regulated transport of the glucose transporter glut4 - CiteSeerX

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REGULATED TRANSPORT OF THE GLUCOSE TRANSPORTER 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 TRANSPORTER

A polytopic membrane protein that transports sugars down a concentration gradient in an energy-independent manner. TYPE II DIABETES

Also known as non-insulindependent diabetes or maturity onset diabetes.

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

Glucose is a fundamental source of energy for all eukaryotic cells. In humans, although all cells use glucose for their energy needs, the main consumer under basal conditions is the brain, which accounts for as much as 80% of whole-body consumption. The energy is provided by the breakdown of endogenous glycogen stores that are primarily in the liver. These whole-body energy stores are replenished from glucose in the diet, which, after being digested and absorbed across the gut wall, is distributed among the various tissues of the body (reviewed in REF. 1). This distribution process involves a family of transport proteins — called GLUTs — which act as shuttles to move sugar across the cell surface. These polytopic membrane proteins (FIG. 1) form an aqueous pore across the membrane through which glucose can move. A large family of FACILITATIVE SUGAR TRANSPORTERS exists in mammals, the individual members of which differ in their tissue distribution and kinetic properties, as well as in their intracellular localization. The latter property is of particular interest for glucose transport in specialized cell types, and provides the basis for polarized glucose transport in epithelial cells, such as those in the gut, or for acute regulation of glucose transport, as is observed in muscle and fat cells after a meal. Many mammalian tissues, such as the brain, have a constitutively high glucose requirement and have been endowed with transporters that are constitutively targeted to the cell surface (for example, GLUTs 1–3). By contrast, certain tissues, such as muscle and adipose tissue, have acquired a highly specialized

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY

glucose-transport system, the activity of which can be rapidly upregulated to allow these tissues to increase their rate of glucose transport by 10–40-fold within minutes of exposure to a particular stimulus (reviewed in REF. 1). This system is crucial during exercise, when the metabolic demands of skeletal muscle can increase more than 100-fold, and during the absorptive period (after a meal), to facilitate the rapid insulin-dependent storage of glucose in muscle and adipose tissue, so preventing large fluctuations in blood glucose levels. Dysfunctional glucose uptake into muscle and fat cells contributes 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 transporter that is found in these cells from an intracellular store to the plasma membrane2,3. This translocation hypothesis was later confirmed when GLUT4 was identified as the main glucose transporter in these cells. GLUT4, which is expressed primarily in muscle and fat cells, is found in a complex intracellular tubulo–vesicular network that is connected to the endosomal–trans-Golgi network (TGN) system. In the absence of stimulation, GLUT4 is almost completely excluded from the plasma membrane (FIG. 2). The addition of insulin, or exercise in the case of muscle cells, causes GLUT4 to shift from its intracellular location to the plasma membrane (FIG. 2). Several observations indicate that GLUT4 has a crucial role in whole-body glucose homeostasis. First, insulinstimulated glucose transport is an important rate-limiting step for glucose metabolism in both muscle and fat

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REVIEWS an insulin-regulated step(s). Although many important signalling molecules that are integral to the insulin regulation 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 cell-biological understanding of GLUT4 transport, and highlight potential regulatory sites of the insulinsignalling cascade.

Sugar moiety

Plasma membrane Cytoplasm NH2 COOH H

GLUT4 transport

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

tissue, and is severely disrupted in type II diabetes1 (BOX 1); second, disruption of GLUT4 expression in mice results in insulin resistance4; and overexpression of GLUT4 ameliorates diabetes in the DB/DB MOUSE model5. Analysing the molecular and cellular regulation of GLUT4 transport not only promises to provide new insights into protein sorting, but could also yield new targets for therapeutic intervention in what could well be one of the most prevalent diseases that we will have to confront in the future. Understanding the regulation of GLUT4 and glucose transport has proved to be extremely challenging, principally because it involves several signal-transduction pathways that are superimposed on a complex series of vesicle transport processes. Insulin binds to a surface receptor on muscle and fat cells and triggers a cascade of signalling events (BOX 2) that culminates in GLUT4 translocation. Studies of this process have been carried out using two approaches.‘Outside–in’ approaches have largely focused on mapping insulin-specific signalling pathways in muscle and fat cells with the view to identifying downstream targets that directly control GLUT4 translocation. Conversely,‘inside–out’ approaches have used cell-biological studies to map the intracellular transport itinerary of GLUT4 with the aim of identifying

GLUT4 is found in many organelles, including the plasma membrane, sorting endosomes, recycling endosomes, the TGN and vesicles that mediate the transport of GLUT4 between these compartments (FIG. 3). Presumably this localization represents a complex and dynamic transport itinerary, and it raises several important questions. How does GLUT4 transport from one organelle to another, what is the relationship between these pathways and the intracellular sequestration of GLUT4 in basal cells, and which of these steps does insulin influence to affect GLUT4 exocytosis? In non-stimulated adipocytes, the rate of GLUT4 exocytosis is 10-fold slower than that of the transferrin receptor (TfR) — one of the most well-studied constitutive recycling proteins in mammalian cells6,7. To account for this, GLUT4 must be selectively retained in one of its intracellular locations, packaged into a specialized compartment that remains static in the absence of insulin, or involved in a dynamic intracellular transport loop that excludes it from recycling endosomal vesicles. Current evidence favours a role for all three mechanisms, which emphasizes the complexity of this process. Another protein, the insulin-responsive aminopeptidase (IRAP), which was recently described as a receptor for angiotensin IV (REF. 8), colocalizes with GLUT4 and is transported in a very similar manner9. Below, we discuss studies concerning the transport of either GLUT4 or IRAP, with a particular focus on adipocytes and muscle cells. We also compare this with transport in cells such as fibroblasts that are not, or only mildly, responsive to insulin, as key differences have been identified that provide new insights into our understanding of insulin action. Endosomal sorting of GLUT4

Morphological studies in both muscle and fat cells indicate that, although there is some overlap of GLUT4 with markers of the endocytic system such as the TfR, a

Box 1 | Type II diabetes

DB/DB MOUSE

A genetic mouse model of type II diabetes and obesity. The defect has been mapped to the gene for the leptin receptor.

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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 conjunction with an oversupply of glucose from the liver, which results in high circulating plasma glucose levels. This causes many of the complications of type II diabetes, including eye, nerve and kidney disease. The highest contributor to morbidity and mortality in type II diabetes is heart disease and, strikingly, type II diabetes is one of the main causes of heart disease in the Western world.

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EVANESCENCE WAVE MICROSCOPY

A technique in which only fluorophores within a 100–220 nm field above a glass coverslip are excited, which allows localization of molecules very close to the cell surface. ATRIAL CARDIOMYOCYTE

A heart muscle cell. AP-1

(Adaptor protein complex 1). Adaptor proteins link cargo molecules on membranes with coat proteins such as clathrin. Several classes of adaptor proteins have been identified and shown to be involved in different transport steps. AP-1 is thought to regulate transport from the trans-Golgi network to endosomes. SNARE

(soluble N-ethylmaleimide sensitive factor attachment protein receptor). A family of membrane-tethered coiled-coil proteins that regulate fusion reactions and target specificity in the vacuolar system. They can be divided into v-SNAREs (vesicle) and t-SNAREs (target) on the basis of their localization, or into Q-SNAREs and R-SNAREs on the basis of a highly conserved amino acid. CONVERTASE

An enzyme that is responsible for protein activation through proteolytic activity. COAT-ASSOCIATED PROTEIN

A protein that links cargo molecules to vesicle coats.

significant pool of GLUT4 is not localized to 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 endosomal 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 — including the TfR — to the plasma membrane, endosomes do not seem to be the main insulin-sensitive GLUT4 storage compartment. Intriguingly, in fibroblasts, most GLUT4 and IRAP colocalizes with the TfR in endosomes13. This might explain the relatively small insulin effect that is observed in these cells (twofold increase), and also indicates that the transport of GLUT4 is more specialized in bona fide insulin-responsive cell types. Nevertheless, there is evidence for a selective retention mechanism of GLUT4 and IRAP in fibroblasts, but this is clearly insufficient to generate the robust insulin effect that is observed in adipocytes. Intriguingly, studies that use EVANESCENCE 13 WAVE MICROSCOPY in 3T3-L1 fibroblasts , and an in vitro assay that reconstitutes budding of transport vesicles from endosomes in Chinese hamster ovary (CHO) cells14, have shown that GLUT4 is packaged into endosomal transport vesicles that are distinct from those that contain the TfR. However, in fibroblasts, the GLUT4 transport vesicles that bud from endosomes are very short-lived, presumably because they fuse rapidly with the plasma membrane, even in the absence of insulin. This is clearly not the case in adipocytes, though, as there is little exocytosis of GLUT4 under basal conditions6. Collectively, these studies implicate an important role for the segregation of GLUT4 from the endosomal system in the insulin-responsive transport of GLUT4. The role of the trans-Golgi network

What is the fate of GLUT4-containing endosomal transport vesicles in adipocytes? One possibility is that they are somehow retained in the cytosol in the absence of insulin, and are discharged in response to insulin. This simple model, however, overlooks the fact that a proportion of GLUT4, which does not represent newly synthesized protein, is present in the TGN of both muscle and fat cells15,16. Interestingly, recent evidence indicates that GLUT4 recycles rapidly between the TGN and endosomes. First, morphological studies in ATRIAL CARDIOMYOCYTES indicate that 60% of all the GLUT4 expressed in this cell type is localized to secretory granules that contain atrial natriuretic factor (ANF)17. GLUT4 seems to enter these granules at the TGN, and as the localization of GLUT4 here is not blocked by protein-synthesis inhibitors, these studies indicate that a GLUT4 recycling pathway, possibly from endosomes, merges with the secretory pathway at the TGN. Second, GLUT4 has been shown to localize to adaptor-protein-1 (AP-1)positive, clathrin-coated, vesicles in the vicinity of the TGN in adipocytes18. Third, by following the internalization of


– Insulin

+ Insulin

Figure 2 | Insulin triggers the translocation of GLUT4 from an intracellular location to the plasma membrane of adipocytes. The figure shows a confocal image of 3T3-L1 adipocytes incubated either with (right panel), or without (left panel) 100 nM insulin for 15 mins. The location of GLUT4 in these cells is shown using an antibody that specifically recognizes GLUT4 and a secondary antibody conjugated to Alexa-488 (shown in green). Confocal-laser-scanned sections were obtained from the base of the cells to the perinuclear region, which were then stacked to create a three-dimensional reconstruction. Images courtesy of Timo Meerloo, Garvan Institute of Medical Research, Darlinghurst, Australia.

GLUT4 from the cell surface of adipocytes, it has been shown that the transporter is transported through endosomes into a perinuclear compartment that is distinct from recycling endosomes10. Using a similar approach, we have recently shown that this perinuclear compartment represents a subdomain of the TGN that also contains the SNARE proteins Syntaxin 6 and Syntaxin 16 (A. Shewan, S. Martin, D. E. J., unpublished observations). The transport of GLUT4 between endosomes and the TGN is regulated by a unique acidic targeting motif in the carboxyl terminus of GLUT4 (REF. 19). Intriguingly, the transport of other proteins, such as the pro-protein CONVERTASES furin and PC6B, between endosomes and the TGN is also regulated by acidic targeting motifs20,21. The COAT-ASSOCIATED PROTEIN phosphofurin acidic cluster sorting protein 1 (PACS1) has been found to bind to the acidic motif in the furin tail22. So far, we have been unable to detect an interaction between PACS1 and GLUT4 (S. Rea, D. E. J., unpublished observations), which indicates that other, related coat proteins might function in this specific region of the cell. The recycling of membrane proteins through the TGN is unusual in that most endosomal proteins do not take this route. However, several molecules, including certain bacterial toxins23, mannose-6-phosphate receptors, TGN38 and pro-protein convertases (reviewed in REF. 20), have been shown to follow this pathway. Once in the TGN, these molecules are sorted to one of many different destinations — this is an important function of this organelle. For example, Shiga toxin is transported to the endoplasmic reticulum (ER), the mannose-6-phosphate receptors are transported back to endosomes, and the pro-protein convertases enter the secretory pathway. Once GLUT4 arrives at the TGN, its fate is uncertain. Despite the fact

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ARNO

(ARF nucleotide binding-site opener). This activates ADPribosylating factors (ARFs), which are known to have a role in protein sorting and vesicle budding. γ-EAR-CONTAINING

This represents a protein domain within the γ-subunit of coat adaptor proteins.

that GLUT4 re-enters the secretory pathway in atrial cardiomyocytes, it is subsequently retrieved from these granules before they are delivered to the cell surface, possibly by an AP-1-mediated pathway17. Consistent with this, GLUT4 does not colocalize with other secretory proteins, such as the 30 kDa adipocyte complement-related protein (ACRP30), leptin or adipsin, in adipocytes24–26. Nevertheless, it seems evident that transit through the TGN probably precedes the packaging of GLUT4 into its insulin-responsive compartment because prolonged incubation of adipocytes at 19°C — a temperature that blocks exit from the TGN — inhibits insulin action27.

The TGN is clearly a complex and central sorting station in which key sorting decisions are made. Many coatprotein complexes, including AP-1, AP-3, AP-4, as well as the Golgi-localized, γ-EAR-CONTAINING, ARF-binding (GGA) family of coat proteins28 have been localized to the TGN and might regulate transport into or out of this organelle. Moreover, these coats are multisubunit protein complexes, and it has been shown that unique isoforms of just one component of a particular coat is sufficient to generate cell-type specific sorting. At least a portion of GLUT4 must be delivered back to endosomes to account for the relatively large pool (~30–40%) that is found in this compartment. 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 Insulin receptor Lipid raft the lipid kinase phosphatidylinositol 3-kinase (PI3K), and the 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 located within the intracellular portion of its β-subunits. On GDP activation, the receptor phosphorylates several proximal PDK CAP substrates, including members of the insulin-receptorIRS-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 PKCζ IRS proteins, which are thought to be held in close proximity to the plasma membrane through association with the underlying cytoskeleton, recruit more effector molecules, Insulin + Insulin such as PI3K, to this location. Substantial evidence indicates 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 TC10 atypical protein kinase C (PKC) isoform, PKCζ. PI3K PI3K PDK CAP activates Akt by generating polyphosphoinositides in the GTP PKCζ C3G AKT IRS-1 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-nucleotideexchange 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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REVIEWS have a role in sorting GLUT4 to endosomes. This would explain the fact that GLUT4 colocalizes with molecules, such as the cation-dependent mannose-6-phosphate receptor, (CD-MPR)18 that also follow this route.

CD-MPR

(Cation-dependent mannose-6phosphate receptor). This protein shuttles between the trans-Golgi network and endosomes.

GLUT4 storage vesicles

Despite the fact that GLUT4 is obviously engaged in a recycling loop between endosomes and the TGN, there is clear evidence for the existence of a more static secretory pool of GLUT4 that can move directly to the cell surface in response to insulin. Using both morphological and biochemical methods, a discrete population of small (50 nm diameter) vesicles have been identified29–31. These vesicles exclude other recycling proteins, such as the TfR and the CD-MPR, and are highly responsive to insulin. Importantly, these vesicles contain the v-SNARE, vesicle associated membrane protein (VAMP2) — the same v-SNARE that is found in synaptic vesicles and aquaporin-2-containing vesicles — which indicates a generic role for this molecule in regulated exocytosis in many cell types. This v-SNARE has been shown to form a complex with the t-SNAREs Syntaxin 4 and SNAP23 (BOX 3), which are highly enriched in the plasma membrane of fat and muscle cells. The identification of these SNAREs has provided important clues about the mechanism of GLUT4 translocation. A model for GLUT4 transport

Several models, based on the studies described above, have been proposed to explain the transport of GLUT4.

Basal + Insulin

1

2 3

5

4

6 7 8

9 10 11

12

0

5

10

15

20

25

30

35

GLUT4 distribution (%)

Figure 3 | Relative GLUT4 distribution throughout organelles of cells from non-stimulated and insulin-stimulated brown adipose tissue. Cryosections of brown adipose tissue were immunolabelled with anti-GLUT4 antibody and gold-conjugated Protein A. Gold particles were counted 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–V 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) non-coated invaginations of the plasma membrane; (10) coated pits and vesicles; (11) plasma membrane; (12) cytoplasm. The graph (right) shows the relative distribution of GLUT4 throughout these organelles. Reproduced with permission from REF. 16 ©1991 The Rockefeller University Press.


These include retention mechanisms, dynamic sorting events and the packaging of GLUT4 into a more stationary population of secretory-type vesicles. It now seems likely that these different models are not mutually exclusive, and indeed facets of each of them must be incorporated into a working model. We propose such a model in FIG. 4. This model accommodates many of the apparently contradictory observations, and proposes that GLUT4 transport is controlled by retention mechanisms and dynamic sorting, as well as by being packaged into a more stationary population of secretorytype vesicles. The main feature of this model is that GLUT4 is selectively targeted to an intracellular transport loop between the TGN and the endosomes (cycle 2 in FIG. 4). The entry of GLUT4 into this intracellular, seemingly futile, cycle probably excludes it from the cellsurface recycling pathway (cycle 1 in FIG. 4). This would account for the very low levels of GLUT4 at the cell surface in basal adipocytes compared with other proteins such as the TfR that do not enter this cycle. An essential feature of this model is that there is an intracellular store of GLUT4 that represents a slowly exchanging pool that moves between the TGN and endosomes. This intracellular store can presumably fuse with either endosomes (in the absence of insulin), or with the cell surface (in the presence of insulin). Several lines of evidence support the existence of this unique pool of GLUT4 vesicles and the idea that it can fuse directly with the cell surface in response to insulin. In 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 most enlightening. Disrupting the function of the Syntaxin 4–SNAP23–VAMP2 SNARE complex selectively inhibits the insulin-stimulated translocation of GLUT4 to the cell surface, but not other recycling proteins such as GLUT1. These data argue strongly in 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 the absence of an insulin signal, as the endosomal and TGN pools of GLUT4 would become depleted and all the GLUT4 would be present in GSVs if this were the case. It therefore seems likely that the GSVs slowly fuse with endosomes, and allow GLUT4 to re-enter the endosomal system. This TGN–endosomal recycling pathway is not unique to insulin-responsive cells, but probably exists in all cell types. Numerous examples of proteins that are transported to the cell surface from an intracellular pool in response to external stimuli have been identified (TABLE 1). For example, aquaporin-2 — a water channel that is normally found in the TGN region of renal epithelial cells — translocates to the plasma membrane in response to the peptide hormone vasopressin. Intriguingly, the SNARE complex that controls GLUT4 translocation is also responsible for the translocation of aquaporin-2. A similar TGN–endosomal transport pathway has been described in the budding yeast Saccharomyces cerevisiae to account for the upregulation of amino-acid 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 SNAREcomplex 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 SNAREdependent 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 Rab

v-SNARE Tether Tethering

Docking

Fusion

Transport vesicle

GTPγS

A non-hydrolysable analogue of GTP.

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external growth conditions. On rich nitrogen sources, the general amino-acid permease Gap1 is transported to the vacuole, where it is degraded. By contrast, when cells are grown on low nitrogen sources, Gap1 is transported from an intracellular storage pool to the cell surface32. Intriguingly, a di-leucine-containing motif in the carboxyl-terminus of Gap1, which is required for its regulated transport, resembles a motif that is required for the insulin-sensitive transport of GLUT4 (REF. 33). The regulated transport of Gap1 is controlled by the addition of ubiquitin to the amino terminus of Gap1, which seems to occur in the TGN. This is intriguing, as it has been reported that GLUT4 is modified by the addition of the ubiquitin-like molecule sentrin, also known as SUMO1, in muscle cells34. So, it is tempting to speculate that there might be a role for sumoylation and/or ubiquitylation in regulating the transport of GLUT4 between the TGN and endosomes. Ubiquitylation has recently been shown to regulate the entry of membrane proteins into multivesicular bodies, which targets them for degradation35. Intriguingly, chronic insulin treatment markedly reduces the stability of GLUT4 in

adipocytes36. It remains to be seen if this is linked to its ubiquitylation or if there is a role for this process in insulin resistance. The futile cycle that is depicted in FIG. 4 might explain several observations that are related to GLUT4 transport. First, such a cycle might provide the basis for the considerable increase in the rate of GLUT4 exocytosis — compared with that of other proteins — in response to insulin. This would explain the very large increase in cell-surface levels of GLUT4. Second, it might explain how different stimuli mobilize discrete intracellular pools of GLUT4. Most notably, in skeletal muscle exercise causes a large increase in GLUT4 translocation to the plasma membrane, mainly from the endosomal pool rather than the GSVs (REF. 37). Similar observations have been made using other agonists such as GTPγS (REF. 38). Intriguingly, the regulation of GLUT4 movement from these different compartments seems to be quite unique. Whereas wortmannin, which inhibits phosphatidylinositol 3-kinase (PI3K) activity, completely inhibits insulin-stimulated GLUT4 translocation, it has no effect on the translocation of GLUT4 that occurs in


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Plasma membrane Cytoplasm Cycle 1 Munc18c

Early endosome

Syntaxin4 + Insulin

Recycling endosome

SNAP23 VAMP2

GLUT4 storage vesicles

Cycle 2

Transport vesicle

Trans-Golgi network

Figure 4 | A model that depicts the transport of GLUT4 in insulin-responsive cells. The model depicts two main intracellular-recycling pathways: cycle 1, between the cell surface and endosomes; and cycle 2, between the trans-Golgi network (TGN) and endosomes. GLUT4 transport is intricately controlled at several points along these cycles. On entry into the endosomal system, GLUT4 is selectively retained at the expense of other recycling transport, such as the transferrin receptor that constitutively moves through cycle 1. This retention mechanism might 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 probably involves a specialized population of secretory vesicles that excludes other secretory cargo, and that does not fuse constitutively with the plasma membrane. Vesicles that emerge from this sorting step, which we have previously referred to as GLUT4 storage vesicles or GSVs, might constitute most of the GLUT4 that is excluded from the endosomal system. In the absence of insulin, GSVs might slowly fuse with endosomes, thereby accounting for the presence of a significant but small pool of GLUT4 in endosomes, even in the absence of insulin. Insulin would then shift GLUT4 from this TGN–endosome cycle to a pathway that takes GLUT4 directly to the cell surface. The inset shows the SNARE proteins that are thought to regulate docking and fusion of GSVs with the cell surface (reviewed in REF. 73). The t-SNAREs Syntaxin 4 and SNAP23 in the plasma 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.

referred to as ‘intrinsic activation’. First, kinetic studies in L6 myotubes have indicated that the insulin-dependent arrival of GLUT4 at the cell surface precedes the increase in glucose uptake by several minutes41. Intriguingly, a similar difference is not observed in adipocytes42,43, which raises the possibility that transporters that translocate more slowly might account for the increase in glucose uptake in L6 cells. Second, a discrepancy in the dose-response effects of wortmannin on insulin-stimulated glucose transport compared with GLUT4 translocation have been observed in both 3T3-L1 adipocytes44 and L6 cells45. In both of these studies, glucose uptake 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 any apparent effect on GLUT4 translocation46. In addition to these studies, several agents such as leptin47, isoproterenol48 and dibutyryl cyclic AMP49 decrease glucose uptake, whereas cycloheximide50 and adenosine48 increase glucose uptake without affecting the amount of GLUT4 at the plasma membrane. An important limitation of the intrinsic-activation hypothesis is that a plausible biochemical mechanism for intrinsic activation of GLUT4 is yet to be described. It is most likely that intrinsic activation involves some type of covalent or structural change in GLUT4. Several possible mechanisms, such as phosphorylation51, nucleotide binding52 and (at least in the case of GLUT1) the formation of homooligomers53 have been proposed. Moreover, it has been reported that GLUT4 can be detected in both clathrincoated pits54 and caveolae55 at the cell surface in adipocytes, and it is possible that within these subdomains, the structure of GLUT4, and consequently its activity, is constrained in some way. Integrating the transport with the signals

response to exercise or GTPγS (REF. 39). Furthermore, in adipocytes, overexpression of constitutively active mutants of a downstream target of PI3K, Akt, stimulate the exocytosis of GSVs but not the endosomal pool40. These studies indicate that the exocytic cues that regulate the movement of GLUT4 from different locations are quite distinct, and so specificity is probably achieved by the use of a combination of discrete pools of intracellular GLUT4, each of which are coupled to unique regulatory mechanisms. These studies also indicate that, at least as far as insulin action is concerned, the regulation might be quite similar in both muscle and fat cells. Intrinsic activation

Can translocation of GLUT4 to the plasma membrane account for the stimulatory effects of insulin on glucose transport in muscle and fat cells, or is its ability to transport glucose also subject to regulation? Several recent studies have shown that insulin-stimulated transport and GLUT4 translocation can be dissociated from each other under certain conditions, which indicates that there might be further means of regulating the transport properties of GLUT4 — a phenomenon previously


As discussed above, there are several steps that are involved in maintaining the intracellular pool of GLUT4 in the absence of insulin, any one of which could be a target of insulin action. GLUT4 is transported between several intracellular compartments even in the absence of insulin, and this alone involves selective retention mechanisms, vesicle-budding reactions that involve the binding of coat proteins such as AP-1 to GLUT4, the movement of vesicles along cytoskeletal elements and the docking and fusion of transport vesicles with their relevant target membrane. Organelles that are potential targets of insulin action include the plasma membrane, endosomes and the TGN, which shows that a vast amount of transport machinery is involved. An important question is: which step does insulin modulate to increase GLUT4 translocation to the cell surface? The lack of in vitro assays that recapitulate some of these stages of GLUT4 transport has been a major limitation in answering this and other questions. Recently, an assay for the in vitro fusion of intracellular vesicles that contain GLUT4 with plasma membranes has been described56. Using this system, it was 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 localization

Remarks

References

General amino acid permease Gap1

S. cerevisiae

Poor nitrogen source (ammonia/urea)

Golgi

Sec13 dependent

Aquaporin-1

Rat peritoneal mesothelial cells

Hyperosmotic stimulus Endosomal

Cholangiocytes

Secretin

Unknown

Inhibited at 20ºC and by colchicine

Aquaporin-2

Renal epithelium Arginine vasopressin/ (renal inner medullary forskolin collecting-duct cells)

trans-Golgi network (TGN)

Translocation blocked at 20ºC and by bafilomycin 77–84 A1; VAMP2, Syntaxin-4 and SNAP23 probably 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 channel (ENaC)

Renal epithelium

cAMP agonists

Unknown

Process inhibited at 15ºC; PPPXY sequence is involved

85,86

Na+-K+-ATPase

Kidney epithelium

Insulin/arginine vasopressin

Unknown

Translocation accompanied by subunit dephosphorylation (insulin + AVP); inhibited by wortmannin (insulin)

87–91

Skeletal muscle

Exercise/insulin

Unknown

Na+/H+ exchanger NHE3

Renal and intestinal epithelial cells

bFGF

Recycling endosomes

Blocked by PI3K inhibitors

92,93

Calcium channel GRC

Neuronal cells

IGF-1, PDGF, head Unknown activator (neuropeptide)

Translocation is wortmannin sensitive

94,95

N-type calcium channel

Neuronal cell

KCl, ionomycin, PKC activation

Unknown

Translocation is BFA-insensitive

96

8 pS chloride channel

Renal epithelium

PKA activation

Unknown

Translocation is BFA-sensitive

97

H+/K+-ATPase

Gastric parietal cells

Histamine

Vesicles

Menkes protein MNK

Ubiquitously expressed

Copper

TGN

GABA transporter GAT1 Neuronal cells

PKC activation (PMA)

Unknown

Glutamate transporter EAAC1

Neuronal cells

PDGF

Unknown

Translocation inhibited by wortmannin and LY 294002, not by PKC inhibitor BisII

Flt3 ligand (growth factor for haematopoietic cells)

T lymphocytes

Bone-marrow failure (chemotherapy); IL-2, -4, -7, -15

Perinuclear

Translocation not due to de novo protein synthesis

κ opioid receptor KOR1

Magnocellular neurosecretory neurons

Salt loading

AVP-containing Occurs during neuropeptide release; removed from secretory plasma membrane within 1 hr of stimulation vesicles

32 75 76

98 99,100 101 102 103,104

105

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 notion that there are several signalling pathways that converge on different aspects of GLUT4 transport. In support of this, whereas Akt is activated at the plasma membrane, another downstream target of PI3K, protein kinase Cζ (PKCζ), is selectively activated in endosomes57. So, it might be of interest to look for specific substrates of each of these kinases at these discrete cellular locations. The most likely targets of insulin action at the cell surface are the SNARE proteins (BOX 3). Several proteins have been implicated in regulating the formation of the ternary complex — which consists of Syntaxin 4, SNAP23 and VAMP2 — in response to insulin. For example, Synip binds to Syntaxin 4 and prevents VAMP2, but not SNAP23, binding. The association of

274


Synip with Syntaxin 4 is reduced on stimulation with insulin58, but how this dissociation is achieved remains unknown. Similarly, the VAMP2-binding proteins pantophysin59 and vesicle-associated protein 33 (VAP33)60 have been proposed to prevent the entry of the v-SNARE into the ternary complex in the absence of insulin, but again, the signal that transduces this is not known. Intriguingly, insulin stimulates the GTP-loading of Rab4, and GTP–Rab4 is known to bind Syntaxin 4 (REFS 61,62). Furthermore, insulin or overexpression of PKCζ induces serine phosphorylation of VAMP2 in primary cultures of rat skeletal muscle63. So, we can imagine that GLUT4 vesicles that are formed from either endosomes or the TGN constantly sample the cell surface, but that their fusion is limited by the availability of tethering and/or docking sites. Insulin might overcome


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BREFELDIN A

A fungal metabolite that affects membrane transport and the structure of the Golgi apparatus.

this barrier by modulating auxiliary regulatory proteins such as the Rab proteins or Synip. If each of the signalling pathways that are implicated in insulin action (BOX 2) were assigned discrete functions in the GLUT4-recruitment process, we would predict that activation of each signalling intermediate on its own would have little or no effect compared with that of insulin. However, in the case of the TC10 pathway this does not seem to be the case. Overexpression of constitutively active forms of PI3K (REF. 64), Akt65 or PKC63, but not TC10 (REF. 66), has a robust stimulatory effect on GLUT4 translocation that is similar to that observed with insulin. One interpretation of these data is that the TC10 pathway regulates a factor, or process, that is permissive for GLUT4 translocation to the cell surface. One such process that has recently been proposed is the regulation of the actin cytoskeleton67, which is consistent with the generalized role of Rho family members in actin rearrangement. Considerable evidence supports a role for the actin cytoskeleton in insulin-stimulated glucose transport. Agents that depolymerize actin inhibit GLUT4 translocation68 and, although controversial, it has been suggested that insulin might modulate the cortical actin cytoskeleton in adipocytes69. This leads to a model in which actin might be involved in tethering the GLUT4 vesicles at the cell surface, and this might precede the docking/fusion step. More recently, it has been shown that insulin stimulates the formation of actin tails that are associated with GLUT4-containing membranes70, which raises the possibility that actin might be involved in propelling the vesicles towards the cell surface. In either case, we can imagine that this step, which is regulated by the TC10 pathway, might not be sufficient to activate GLUT4 translocation, and this might also explain why activation of either the Akt or PKC pathways on their own might overcome the need for this pathway. So, until the function of TC10 has been more clearly defined, with particular attention to the identification of its downstream targets in muscle and fat cells, it is difficult to assign an important role for this pathway in insulin action. Conclusions and perspectives

So, to return to the central questions that we posed at the beginning of this review: how is GLUT4 transported from one organelle to another, and what is the relationship between these pathways and the intracellular sequestration of GLUT4 in the absence of insulin? The intracellular movement of GLUT4 is complex, and involves many organelles and perhaps also a unique storage compartment — GSVs. In the absence of insulin, GLUT4 is trapped in an intracellular circuit between endosomes and the TGN as a diversionary

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3.

4.

tactic to avoid appearing at the cell surface. Elements of this system are absent from ‘non-insulin-responsive’ cell types. So, the adaptations that occur during muscle and fat differentiation to allow the entry of GLUT4 into this intracellular loop are clearly an important area for future study. We imagine that the complex transport itinerary of GLUT4 is governed by the protein encountering different coat complexes throughout the cell. Some of these we know, including AP-2 at the cell surface, and AP-1 at the TGN. But the coats that regulate transport between endosomes and the TGN are not yet known. These are probably somewhat specialized, perhaps by being expressed uniquely in insulin-responsive cells and/or by being resistant to the effects of BREFELDIN A (BFA). In addition to GLUT4, GSVs also contain IRAP and VAMP2. The identification of the latter has allowed huge inroads to our understanding of the docking and fusion of GSVs with the plasma membrane. The SNAREs and some of their associated proteins are now known. Such discoveries will provide a template for the discovery of new molecules that might be unique to the insulin-stimulated transport of GLUT4 in muscle and fat cells. A large gap in our current knowledge of the exocytosis of GLUT4 is the identity of the Rab protein that is involved in delivery of the transport to the cell surface. Although Rab4 has been implicated in this process, it might be involved in less specialized aspects of the transport itinerary of GLUT4, in which case the Rab that is responsible for the delivery of GSVs to the plasma membrane remains to be identified. Perhaps the ultimate question is, what does insulin do? Without more complete answers to the above two questions, this question will probably remain unanswered. Although our knowledge of signalling has advanced tremendously over the past few years, we have not, as yet, identified the intersection point of the insulin-signalling pathway with the GLUT4 transport pathway. This intersection point might be governed by coat proteins, cytoskeletal elements, the SNARE proteins or, more probably, a combination of all three. Identification of this intersection point will require a convergence of different approaches. New approaches such as DNA microarrays will provide knowledge of the genes that are uniquely expressed in muscle and fat cells, and will offer new insights into the dynamic and regulated characteristics of GLUT4 transport. Finally, the development of in vitro assays that reconstitute various aspects of insulin-stimulated GLUT4 translocation will be required for the discovery and characterization of key molecules that are involved in this process. All of this knowledge will contribute to our understanding of both cell biology and type II diabetes.

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Online links DATABASES The following terms in this article are linked online to: Interpro: http://www.ebi.ac.uk/interpro/ pleckstrin homology domain LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink Akt | TfR OMIM: http://www.ncbi.nlm.nih.gov/Omim Type II diabetes Swiss-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 | VAP33 Access to this interactive links box is free online.

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