosomal sorting of GLUT4 translocation. Conversely,‘inside–out’ approaches have Morphological studies in both muscle and fat cells used cell-biological studies to map the intracellular indicate that, although there is some overlap of GLUT4 transport itinerary of GLUT4 with the aim of identifying with markers of the endocytic system such as the TfR, a

Box 1 | Type II diabetes The prevalence of type II diabetes is increasing at an alarming rate. In 1998, 143 million people worldwide suffered from this disease, and it is likely that this number will double over the next 20–30 years71. The incidence of type II diabetes increases sharply with age, and it is highly prevalent in certain ethnic groups. For example, 10–30% of Australian aborigines are currently thought to have type II diabetes, and this number is predicted to increase to more than 50% in the next ten years. The disease is characterized by defective insulin action, a condition that is referred to as insulin resistance. Insulin resistance is characterized by dysfunctional glucose uptake into muscle and adipose tissue, in DB/DB MOUSE conjunction with an oversupply of glucose from the liver, which results in high circulating plasma glucose levels. This A genetic mouse model of type causes many of the complications of type II diabetes, including eye, nerve and kidney disease. The highest contributor II diabetes and obesity. The to morbidity and mortality in type II diabetes is disease and, strikingly, type II diabetes is one of the main causes defect has been mapped to the of heart disease in the Western world. gene for the leptin receptor.

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significant pool of GLUT4 is not localized to – Insulin + Insulin endosomes10. Endosomes can be chemically ablated on uptake of horseradish peroxidase (HRP)-conjugated transferrin11. This procedure can be used to determine the proportion of a protein that is localized to the endo- somal system, and has shown that only 30–40% of GLUT4 is found in endosomes under basal conditions11. Furthermore, chemical ablation of endosomes does not block insulin-stimulated GLUT4 translocation in adipocytes12. So, although insulin has a modest effect on general recycling through the endosomal system, which results in the translocation of many molecules — includ- ing the TfR — to the plasma membrane, endosomes do not seem to be the main insulin-sensitive GLUT4 storage Figure 2 | Insulin triggers the translocation of GLUT4 from compartment. Intriguingly, in fibroblasts, most GLUT4 an intracellular location to the plasma membrane of and IRAP colocalizes with the TfR in endosomes13.This adipocytes. The figure shows a confocal image of 3T3-L1 might explain the relatively small insulin effect that is adipocytes incubated either with (right panel), or without (left observed in these cells (twofold increase), and also indi- panel) 100 nM insulin for 15 mins. The location of GLUT4 in these cells is shown using an antibody that specifically cates that the transport of GLUT4 is more specialized in recognizes GLUT4 and a secondary antibody conjugated to bona fide insulin-responsive cell types. Nevertheless, Alexa-488 (shown in green). Confocal-laser-scanned sections there is evidence for a selective retention mechanism of were obtained from the base of the cells to the perinuclear GLUT4 and IRAP in fibroblasts, but this is clearly insuffi- region, which were then stacked to create a three-dimensional EVANESCENCE WAVE cient to generate the robust insulin effect that is observed reconstruction. Images courtesy of Timo Meerloo, Garvan MICROSCOPY Institute of Medical Research, Darlinghurst, Australia. A technique in which only in adipocytes. Intriguingly, studies that use EVANESCENCE fluorophores within a WAVE MICROSCOPY in 3T3-L1 fibroblasts13, and an in vitro 100–220 nm field above a glass assay that reconstitutes budding of transport vesicles coverslip are excited, which from endosomes in Chinese hamster ovary (CHO) GLUT4 from the cell surface of adipocytes, it has been allows localization of molecules 14 very close to the cell surface. cells , have shown that GLUT4 is packaged into endoso- shown that the transporter is transported through endo- mal transport vesicles that are distinct from those that somes into a perinuclear compartment that is distinct ATRIAL CARDIOMYOCYTE contain the TfR. However, in fibroblasts, the GLUT4 from recycling endosomes10. Using a similar approach, A heart muscle cell. transport vesicles that bud from endosomes are very we have recently shown that this perinuclear compart- short-lived, presumably because they fuse rapidly with ment represents a subdomain of the TGN that also AP-1 (Adaptor protein complex 1). the plasma membrane, even in the absence of insulin. contains the SNARE proteins Syntaxin 6 and Syntaxin 16 Adaptor proteins link cargo This is clearly not the case in adipocytes, though, as there (A. Shewan, S. Martin, D. E. J., unpublished observa- molecules on membranes with is little exocytosis of GLUT4 under basal conditions6. tions). The transport of GLUT4 between endosomes coat proteins such as clathrin. Collectively, these studies implicate an important role for and the TGN is regulated by a unique acidic targeting Several classes of adaptor proteins have been identified the segregation of GLUT4 from the endosomal system in motif in the carboxyl terminus of GLUT4 (REF. 19). and shown to be involved in the insulin-responsive transport of GLUT4. Intriguingly, the transport of other proteins, such as the different transport steps. AP-1 is pro-protein CONVERTASES furin and PC6B, between endo- thought to regulate transport The role of the trans-Golgi network somes and the TGN is also regulated by acidic targeting from the trans-Golgi network to What is the fate of GLUT4-containing endosomal trans- motifs20,21. The COAT-ASSOCIATED PROTEIN phosphofurin endosomes. port vesicles in adipocytes? One possibility is that they acidic cluster sorting protein 1 (PACS1) has been found 22 SNARE are somehow retained in the cytosol in the absence of to bind to the acidic motif in the furin tail . So far, we (soluble N-ethylmaleimide insulin, and are discharged in response to insulin. This have been unable to detect an interaction between sensitive factor attachment simple model, however, overlooks the fact that a propor- PACS1 and GLUT4 (S. Rea, D. E. J., unpublished obser- protein receptor). A family of membrane-tethered coiled-coil tion of GLUT4, which does not represent newly synthe- vations), which indicates that other, related coat proteins proteins that regulate fusion sized protein, is present in the TGN of both muscle and might function in this specific region of the cell. reactions and target specificity in fat cells15,16. Interestingly, recent evidence indicates that The recycling of membrane proteins through the the vacuolar system. They can be GLUT4 recycles rapidly between the TGN and endo- TGN is unusual in that most endosomal proteins do divided into v-SNAREs (vesicle) somes. First, morphological studies in ATRIAL CARDIOMY- not take this route. However, several molecules, includ- and t-SNAREs (target) on the 23 basis of their localization, or into OCYTES indicate that 60% of all the GLUT4 expressed in ing certain bacterial toxins , mannose-6-phosphate Q-SNAREs and R-SNAREs on this cell type is localized to secretory granules that con- receptors, TGN38 and pro-protein convertases the basis of a highly conserved tain atrial natriuretic factor (ANF)17. GLUT4 seems to (reviewed in REF. 20), have been shown to follow this amino acid. enter these granules at the TGN, and as the localization pathway. Once in the TGN, these molecules are sorted

CONVERTASE of GLUT4 here is not blocked by protein-synthesis to one of many different destinations — this is an An that is responsible inhibitors, these studies indicate that a GLUT4 recycling important function of this organelle. For example, for protein activation through pathway, possibly from endosomes, merges with the Shiga toxin is transported to the endoplasmic reticu- proteolytic activity. secretory pathway at the TGN. Second, GLUT4 has been lum (ER), the mannose-6-phosphate receptors are shown to localize to adaptor-protein-1 (AP-1)positive, transported back to endosomes, and the pro-protein COAT-ASSOCIATED PROTEIN A protein that links cargo clathrin-coated, vesicles in the vicinity of the TGN in convertases enter the secretory pathway. Once GLUT4 molecules to vesicle coats. adipocytes18. Third, by following the internalization of arrives at the TGN, its fate is uncertain. Despite the fact

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ARNO that GLUT4 re-enters the secretory pathway in atrial The TGN is clearly a complex and central sorting sta- (ARF nucleotide binding-site cardiomyocytes, it is subsequently retrieved from these tion in which key sorting decisions are made. Many coat- opener). This activates ADP- granules before they are delivered to the cell surface, protein complexes, including AP-1, AP-3, AP-4, as well as ribosylating factors (ARFs), possibly by an AP-1-mediated pathway17. Consistent the Golgi-localized, γ-EAR-CONTAINING, ARF-binding (GGA) which are known to have a role 28 in protein sorting and vesicle with this, GLUT4 does not colocalize with other secre- family of coat proteins have been localized to the TGN budding. tory proteins, such as the 30 kDa adipocyte comple- and might regulate transport into or out of this organelle. ment-related protein (ACRP30), leptin or adipsin,in Moreover, these coats are multisubunit protein com- γ-EAR-CONTAINING adipocytes24–26. Nevertheless, it seems evident that tran- plexes, and it has been shown that unique isoforms of just This represents a protein domain within the γ-subunit of sit through the TGN probably precedes the packaging one component of a particular coat is sufficient to gener- coat adaptor proteins. of GLUT4 into its insulin-responsive compartment ate cell-type specific sorting. At least a portion of GLUT4 because prolonged incubation of adipocytes at 19°C — must be delivered back to endosomes to account for the a temperature that blocks exit from the TGN — relatively large pool (~30–40%) that is found in this com- inhibits insulin action27. partment. AP-1 coated vesicles have been proposed to

Box 2 | Insulin signalling pathways that control glucose transport in muscle and fat cells At least two discrete signalling pathways have been implicated – Insulin Flotillin in insulin-regulated GLUT4 translocation. The first involves the lipid kinase phosphatidylinositol 3-kinase (PI3K), and the Insulin receptor Lipid raft second involves the proto-oncoprotein c-Cbl. Insulin binds to its receptor — a heterotetramer that is comprised of two α- and two β-subunits — on the surface of target cells. This binding induces a conformational change in the receptor, and leads to activation of its tyrosine-kinase domain, which is TC10 β located within the intracellular portion of its -subunits. On GDP activation, the receptor phosphorylates several proximal PDK CAP substrates, including members of the insulin-receptor- IRS-1 substrate family (IRS-1 and IRS-2 being the most important Cbl PI3K in muscle and fat cells) and c-Cbl. Tyrosine-phosphorylated AKT IRS proteins, which are thought to be held in close proximity PKCζ to the plasma membrane through association with the underlying cytoskeleton, recruit more effector molecules, Insulin such as PI3K, to this location. Substantial evidence indicates + Insulin that the Class 1a PI3K might have an important role in insulin-stimulated GLUT4 translocation, although a role for Polyphosphoinositides other PI3K isoforms cannot be excluded. Two important targets of PI3K in muscle and fat cells that have been shown to have a role in insulin-stimulated GLUT4 translocation are the serine/threonine kinase Akt/protein kinase B (PKB) and the atypical protein kinase C (PKC) isoform, PKCζ.PI3K TC10 PI3K PDK CAP activates Akt by generating polyphosphoinositides in the PKCζ GTP IRS-1 AKT C3G inner leaflet of the plasma membrane. This acts as a docking Cbl site for Akt through its pleckstrin homology domain,thereby CrkII bringing it in close proximity to its upstream regulatory kinase, phosphatidylinositol-dependent kinase-1 (PDK-1). The mechanism of activation of PKCζ, although not clear, PKCζ might involve its recruitment to intracellular membranes, and AKT indeed it has been shown to be present in intracellular GLUT4-containing vesicles. Although Akt and PKCζ have both been implicated in insulin action, there are numerous downstream targets of PI3K — including proteins such as GLUT4 translocation ARNO that have a role in membrane transport — that might also be involved in the insulin regulation of GLUT4 translocation. The second, putative signalling pathway that has been shown to have a role in insulin-stimulated GLUT4 translocation 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 tyrosine phosphorylation and the expression of CAP,seem to be unique to muscle and fat cells. Insulin triggers the movement of this dimeric c-Cbl–CAP complex into cell-surface lipid rafts through association with the raft protein flotillin. Inhibition of this process inhibits insulin-stimulated GLUT4 translocation in adipocytes72. 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.

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CD-MPR have a role in sorting GLUT4 to endosomes. This would These include retention mechanisms, dynamic sorting (Cation-dependent mannose-6- explain the fact that GLUT4 colocalizes with molecules, events and the packaging of GLUT4 into a more sta- phosphate receptor). This such as the cation-dependent mannose-6-phosphate tionary population of secretory-type vesicles. It now protein shuttles between the receptor, (CD-MPR)18 that also follow this route. seems likely that these different models are not mutually trans-Golgi network and endosomes. exclusive, and indeed facets of each of them must be GLUT4 storage vesicles incorporated into a working model. We propose such a Despite the fact that GLUT4 is obviously engaged in a model in FIG. 4. This model accommodates many of the recycling loop between endosomes and the TGN, there is apparently contradictory observations, and proposes clear evidence for the existence of a more static secretory that GLUT4 transport is controlled by retention mecha- pool of GLUT4 that can move directly to the cell surface nisms and dynamic sorting, as well as by being pack- in response to insulin. Using both morphological and aged into a more stationary population of secretory- biochemical methods, a discrete population of small type vesicles. The main feature of this model is that (50 nm diameter) vesicles have been identified29–31. These GLUT4 is selectively targeted to an intracellular trans- vesicles exclude other recycling proteins, such as the TfR port loop between the TGN and the endosomes (cycle 2 and the CD-MPR, and are highly responsive to insulin. in FIG. 4). The entry of GLUT4 into this intracellular, Importantly, these vesicles contain the v-SNARE, vesi- seemingly futile, cycle probably excludes it from the cell- cle associated membrane protein (VAMP2) — the surface recycling pathway (cycle 1 in FIG. 4). This would same v-SNARE that is found in synaptic vesicles and account for the very low levels of GLUT4 at the cell sur- aquaporin-2-containing vesicles — which indicates a face in basal adipocytes compared with other proteins generic role for this molecule in regulated exocytosis in such as the TfR that do not enter this cycle. An essential many cell types. This v-SNARE has been shown to form feature of this model is that there is an intracellular store a complex with the t-SNAREs Syntaxin 4 and SNAP23 of GLUT4 that represents a slowly exchanging pool that (BOX 3), which are highly enriched in the plasma mem- moves between the TGN and endosomes. brane of fat and muscle cells. The identification of these This intracellular store can presumably fuse with SNAREs has provided important clues about the mech- either endosomes (in the absence of insulin), or with anism of GLUT4 translocation. the cell surface (in the presence of insulin). Several lines of evidence support the existence of this unique A model for GLUT4 transport pool of GLUT4 vesicles and the idea that it can fuse Several models, based on the studies described above, directly with the cell surface in response to insulin. In have been proposed to explain the transport of GLUT4. particular, studies of the SNARE proteins that are involved in the docking and fusion of GLUT4 storage vesicles (GSVs) with the cell surface (BOX 3) have been 1 Basal most enlightening. Disrupting the function of the + Insulin Syntaxin 4–SNAP23–VAMP2 SNARE complex selec- tively inhibits the insulin-stimulated translocation of 2 GLUT4 to the cell surface, but not other recycling pro- 4 teins such as GLUT1. These data argue strongly in 5 3 favour of a model in which a population of vesicles is ready to move directly to the cell surface. Once formed, it is highly unlikely that these vesicles remain static in 6 the absence of an insulin signal, as the endosomal and 7 TGN pools of GLUT4 would become depleted and all the GLUT4 would be present in GSVs if this were the 8 case. It therefore seems likely that the GSVs slowly fuse with endosomes, and allow GLUT4 to re-enter the 9 endosomal system. This TGN–endosomal recycling pathway is not unique to insulin-responsive cells, but 10 probably exists in all cell types. Numerous examples of 11 12 proteins that are transported to the cell surface from an intracellular pool in response to external stimuli have

0 5 10 15 20 25 30 35 been identified (TABLE 1). For example, aquaporin-2 — a GLUT4 distribution (%) water channel that is normally found in the TGN region Figure 3 | Relative GLUT4 distribution throughout organelles of cells from non-stimulated of renal epithelial cells — translocates to the plasma and insulin-stimulated brown adipose tissue. Cryosections of brown adipose tissue were membrane in response to the peptide hormone vaso- immunolabelled with anti-GLUT4 antibody and gold-conjugated Protein A. Gold particles were pressin. Intriguingly, the SNARE complex that controls counted and assigned to the following organelles: (1) trans-Golgi network (TGN); (2) tubulo–vesicular GLUT4 translocation is also responsible for the translo- (T–V) elements located underneath the plasma membrane; (3) clusters of T–V elements; (4) T–V cation of aquaporin-2. elements distributed throughout the cytoplasm; (5) T–V elements connected or close to late endosomal vacuoles (6); (7) T–V elements connected or close to early endosomal vacuoles (8); (9) A similar TGN–endosomal transport pathway has non-coated invaginations of the plasma membrane; (10) coated pits and vesicles; (11) plasma been described in the budding yeast Saccharomyces cere- membrane; (12) cytoplasm. The graph (right) shows the relative distribution of GLUT4 throughout visiae to account for the upregulation of amino-acid these organelles. Reproduced with permission from REF.16 ©1991 The Rockefeller University Press. permeases on the cell surface that occurs in response to

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Box 3 | The SNARE hypothesis The multitude of membrane transport events that occurs in eukaryotic cells are controlled by families of proteins known as SNAREs and SNARE-associated proteins. v-SNAREs (membrane proteins that are found in transport vesicles) bind in a highly specific manner to t-SNAREs (membrane proteins that are found on the relevant target membrane). The formation of a stable, ternary complex between the correct set of SNARE proteins brings transport vesicles and target membranes into close proximity, and ultimately leads to their fusion. Although the precise role of SNAREs in membrane docking and fusion is still debated, these molecules and their associated proteins clearly have an important role in membrane 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 the appropriate target membrane. Rab proteins bind to specific transport vesicles and seem to function — through their GTPase activity — as molecular switches, to recruit cytosolic effector molecules that are required for vesicle tethering to docking 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 SNARE complex docks the transport vesicle onto the target membrane. The Sec1-like/Munc18 (SM) family adds a further level of regulation 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 a stable 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. A situation is emerging in many cellular systems, in which different members of these families mark different transport vesicles and target (or acceptor) membranes. The coordination of the various families of proteins that are involved in membrane fusion results in a highly regulated process.

t-SNARES

Target membrane SNAP23 v-SNARE Rab Tether Tethering Docking Fusion

Transport vesicle

external growth conditions. On rich nitrogen sources, adipocytes36. It remains to be seen if this is linked to its the general amino-acid permease Gap1 is transported to ubiquitylation or if there is a role for this process in the vacuole, where it is degraded. By contrast, when cells insulin resistance. are grown on low nitrogen sources, Gap1 is transported The futile cycle that is depicted in FIG. 4 might explain from an intracellular storage pool to the cell surface32. several observations that are related to GLUT4 trans- Intriguingly, a di-leucine-containing motif in the car- port. First, such a cycle might provide the basis for the boxyl-terminus of Gap1, which is required for its regu- considerable increase in the rate of GLUT4 exocytosis lated transport, resembles a motif that is required for — compared with that of other proteins — in response the insulin-sensitive transport of GLUT4 (REF. 33).The to insulin. This would explain the very large increase in regulated transport of Gap1 is controlled by the addi- cell-surface levels of GLUT4. Second, it might explain tion of ubiquitin to the amino terminus of Gap1, which how different stimuli mobilize discrete intracellular seems to occur in the TGN. This is intriguing, as it has pools of GLUT4. Most notably, in skeletal muscle exer- been reported that GLUT4 is modified by the addition cise causes a large increase in GLUT4 translocation to of the ubiquitin-like molecule sentrin, also known as the plasma membrane, mainly from the endosomal SUMO1, in muscle cells34. So, it is tempting to speculate pool rather than the GSVs (REF. 37). Similar observa- that there might be a role for sumoylation and/or ubiq- tions have been made using other agonists such as GTPγS uitylation in regulating the transport of GLUT4 (REF.38). Intriguingly, the regulation of GLUT4 move- between the TGN and endosomes. Ubiquitylation has ment from these different compartments seems to be recently been shown to regulate the entry of membrane quite unique. Whereas wortmannin, which inhibits proteins into multivesicular bodies, which targets them phosphatidylinositol 3-kinase (PI3K) activity,completely GTPγS 35 A non-hydrolysable analogue of for degradation . Intriguingly, chronic insulin treat- inhibits insulin-stimulated GLUT4 translocation, it has GTP. ment markedly reduces the stability of GLUT4 in no effect on the translocation of GLUT4 that occurs in

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referred to as ‘intrinsic activation’.First, kinetic studies in L6 myotubes have indicated that the insulin-dependent Plasma membrane arrival of GLUT4 at the cell surface precedes the increase Cytoplasm in glucose uptake by several minutes41. Intriguingly, a similar difference is not observed in adipocytes42,43, Cycle 1 Early which raises the possibility that transporters that translo- endosome Munc18c cate more slowly might account for the increase in glu- Syntaxin4 cose uptake in L6 cells. Second, a discrepancy in the + Insulin Recycling dose-response effects of wortmannin on insulin-stimu- SNAP23 endosome lated glucose transport compared with GLUT4 translo- VAMP2 cation have been observed in both 3T3-L1 adipocytes44 GLUT4 Cycle 2 45 storage Transport and L6 cells . In both of these studies, glucose uptake vesicles vesicle was inhibited at a much lower dose of wortmannin than GLUT4 translocation, which indicates that these two processes are clearly dissociated. Finally, an inhibitor of the mitogen-activated protein kinase (MAPK) isoform p38 inhibits insulin-stimulated glucose uptake without Trans-Golgi network any apparent effect on GLUT4 translocation46. In addi- 47 Figure 4 | A model that depicts the transport of GLUT4 in insulin-responsive cells. The tion to these studies, several agents such as leptin , iso- 48 49 model depicts two main intracellular-recycling pathways: cycle 1, between the cell surface and proterenol and dibutyryl cyclic AMP decrease glucose endosomes; and cycle 2, between the trans-Golgi network (TGN) and endosomes. GLUT4 uptake, whereas cycloheximide50 and adenosine48 transport is intricately controlled at several points along these cycles. On entry into the endosomal increase glucose uptake without affecting the amount of system, GLUT4 is selectively retained at the expense of other recycling transport, such as the GLUT4 at the plasma membrane. An important limita- transferrin receptor that constitutively moves through cycle 1. This retention mechanism might tion of the intrinsic-activation hypothesis is that a plau- predispose GLUT4 for sorting into transport vesicles that bud slowly from the endosome and that are targeted to the TGN. GLUT4 is sorted into a secretory pathway in the TGN. This sorting step sible biochemical mechanism for intrinsic activation of probably involves a specialized population of secretory vesicles that excludes other secretory GLUT4 is yet to be described. It is most likely that intrin- cargo, and that does not fuse constitutively with the plasma membrane. Vesicles that emerge from sic activation involves some type of covalent or struc- this sorting step, which we have previously referred to as GLUT4 storage vesicles or GSVs, might tural change in GLUT4. Several possible mechanisms, constitute most of the GLUT4 that is excluded from the endosomal system. In the absence of such as phosphorylation51, nucleotide binding52 and (at insulin, GSVs might slowly fuse with endosomes, thereby accounting for the presence of a least in the case of GLUT1) the formation of homo- significant but small pool of GLUT4 in endosomes, even in the absence of insulin. Insulin would 53 then shift GLUT4 from this TGN–endosome cycle to a pathway that takes GLUT4 directly to the oligomers have been proposed. Moreover, it has been cell surface. The inset shows the SNARE proteins that are thought to regulate docking and fusion reported that GLUT4 can be detected in both clathrin- 54 55 of GSVs with the cell surface (reviewed in REF.73). The t-SNAREs Syntaxin 4 and SNAP23 in the coated pits and caveolae at the cell surface in plasma membrane of fat and muscle cells form a ternary complex with the v-SNARE VAMP2, adipocytes, and it is possible that within these subdo- which is present on GSVs. Munc18c has been identified as the SM (Sec1-like/Munc18 family) mains, the structure of GLUT4, and consequently its protein (BOX 3) that controls the formation of this ternary complex. activity, is constrained in some way.

Integrating the transport with the signals response to exercise or GTPγS (REF.39). Furthermore, in As discussed above, there are several steps that are adipocytes, overexpression of constitutively active involved in maintaining the intracellular pool of mutants of a downstream target of PI3K, Akt, stimulate GLUT4 in the absence of insulin, any one of which the exocytosis of GSVs but not the endosomal pool40. could be a target of insulin action. GLUT4 is trans- These studies indicate that the exocytic cues that regu- ported between several intracellular compartments late the movement of GLUT4 from different locations even in the absence of insulin, and this alone involves are quite distinct, and so specificity is probably achieved selective retention mechanisms, vesicle-budding reac- by the use of a combination of discrete pools of intracel- tions that involve the binding of coat proteins such as lular GLUT4, each of which are coupled to unique regu- AP-1 to GLUT4, the movement of vesicles along latory mechanisms. These studies also indicate that, at cytoskeletal elements and the docking and fusion of least as far as insulin action is concerned, the regulation transport vesicles with their relevant target membrane. might be quite similar in both muscle and fat cells. Organelles that are potential targets of insulin action include the plasma membrane, endosomes and the Intrinsic activation TGN, which shows that a vast amount of transport Can translocation of GLUT4 to the plasma membrane machinery is involved. An important question is: which account for the stimulatory effects of insulin on glucose step does insulin modulate to increase GLUT4 translo- transport in muscle and fat cells, or is its ability to trans- cation to the cell surface? The lack of in vitro assays that port glucose also subject to regulation? Several recent recapitulate some of these stages of GLUT4 transport studies have shown that insulin-stimulated transport has been a major limitation in answering this and other and GLUT4 translocation can be dissociated from each questions. Recently, an assay for the in vitro fusion of other under certain conditions, which indicates that intracellular vesicles that contain GLUT4 with plasma there might be further means of regulating the transport membranes has been described56. Using this system, it was properties of GLUT4 — a phenomenon previously shown that insulin modulates targets in both the vesicle

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Table 1 | Proteins that translocate after stimulus (in addition to GLUT4 and IRAP) Name Cell type Stimulus Intracellular Remarks References localization General amino acid S. cerevisiae Poor nitrogen source Golgi Sec13 dependent 32 permease Gap1 (ammonia/urea) Aquaporin-1 Rat peritoneal Hyperosmotic stimulus Endosomal 75 mesothelial 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 77–84 (renal inner medullary forskolin network A1; VAMP2, Syntaxin-4 and SNAP23 probably collecting-duct cells) (TGN) involved; cyclic AMP and PKA involved (PKA-mediated phosphorylation of AQP-2 is probably required for translocation); mutation of phosphorylation site Ser256 blocks translocation; might involve heterotrimeric G proteins (Gαi); okadaic acid induces translocation 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 85,86 channel (ENaC) sequence is involved Na+-K+-ATPase Kidney epithelium Insulin/arginine Unknown Translocation accompanied by subunit 87–91 vasopressin dephosphorylation (insulin + AVP); inhibited by wortmannin (insulin) Skeletal muscle Exercise/insulin Unknown Na+/H+ exchanger Renal and intestinal bFGF Recycling Blocked by PI3K inhibitors 92,93 NHE3 epithelial cells endosomes Calcium channel Neuronal cells IGF-1, PDGF, head Unknown Translocation is wortmannin sensitive 94,95 GRC activator (neuropeptide) N-type calcium channel Neuronal cell KCl, ionomycin, Unknown Translocation is BFA-insensitive 96 PKC 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 99,100 MNK expressed GABA transporter GAT1 Neuronal cells PKC activation (PMA) Unknown 101 Glutamate Neuronal cells PDGF Unknown Translocation inhibited by wortmannin and 102 transporter EAAC1 LY 294002, not by PKC inhibitor BisII Flt3 (growth T lymphocytes Bone-marrow failure Perinuclear Translocation not due to de novo protein 103,104 factor for (chemotherapy); synthesis haematopoietic cells) IL-2, -4, -7, -15 κ opioid Magnocellular Salt loading AVP-containing Occurs during neuropeptide release; removed from 105 receptor KOR1 neurosecretory secretory plasma membrane within 1 hr of stimulation vesicles bFGF, basic fibroblast growth factor; IGF, insulin growth factor; IL, interleukin; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; BFA, brefeldin A.

and the plasma membranes. So, these data support the Synip with Syntaxin 4 is reduced on stimulation with notion that there are several signalling pathways that insulin58, but how this dissociation is achieved remains converge on different aspects of GLUT4 transport. In unknown. Similarly, the VAMP2-binding proteins support of this, whereas Akt is activated at the plasma pantophysin59 and vesicle-associated protein 33 membrane, another downstream target of PI3K, protein (VAP33)60 have been proposed to prevent the entry of kinase Cζ (PKCζ), is selectively activated in the v-SNARE into the ternary complex in the absence of endosomes57. So, it might be of interest to look for spe- insulin, but again, the signal that transduces this is not cific substrates of each of these kinases at these discrete known. Intriguingly, insulin stimulates the GTP-loading cellular locations. of Rab4, and GTP–Rab4 is known to bind Syntaxin 4 The most likely targets of insulin action at the cell (REFS 61,62). Furthermore, insulin or overexpression of surface are the SNARE proteins (BOX 3). Several proteins PKCζ induces serine phosphorylation of VAMP2 in pri- have been implicated in regulating the formation of the mary cultures of rat skeletal muscle63. So, we can imag- ternary complex — which consists of Syntaxin 4, ine that GLUT4 vesicles that are formed from either SNAP23 and VAMP2 — in response to insulin. For endosomes or the TGN constantly sample the cell sur- example, Synip binds to Syntaxin 4 and prevents face, but that their fusion is limited by the availability of VAMP2,but not SNAP23, binding. The association of tethering and/or docking sites. Insulin might overcome

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BREFELDIN A this barrier by modulating auxiliary regulatory proteins tactic to avoid appearing at the cell surface. Elements of A fungal metabolite that affects such as the Rab proteins or Synip. this system are absent from ‘non-insulin-responsive’ cell membrane transport and the If each of the signalling pathways that are implicated types. So, the adaptations that occur during muscle and structure of the Golgi apparatus. in insulin action (BOX 2) were assigned discrete functions fat differentiation to allow the entry of GLUT4 into this in the GLUT4-recruitment process, we would predict intracellular loop are clearly an important area for that activation of each signalling intermediate on its future study. own would have little or no effect compared with that of We imagine that the complex transport itinerary of insulin. However, in the case of the TC10 pathway this GLUT4 is governed by the protein encountering differ- does not seem to be the case. Overexpression of consti- ent coat complexes throughout the cell. Some of these tutively active forms of PI3K (REF.64),Akt65 or PKC63,but we know, including AP-2 at the cell surface, and AP-1 at not TC10 (REF. 66), has a robust stimulatory effect on the TGN. But the coats that regulate transport between GLUT4 translocation that is similar to that observed endosomes and the TGN are not yet known. These are with insulin. One interpretation of these data is that the probably somewhat specialized, perhaps by being TC10 pathway regulates a factor, or process, that is per- expressed uniquely in insulin-responsive cells and/or by missive for GLUT4 translocation to the cell surface. One being resistant to the effects of BREFELDIN A (BFA). such process that has recently been proposed is the reg- In addition to GLUT4, GSVs also contain IRAP and ulation of the actin cytoskeleton67, which is consistent VAMP2. The identification of the latter has allowed with the generalized role of Rho family members in huge inroads to our understanding of the docking and actin rearrangement. Considerable evidence supports a fusion of GSVs with the plasma membrane. The role for the actin cytoskeleton in insulin-stimulated glu- SNAREs and some of their associated proteins are now cose transport. Agents that depolymerize actin inhibit known. Such discoveries will provide a template for the GLUT4 translocation68 and, although controversial, it discovery of new molecules that might be unique to the has been suggested that insulin might modulate the cor- insulin-stimulated transport of GLUT4 in muscle and tical actin cytoskeleton in adipocytes69. This leads to a fat cells. A large gap in our current knowledge of the model in which actin might be involved in tethering the exocytosis of GLUT4 is the identity of the Rab protein GLUT4 vesicles at the cell surface, and this might pre- that is involved in delivery of the transport to the cell cede the docking/fusion step. More recently, it has been surface. Although Rab4 has been implicated in this shown that insulin stimulates the formation of actin process, it might be involved in less specialized aspects tails that are associated with GLUT4-containing mem- of the transport itinerary of GLUT4, in which case the branes70, which raises the possibility that actin might be Rab that is responsible for the delivery of GSVs to the involved in propelling the vesicles towards the cell sur- plasma membrane remains to be identified. face. In either case, we can imagine that this step, which Perhaps the ultimate question is, what does insulin is regulated by the TC10 pathway, might not be suffi- do? Without more complete answers to the above two cient to activate GLUT4 translocation, and this might questions, this question will probably remain unan- also explain why activation of either the Akt or PKC swered. Although our knowledge of signalling has pathways on their own might overcome the need for advanced tremendously over the past few years, we have this pathway. So, until the function of TC10 has been not, as yet, identified the intersection point of the more clearly defined, with particular attention to the insulin-signalling pathway with the GLUT4 transport identification of its downstream targets in muscle and pathway. This intersection point might be governed by fat cells, it is difficult to assign an important role for this coat proteins, cytoskeletal elements, the SNARE pro- pathway in insulin action. teins or, more probably, a combination of all three. Identification of this intersection point will require a Conclusions and perspectives convergence of different approaches. New approaches So, to return to the central questions that we posed at such as DNA microarrays will provide knowledge of the the beginning of this review: how is GLUT4 transported genes that are uniquely expressed in muscle and fat cells, from one organelle to another, and what is the rela- and will offer new insights into the dynamic and regu- tionship between these pathways and the intracellular lated characteristics of GLUT4 transport. Finally, the sequestration of GLUT4 in the absence of insulin? The development of in vitro assays that reconstitute various intracellular movement of GLUT4 is complex, and aspects of insulin-stimulated GLUT4 translocation will involves many organelles and perhaps also a unique be required for the discovery and characterization of storage compartment — GSVs. In the absence of key molecules that are involved in this process. All of insulin, GLUT4 is trapped in an intracellular circuit this knowledge will contribute to our understanding of between endosomes and the TGN as a diversionary both cell biology and type II diabetes.

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