The GLUT4 Glucose Transporter

Cell Metabolism

Review The GLUT4 Glucose Transporter Shaohui Huang1 and Michael P. Czech1,* 1

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA *Correspondence: [email protected] DOI 10.1016/j.cmet.2007.03.006

Few physiological parameters are more tightly and acutely regulated in humans than blood glucose concentration. The major cellular mechanism that diminishes blood glucose when carbohydrates are ingested is insulin-stimulated glucose transport into skeletal muscle. Skeletal muscle both stores glucose as glycogen and oxidizes it to produce energy following the transport step. The principal glucose transporter protein that mediates this uptake is GLUT4, which plays a key role in regulating whole body glucose homeostasis. This review focuses on recent advances on the biology of GLUT4. Elevated glucose levels are rapidly returned to normal (5–6 mM) even after huge caloric ingestions, and they are maintained at only slightly lower levels during longterm starvation. Such control prevents severe dysfunctions such as loss of consciousness due to hypoglycemia and toxicity to peripheral tissues in response to the chronic hyperglycemia of diabetes. The major cellular mechanism for disposal of an exogenous glucose load is insulin-stimulated glucose transport into skeletal muscle. Skeletal muscle both stores glucose as glycogen and oxidizes it to produce energy following the transport step. The principal glucose transporter protein that mediates this uptake is one isoform (Gene name, SLC2A4; protein name: GLUT4) of a family of sugar transporter proteins containing 12-transmembrane domains (Figure 1). The GLUT4 glucose transporter is thus a major mediator of glucose removal from the circulation and a key regulator of whole-body glucose homeostasis. Here, we discuss the known molecular and cellular regulatory mechanisms for GLUT4, its gene regulation, its trafficking pathways and their integration with insulin signaling, and the profound effects GLUT4 exerts on whole-body metabolism. GLUT4 is one of 13 sugar transporter proteins (GLUT1GLUT12, and HMIT) encoded in the human genome (Joost and Thorens, 2001; Wood and Trayhurn, 2003) that catalyzes hexose transport across cell membranes through an ATP-independent, facilitative diffusion mechanism (Hruz and Mueckler, 2001). These sugar transporters display differences in their kinetics and respective substrate specificities, such that GLUT5 and perhaps GLUT11 are likely fructose transporters. GLUT4 is highly expressed in adipose tissue and skeletal muscle, but these tissues also express a selective cohort of these other transporters. In the case of skeletal muscle, GLUT1, GLUT5, and GLUT12 may significantly contribute to sugar uptake in addition to GLUT4 (Stuart et al., 2000, 2006), while in adipose tissue GLUT8, GLUT12, and HMIT are also expressed (Wood et al., 2003; Wood and Trayhurn, 2003). However, GLUT4 displays the unique characteristic of a mostly intracellular disposition in the unstimulated state that is acutely redistributed to the plasma membrane in response to insulin and other stimuli (Bryant et al., 2002; Czech and Corvera, 1999).

GLUT4 contains unique sequences in its N- and COOHterminal cytoplasmic domains that direct its characteristic membrane trafficking capability (Figure 1). These include a distinctive N-terminal sequence with a potentially critical phenylalanine residue (Piper et al., 1993; Araki et al., 1996; Melvin et al., 1999; Al-Hasani et al., 2002), as well as dileucine and acidic motifs in the COOH terminus (Corvera et al., 1994; Garippa et al., 1996; Shewan et al., 2000; Sandoval et al., 2000; Martinez-Arca et al., 2000). These motifs likely govern kinetic aspects of both endocytosis and exocytosis in a continuously recycling trafficking system. The COOH terminus LL and acidic motifs are also present in an aminopeptidase protein (IRAP) that in adipocytes is similarly sequestered within GLUT4-enriched intracellular membranes and highly responsive to insulin action (Johnson et al., 1998, 2001; Hou et al., 2006). These considerations place GLUT4 at the interface of two exciting fields of biology—insulin signaling and membrane trafficking. GLUT4 Is a Key Determinant of Glucose Homeostasis A central role for GLUT4 in whole-body metabolism is strongly supported by a variety of genetically engineered mouse models where expression of the transporter is either enhanced or ablated in muscle or adipose tissue or both. The whole-body GLUT4/ mouse itself may be less informative due to upregulation of compensatory mechanisms that may promote survival of these animals (Katz et al., 1995; Stenbit et al., 1996). However, heterozygous GLUT4+/ mice that display decreased GLUT4 protein in muscle and adipose tissue show the expected insulin resistance and propensity toward diabetes that is consistent with a major role of GLUT4 in glucose disposal (Rossetti et al., 1997; Stenbit et al., 1997; Li et al., 2000). Interestingly, overexpression of GLUT4 expression in skeletal muscle of such GLUT4+/ animals through crosses with transgenic mice normalizes insulin sensitivity and glucose tolerance (Tsao et al., 1999). Transgenic mice expressing high levels of GLUT4 in adipose tissue (Shepherd et al., 1993; Tozzo et al., 1995) or in skeletal muscle (Tsao et al., 1996, 2001) in turn are both highly insulin sensitive and glucose tolerant. Cell Metabolism 5, April 2007 ª2007 Elsevier Inc. 237

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Review

Figure 1. Structural Features of the Insulin-Regulated GLUT4 Glucose Transporter Protein The unique sensitivity of GLUT4 to insulin-mediated translocation appears to derive from sequences shown in the N-terminal (required phenylalanine) and COOH-terminal (required dileucine and acidic residues) regions. These sequences are likely involved in rapid internalization and sorting of GLUT4 in intracellular membranes termed GLUT4 storage vesicles (GSV), as outlined in Figure 3. See text for further details.

Conversely, conditional depletion of GLUT4 in either adipose tissue or skeletal muscle causes insulin resistance and a roughly equivalent incidence of diabetic animals (Zisman et al., 2000; Abel et al., 2001). This was particularly surprising in the former case since adipose tissue accounts for only a small fraction of total body glucose disposal (James et al., 1985). These tissue-specific depletions of GLUT4 have profound metabolic effects on other tissues. For example, mice with muscle-specific GLUT4 deficiency display decreased insulin responsiveness in adipose tissue and liver (Zisman et al., 2000), while those with adipose-specific GLUT4 depletion exhibit muscle and liver insulin resistance (Abel et al., 2001). In the muscle-specific GLUT4 knockout mice, this is at least partially mediated through elevated blood glucose levels that occur in the conditional knockout animals, which secondarily impairs insulin signaling (Kim et al., 2001). Remarkably, overexpression of GLUT4 in the adipose tissue of muscle-specific GLUT4 deficient mice overcomes the glucose intolerance and diabetes (Carvalho et al., 2005). More recently, it has been reported that a retinol binding protein (RBP4) is released into the serum from GLUT4-deficient adipose tissue (Yang et al., 2005), and that RBP4 may contribute to the insulin resistance of obese and diabetic individuals (Graham et al., 2006). The mechanisms by which glucose sensing through GLUT4 may operate to integrate whole-body metabolism have been recently reviewed (Herman and Kahn, 2006). Even though cell surface GLUT4 is highly dependent on insulin in vitro, muscle-specific (MIRKO) or adiposespecific (FIRKO) insulin receptor knockout mice produce surprisingly mild metabolic phenotypes compared to the 238 Cell Metabolism 5, April 2007 ª2007 Elsevier Inc.

conditional GLUT4 knockout mice described above. MIRKO mice have an enlarged fat mass with increased serum triglyceride and free fatty acids, but otherwise have normal whole-body glucose homeostasis (Bruning et al., 1998). Insulin-stimulated glucose uptake is greatly reduced in MIRKO muscle, but muscle glycogen level is normal, indicating possible compensatory mechanisms for glucose import (Kim and Accili, 2002; Fernandez et al., 2001). FIRKO mice have severe insulin resistance in adipose tissue but are protected from age- and hyperphagia-induced glucose intolerance (Bluher et al., 2002). This favorable metabolic phenotype probably results from enhanced adiponectin and leptin secretion and improved whole-body lipid metabolism (Xue and Kahn, 2006). Thus, GLUT4 in muscle and adipose tissue is indispensable for normal global glucose homeostasis, while insulin receptor in these tissues appears much less critical. Analysis of the above mouse models also highlights the potential dissociation between glucose uptake versus triglyceride synthesis and storage in the adipocyte. The adipose-specific GLUT4 deficient mouse has normal fat mass and adipocyte size (Bluher et al., 2002). In the absence of robust glucose import and glycolysis, triglyceride synthesis in adipocytes can proceed via enhanced glyceroneogenesis based on substrates derived from liver and muscle (Reshef et al., 2003). On the other hand, lack of the lipogenic and antilipolytic actions of insulin in adipocytes of the FIRKO mouse leads to reduced fat mass. These observations are consistent with the concept that adipocytes are the major site for energy (triglyceride) storage but not glucose disposal, which mostly occurs in muscle. If this is the case, why did GLUT4-mediated glucose

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Review uptake in the adipocyte evolve to be a critical sensing mechanism for maintaining whole-body glucose homeostasis? Perhaps the answer to this question is that insulin-regulated GLUT4 content on the adipocyte plasma membrane closely reflects nutrient status and is therefore a sensitive and reliable energy sensor. Control of Endogenous GLUT4 Expression The profound effects on whole-body glucose homeostasis observed in mouse models of GLUT4 deficiency or overexpression heighten the potential physiological importance of changes in endogenous GLUT4 expression in different states. Examples of particular interest are the downregulation of GLUT4 expression in adipose tissue in obesity (Olefsky et al., 1988; Garvey et al., 1991; Sinha et al., 1991) and the upregulation of GLUT4 expression in skeletal muscle in response to exercise (Ren et al., 1994; Kraniou et al., 2000; Holmes and Dohm, 2004) or thyroid hormone (Weinstein et al., 1991; Torrance et al., 1997a, 1997b). Also, GLUT4 expression is greatly decreased in muscle atrophy (Didyk et al., 1994; Blakemore et al., 1996) and denervation (Block et al., 1991; Henriksen et al., 1991; Coderre et al., 1992), consistent with decreased energy requirements in these conditions. Signals from neuronal cells in the form of neuregulins may mediate some of these effects (Jo et al., 1995; Suarez et al., 2001). These changes in GLUT4 protein levels could translate into alterations in glucose tolerance if the effects observed in transgenic mice apply to normal physiology. Thus, therapeutic strategies based on enhancing GLUT4 expression may facilitate drug discovery. For these reasons the mechanisms that regulate GLUT4 expression are important to clarify, and there is much fertile territory for exploration in this field. Tissue-specific expression of GLUT4 in adipose tissue, skeletal muscle, and cardiac muscle, as well as its regulation by fasting and refeeding, is conferred within a 2.4 kb DNA segment at the 50 region of the GLUT4 gene (Liu et al., 1992). For skeletal muscle-specific expression, a region between 522 and 420 has been inferred to be important in transgenic mice (Thai et al., 1998). This region contains an apparent myocyte enhancer factor (MEF)2 binding domain at 466 to 457 that is critical for specifying tissue expression (Liu et al., 1994) and increased GLUT4 expression during muscle regeneration (Moreno et al., 2003). Near this same region it has been proposed that thyroid hormone receptor and myoD form a complex with MEF2 to regulate GLUT4 expression (Santalucia et al., 2001). However, modulation of GLUT4 expression by denervation does not apparently require this region (Tsunoda et al., 2000). Another domain that has been implicated in tissue-specific expression is termed Domain I and includes the region 742 to 712 relative to the initiation site for transcription (Oshel et al., 2000). A factor termed GEF appears to operate in this region in association with MEF2A (Knight et al., 2003), but the putative 50kd GEF is identical in sequence to a segment of a larger protein (HDBP1) that regulates the transcription of the Huntington polypeptide in brain (Tanaka et al., 2004).

These and other data have been recently reviewed in detail (Olson and Knight, 2003; Zorzano et al., 2005), and suggest a complex mode of GLUT4 regulation at the transcriptional level that is poorly understood at present. Furthermore, whether there are mechanisms that selectively regulate translation or degradation of GLUT4 protein or mRNA is unknown. Signaling Mechanisms that Acutely Regulate GLUT4 Both insulin and exercise acutely stimulate GLUT4 recruitment to the cell surfaces of muscle and adipose cells independent of transcription or translation (Herman and Kahn, 2006; Rose and Richter, 2005). Nevertheless, these two physiological stimuli initiate distinct signaling mechanisms that lead to enhanced GLUT4 translocation and glucose uptake (Figure 2). The insulin signaling pathway to GLUT4 has been discussed in detail in recent reviews (Bryant et al., 2002; Watson et al., 2004a; Thong et al., 2005). The canonical insulin signaling pathway is triggered by activation of the insulin receptor (IR) tyrosine kinase leading to tyrosine phoshphorylation of insulin receptor substrate proteins (IRS) and their recruitment of PI 3-kinase, which catalyzes conversion of phosphatidylinositol (4,5)P2 to phosphatidylinositol (3,4,5)P3 (denoted PIP3). PIP3 in turn triggers the activation of the protein kinase Akt through the actions of two intermediate protein kinases, PDK1 and Rictor/mTOR (Vanhaesebroeck and Alessi, 2000; Sarbassov et al., 2005b). The requirements of these overall reactions have largely been confirmed in vivo using genetically engineered mouse models (Nandi et al., 2004; Plum et al., 2005), and more recently by siRNA knockdown experiments in cultured cells (Jiang et al., 2003; Mitra et al., 2004; Zhou et al., 2004; Tang et al., 2005). Interestingly, Akt2 rather than the Akt1 or Akt3 isoforms appears to control GLUT4 trafficking in adipose and muscle cells as well as mediate insulin signaling to control glucose output in liver (Cho et al., 2001a; Bae et al., 2003; Jiang et al., 2003), while the Akt1 isoform appears to control cell and body size (Cho et al., 2001b; Whiteman et al., 2002) and Akt3 controls brain size (Easton et al., 2005). Substrates of Akt2 that may mediate insulin effects on the machinery of GLUT4 trafficking are being actively investigated. The GTPase activating protein TBC1D4, denoted AS160, is such a substrate (Kane et al., 2002) that catalyzes inactivation of Rab proteins 2A, 8A, 10 and 14 in vitro. Rab proteins are critical organizers of intracellular membrane trafficking (Zerial and McBride, 2001). Expression of mutant AS160 lacking Akt-specific phosphorylation sites inhibits insulin-stimulated GLUT4 translocation (Sano et al., 2003), suggesting it is a negative regulator that is itself inhibited by insulin through Akt. AS160 binding to IRAP has been demonstrated, although there are contradictory claims regarding whether such interaction is modulated by Akt phosphorylation of AS160 (Larance et al., 2005; Peck et al., 2006). It is also noteworthy that AS160 interaction with 14-3-3 proteins is dependent on Akt phosphorylation of its Thr642 residue (Ramm et al., 2006). AS160 plays a role in insulin-stimulated GLUT4 Cell Metabolism 5, April 2007 ª2007 Elsevier Inc. 239

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Review Figure 2. Convergence of Signaling Pathways Initiated by Insulin and Exercise Leading to GLUT4 Translocation Insulin signaling through the PI 3-kinase pathway and muscle contraction through both elevated AMP/ATP ratios and intracellular [Ca2+] leads to activation of downstream protein kinases (Akt, aPKCl/z, AMPK, CaMKII cPKC) that phosphorylate putative effectors that modulate steps in the GLUT4 trafficking pathways (also see Figure 3). AS160 is one such substrate that appears to negatively regulate an early step in GLUT4 exocytosis. Negative regulation of these pathways by fatty acids, cytokines, and endoplasmic reticulum stress responses are observed in obesity and diabetes, contributing to insulin resistance. Dashed lines imply hypothetical pathways not yet experimentally verified. See text for further details.

exocytosis but not its inhibition of GLUT4 endocytosis (Zeigerer et al., 2004). RNAi knockdown of AS160 increases basal GLUT4 levels on the adipocyte surface, consistent with its proposed role in intracellular GLUT4 retention that is relieved upon insulin stimulation (Eguez et al., 2005; Larance et al., 2005). However, AS160 knockdown only partially releases the pool of intracellular GLUT4 mobilized by insulin, and careful analysis suggests that other unknown Akt substrate proteins must make major contributions to overall GLUT4 regulation by insulin (Eguez et al., 2005; Gonzalez and McGraw, 2006, Bai et al., 2007). Muscle contraction also increases AS160 phosphorylation, but it may do so by a PI 3-kinase independent mechanism (Bruss et al., 2005; Kramer et al., 2006; Deshmukh et al., 2006). Contraction-induced AS160 phosphorylation is mediated through the AMPK pathway, providing a potential convergence of insulin and exercise-mediate signaling to GLUT4 (see Figure 2; Jessen and Goodyear, 2005; Kramer et al., 2006; Treebak et al., 2006). On the other hand, simultaneously disrupting AMPK and Akt failed to completely inhibit contraction-induced AS160 phosphorylation, consistent with additional signals leading to GLUT4 translocation (Figure 2). Interestingly, calmodulin binds to AS160 in a Ca2+-dependent manner (Kane and Lienhard, 2005), and treatment of T cells with calcium ionophore increases AS160 mRNA levels (Matsumoto et al., 2004). Furthermore, it appears that AS160 is a target of metabolic disease related to insulin resistance. Insulin-stimulated AS160 phosphorylation is impaired in skeletal muscle of type II diabetic patients (Karlsson et al., 2005) and in response to TNF-a (Plomgaard et al., 2005). Furthermore, in skeletal muscle of first-degree relatives of diabetic patients, the correlation between AS160 phosphorylation and glucose uptake is lost, predicting an increasing risk for onset of the disease (Karlsson 240 Cell Metabolism 5, April 2007 ª2007 Elsevier Inc.

et al., 2006). However, levels of insulin-induced GLUT4 translocation in the skeletal muscle of type II diabetic patients are typically reduced by 90% (Ryder et al., 2000), while the correspondent impairment in AS160 phosphorylation is down only 39% (Karlsson et al., 2005). Thus, other components of the GLUT4 translocation machinery are most likely also impaired in diabetic patients, and it is not yet clear whether defective AS160 phosphorylation contributes functionally to insulin resistance. Substantial evidence also indicates atypical PKCl/z acts downstream of PI 3-kinase to relay insulin signals to GLUT4 translocation (Farese et al., 2005; Figure 2). However, there are conflicting results using RNAi regarding the importance of PKCl/z on insulin-stimulated glucose uptake in adipocytes (Zhou et al., 2004; Sajan et al., 2006). Thus, the precise roles of atypical PKCl/z in GLUT4 regulation need further clarification, perhaps from tissue-specific knockout mice. A proposed PI 3-kinase-independent pathway including component proteins APS, c-Cbl, CAP, CrkII/C3G, and TC10 has also been proposed to operate in parallel to the PI 3-kinase pathway as a required mechanism that promotes GLUT4 translocation (Saltiel and Kahn, 2001; Saltiel and Pessin, 2003). Indeed, good evidence indicates that APS and CAP proteins do associate with insulin receptor complexes (Ribon et al., 1998; Hu et al., 2003). However, siRNA-mediated gene silencing of several intermediates of this putative signaling pathway in 3T3-L1 adipocytes have challenged this hypothesis (Mitra et al., 2004; Zhou et al., 2004), and other data suggest TC10 does not promote GLUT4 translocation in cultured muscle cells (JeBailey et al., 2004). Furthermore, gene ablation of APS or c-Cbl in mice actually improve insulin sensitivity in peripheral tissues, consistent with the idea that these proteins act instead as negative regulators of the insulin signaling pathway (Minami et al., 2003; Molero et al., 2004). Thus, APS may be involved in insulin receptor

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Review endocytosis, a process potentially regulated by Akt (Kishi et al., 2006; Katsanakis and Pillay, 2005). Perhaps TC10 functions in control of actin which secondarily facilitates GLUT4 movements in 3T3-L1 adipocytes (Chiang et al., 2001; Kanzaki et al., 2002; Chang et al., 2006), but whether this occurs in primary adipocytes is unknown. The fact that ectopic expression in adipocytes of an activated form of Akt alone stimulates GLUT4 translocation is consistent with the concept that the PI 3-kinase pathway may be sufficient for GLUT4 translocation (Kohn et al., 1996; Eyster et al., 2005). However, expressed Akt could have confounding effects in such experiments and further data are required to unequivocally clarify this point. The PI 3-kinase pathway mediates insulin signaling to regulate GLUT4 in skeletal muscle as well (Ploug and Ralston, 2002; Ishiki and Klip, 2005). In addition, muscle contraction acutely increases GLUT4 transporter levels in sarcolemma and T-tubules, and is additive to the effect of insulin stimulation (Nesher et al., 1985; Zorzano et al., 1986). Muscle contraction and insulin stimulation were suggested to target separate pools of intracellular GLUT4-containing membranes, and convincing evidence has established that these processes are regulated by different signaling mechanisms (see recent reviews by Holloszy, 2003; Jessen and Goodyear, 2005; Rose and Richter, 2005). Two cellular consequences of muscle contraction, a transient increase in intracellular calcium concentration ([Ca2+]i) and an increase in [AMP]/[ATP] ratio, are thought to contribute to enhanced GLUT4 translocation to the cell surface (Figure 2). The former signal is mediated through activations of the protein kinase CaMKII and perhaps conventional protein kinase C (Jessen and Goodyear, 2005; Rose and Richter, 2005). In addition, Ca2+/CaM appears to activate the AMPK signaling pathway through upstream kinases CaMKKa and b, at least in cultured cell lines and ex vivo brain slices (Hurley et al., 2005; Hawley et al., 2005; Woods et al., 2005). AMPK acts as a cellular energy gauge that is allosterically activated by increasing [AMP]/[ATP] ratios and perhaps other mechanisms. This transforms AMPK into a better substrate for at least one of its upstream activator kinases, e.g., LKB1, or a potentially poorer substrate for one of its phosphatases (reviewed by Fujii et al., 2006; Jorgensen et al., 2006). Muscle glycogen appears to be potent negative regulator of AMPK activity (Derave et al., 2000; Wojtaszewski et al., 2002), thus providing a negative feedback mechanism for AMPK-mediated glucose uptake. The critical substrates (‘‘effectors’’ in Figure 2) that mediate GLUT4 translocation downstream of these protein kinases are unknown. The role of AMPK in contraction-induced glucose uptake has been questioned. Muscle-specific overexpression of a dominant-inhibitory catalytic subunit of AMPK (i.e., a2-AMPK: the major catalytic isoform in skeletal muscle) reduced contraction-mediated glucose uptake by only 30%–40% (Mu et al., 2001). However, a recent report suggests even that modest effect can be accounted for by a corresponding decrease in force generation by the transgenic muscle (Fujii et al., 2005). On the other hand,

examinations of skeletal muscle isolated from wholebody a1-AMPK or a2-AMPK knock-out mice suggest that in the absence of the a2 subunit, the a1 catalytic domain may mediate contraction-induced glucose uptake (Jorgensen et al., 2004). Furthermore, muscle-specific deletion of LKB1 (the major upstream AMPK kinase in muscle) inhibits contraction-induced glucose uptake, which cannot be explained by reduction in muscle force generation (Sakamoto et al., 2005). However, since LKB1 also phosphorylates and activates many other AMPKrelated kinases (Lizcano et al., 2004), AMPK involvement is unclear. Interestingly, muscle-specific LKB1 knockout mice have enhanced insulin sensitivity and improved whole-body glucose homeostasis (Koh et al., 2006), suggesting AMPK also regulates the insulin signaling pathway in vivo (Sarbassov et al., 2005a; Um et al., 2006). Nevertheless, enhanced muscle glucose uptake in response to activation of AMPK by AICAR (i.e., an AMP analog) is not additive to that achieved through muscle contraction, indicating AMPK is a component of the contraction signaling mechanism (Hayashi et al., 1998). As discussed above, Ca2+/CaM-mediated signaling pathways also lead to GLUT4 translocation (Figure 2). Recently, increases in cytosolic Ca2+ were found to induce metalloproteinasemediated release of neuregulins, which activates receptor tyrosine kinase ErbB4 (Canto et al., 2006). Importantly, blocking ErbB4 activation impairs contraction-induced glucose uptake in slow twitch muscle fibers where alternative AMPK signaling is minimal. In addition, muscle cell depolarization increases surface GLUT4 through reduced endocytosis independent of AMPK activity (Wijesekara et al., 2006). Thus, existing evidence indicates an AMPK-mediated pathway may be one of several redundant contraction-induced signaling mechanisms leading to GLUT4 translocation to the cell surface. A variety of other cellular stress signals also enhance glucose uptake in skeletal muscle. Hypoxia and inhibitors of cellular metabolism (e.g., glycolysis inhibitor arsenate; electron transport inhibitor rotenone; oxidative phosphorylation uncoupler 2,4-dinitrophenol) signal at least partially through AMPK by decreasing the cellular energy supply and increasing AMP/ATP ratios (Figure 2). Hyperosmolality also promotes GLUT4 translocation by activating AMPK in muscle or by activating a Gab-1 dependent signaling pathway in adipocytes (Gual et al., 2003). Interestingly, osmotic shock also activates PIKfyve, a recently identified Akt substrate (Berwick et al., 2004; Sbrissa and Shisheva, 2005). The lipid product of PIKfyve activity [i.e., PtdIns(5)P] has been shown to promote GLUT4 exocytosis to the adipocyte plasma membrane (Sbrissa et al., 2004). Paradoxically, chronic hyperosmotic stress results in insulin resistance in isolated or cultured adipocytes, which is apparently mediated through the mTOR signaling pathway (Gual et al., 2003). Recent breakthroughs in mTOR signaling have revealed intricate negative feedback mechanisms that target the insulin and AMPK signaling pathways (reviewed by Sarbassov et al., 2005a; Um et al., 2006). In addition, impaired responsiveness of GLUT4 to the insulin signaling pathway occurs in obesity and Cell Metabolism 5, April 2007 ª2007 Elsevier Inc. 241

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Review diabetes through the actions of fatty acids (Kim et al., 2004), cytokines (Kanda et al., 2006; Weisberg et al., 2003), and the endoplasmic reticulum stress response (Ozcan et al., 2006). These negative regulators appear to act in part through the activation of stress protein kinases that phosphorylate IRS proteins on serine residues, thus attenuating the tyrosine phosphorylation of IRS by insulin (see Figure 2). The detailed mechanisms through which insulin resistance may be induced in obesity and diabetes has been recently reviewed (Petersen and Shulman, 2006; Marciniak and Ron, 2006; Tilg and Moschen, 2006). Regulated Steps in the Membrane Recycling of GLUT4 Newly synthesized and glycosylated GLUT4 enters a continuously recycling pathway that concentrates most of the GLUT4 in intracellular membrane systems in unstimulated adipocytes or muscle cells (Cushman and Wardzala, 1980; Suzuki and Kono, 1980; Oka et al., 1984; for recent reviews see Bryant et al., 2002; Dugani and Klip, 2005). Insulin markedly stimulates GLUT4 exocytic pathways (arrows 1, 2, and 5 in Figure 3) while also significantly inhibiting its endocytosis from the PM (arrow 4 in Figure 3), which together cause the overall redistribution of GLUT4 to the cell surface (Czech and Buxton, 1993; Holman and Cushman, 1994; Zeigerer et al., 2004). Recently, Blot and McGraw, 2006 reported that endocytosis of GLUT4 in unstimulated cells occurs mostly through a cholesteroldependent, clathrin adaptor AP-2-independent pathway, while insulin selectively inhibits this. Thus, GLUT4 recycling in insulin-stimulated adipocytes and muscle may be a specialized case of the clathrin-dependent recycling pathway which occurs in all cells, best studied in reference to the transferrin receptor (TfR) (for review, see Maxfield and McGraw, 2004, and Figure 3). Iron-bound transferrin (Tf) is taken up by TfR and endocytosed through a clathrinmediated mechanism in the plasma membrane (PM) which requires the Rab5 GTPase. Newly endocytosed vesicles merge with each other to form sorting endosomes/early endosomes (SE/EE) or are delivered directly to existing SE/EE. Proteins or lipids can be rapidly recycled back to the cell surface through narrow tubular structures derived from SE/EE (thin blue dotted line in Figure 3). SE/ EE are transient structures that appear to gradually mature into later endosomes (LE) to form lysosomes (LY). During this process, ferric ions dissociate from Tf-TfR in the increasingly acidic luminal environment, while the receptor is sorted to the endosomal recycling compartment (ERC) through tubular-vesicular structures. ERC is a stable cellular organelle, from which TfR escapes (thin blue solid line in Figure 3) with a slower rate than its influx rate. In the case of adipocytes, a highly insulin-responsive, specialized GLUT4 sequestration compartment (GSV) is thought to be downstream of the ERC (Figure 3). Skeletal and heart muscle have uniquely structured membrane systems through which insulin-sensitive GLUT4 trafficking proceeds, but the mechanisms for stimulation of GLUT4 translocation to the cell surface may be very similar (for reviews see Ploug and Ralston, 2002; Ishiki and Klip, 2005). 242 Cell Metabolism 5, April 2007 ª2007 Elsevier Inc.

The GSV membranes in adipocytes that selectively sequester GLUT4 and certain other proteins such as IRAP can be experimentally distinguished from the ERC (Figure 3). This concept is derived from studies using horseradish peroxidase-conjugated transferrin (Tf-HRP) to ablate membrane compartments constituting the general recycling pathway. Thus it was found that in unstimulated cultured adipocytes, more than 50% of the intracellular GLUT4 escapes Tf-HRP ablation, apparently by localizing in specialized membranes (GSV) sensitive to insulin (Livingstone et al., 1996; Martin et al., 1997, 1998; Millar et al., 1999; Zeigerer et al., 2002). The transition of endocytosed GLUT4 from endosomes to GSVs can be monitored by time-dependent Tf-HRF ablation, which is assisted by Rab11 (Zeigerer et al., 2002) and requires the sorting signal FQQI on the GLUT4 N terminus (Melvin et al., 1999; Palacios et al., 2001; see Figure 1). Interestingly, GLUT4 accumulates with TfR in ERC when exogenously expressed in fibroblasts, and subsequent exodus of both GLUT4 and TfR to the PM can be stimulated by insulin (Lampson et al., 2000, 2001). Nevertheless, GLUT4 is more effectively sequestered than TfR and responds more strongly to insulin stimulation, due in part to the dileucine sorting motif on the GLUT4 COOH terminus (Johnson et al., 2001; arrow 2 in Figure 3). Furthermore, TfR and GLUT4 are sorted into the ERC through different mechanisms and subsequently escape in distinct secretory vesicles, indicating GLUT4 is uniquely sorted even in fibroblasts (Wei et al., 1998; Lampson et al., 2001). The fact that ERC also likely contributes to insulin-enhanced glucose uptake in adipose and muscle cells, as evidenced by TfR sensitivity to insulin, suggest that GSVs could be evolutionarily derived from recycling endosomes to confer greater insulin sensitivities to these cells (Kandror and Pilch, 1998; Hashiramoto and James, 2000; Lampson et al., 2001; Becker et al., 2001; Zeigerer et al., 2002). It should also be noted that the rapid recycling pathway directly from SE/EE to the PM (thin blue dotted line in Figure 3) defined for TfR recycling (Sheff et al., 2002; van Dam et al., 2002) has not yet been tested for its potential responsiveness to insulin (arrow 3 in Figure 3). Taken together, these and other data support the hypothesis presented in Figure 3 that insulin signaling directly modulates the GSV (arrow 1) and to a lesser extent the ERC (arrow 2) to promote translocation of GLUT4 and other recycling proteins to the PM. Multiple studies have established GSVs as major targets of insulin-mediated mobilization in insulin-sensitive tissues (Aledo et al., 1997; Martin et al., 1998, 2000; Millar et al., 1999; Hashiramoto and James, 2000). Furthermore, recent evidence supports the concept that GLUT4 continuously recycles through this compartment, albeit slowly in the basal state, as opposed to being stored in a static site that is not in equilibrium with the PM (see references for contrasting views: Coster et al., 2004; Govers et al., 2004; Karylowski et al., 2004; Martin et al., 2006). Biochemically, GSVs lack common endosomal markers such as TfR, Rab4, annexin II and cellubrevin (Aledo et al., 1997; Hashiramoto and James, 2000), but are

Cell Metabolism

Review Figure 3. Combinatorial Regulation by Insulin of Multiple Steps in the Membrane Trafficking Pathways of GLUT4 GLUT4 continuously recycles through several membrane systems of adipose and muscle cells and accumulates in sequestration vesicles (GSV) and perhaps other intracellular membranes (e.g., ERC, TGN) due to very slow exocytosis rates in the absence of stimulation. Insulin stimulates exocytosis of GSV (arrow 1) and ERC (arrow 2) and may also enhance a rapid short circuit pathway from the sorting endosome/early endosome (SE/EE) to the plasma membrane (PM) (dashed arrow 3). Recent data indicate that insulin signaling also directly enhances the fusion of docked GLUT4-containing vesicles by decreasing docking duration (arrow 5), and a preceding tethering step may also be insulin regulated (dashed arrow 6). In addition, insulin signaling attenuates GLUT4 endocytosis (arrow 4), which in combination with its stimulatory effects on exocytic steps promotes redistribution of GLUT4 to the cell surface. Insulin-responsive Akt (see Figure 2) may operate in the early steps of exocytosis (arrows 1 and 2), while other PI 3-kinase-dependent mediators may operate at the fusion step (arrow 5), but further work is necessary to confirm this hypothesis. Dashed arrows emanating from insulin indicate hypotheses that await definitive experimental approaches. Insulin may also regulate the interchange (blue dashed bidirectional arrows) between membranes deriving from endosomal recycling compartment (ERC), the trans-Golgi (TGN), and GSV (circle 7). All of the above membrane trafficking pathways are thought to be governed by small Rab GTPases or related proteins, which have been reviewed in detail elsewhere (Zerial and McBride, 2001).

enriched in GLUT4 (but not GLUT1), IRAP and the v-SNARE protein VAMP2 (Martin et al., 1996). They are a subpopulation of GLUT4-containing vesicles that can be isolated based on lack of cellugyrin content and are mostly sensitive to insulin mobilization (Kupriyanova et al., 2002). There is also an ongoing dispute on whether the TGN itself is the GSV compartment. The existence of retrograde transport from endosomes to TGN that is utilized by TGN proteins MPR, furin and TGN38 supports the TGN hypothesis (Bonifacino and Rojas, 2006). Electron microscopy shows a sizable GLUT4 population is located in endosomes and TGN (Slot et al., 1991; Martin et al., 1997). Based on quantitative colocalization analysis between GLUT4 and CD-MPR (cation-dependent manose-6-phosphate receptor) before and after insulin stimulation, it was proposed that TGN interacts with recycling endosomes to generate GSVs (Martin et al., 2000; Ramm et al., 2000; Bryant et al., 2002). After passing through endosomal compartments in the 3T3-L1 adipocyte, GLUT4 apparently transits into perinuclear vesicular-tubular structures that are partially labeled by endosome/TGN t-SNAREs syntaxins 6 and 16 (Shewan et al., 2003). These syntaxins appear to be important for transporting GLUT4 to GSVs, and an acidic targeting motif near the dileucine on the COOH terminus of GLUT4 is proposed to be the sorting signal involved in this process (Shewan et al., 2003; Perera et al., 2003; Proctor et al., 2006). On the other hand, endocytosed GLUT4 localizes poorly with TGN38 and furin, suggesting that TGN plays a minimal role in intracellular GLUT4 recycling (Martin et al., 1994; Karylowski et al., 2004). These seemingly contradictory results could be due to complexity in TGN organization (Gu et al., 2001; Gleeson

et al., 2004), such that GLUT4 trafficking proceeds through a subdomain of the TGN that is labeled by CDMPR, syntaxins 6 and 16, but not by TGN38 and furin. It is also known that Golgi/TGN morphology and function change dramatically depending on cell type and physiological state (Polishchuk and Mironov, 2004). Thus, in specialized adipocytes the boundary between TGN and endosomes may be altered and a conventional TGN protein may perform a trafficking role outside the classical TGN. For example, the SNARE protein Vti1a is involved in vacuole-TGN trafficking in yeast but interacts with Syntaxins 6 and 16 in endosome-TGN trafficking in Hela cell (von Mollard et al., 1997; Mallard et al., 2002). Vti1a is a constitutive protein on GLUT4-containing membranes, and colocalizes extensively with GLUT4 in perinuclear structures in basal 3T3-L1 adipocytes (Bose et al., 2005). However, Vti1a is completely segregated from other Golgi/TGN proteins such as b-COP, p115 and g-adaptin, and its intracellular localization is not disrupted by the Golgi-specific drug brefeldin A. Instead Vti1a appears required for insulin-stimulated GLUT4 translocation (Bose et al., 2005), a function that is consistent with Vti1a-b localization in brain synaptic vesicles (Antonin et al., 2000). Nonetheless, it appears that whatever the nature of the insulin-sensitive GLUT4 sequestration compartment, it does not statically retain GLUT4 away from a continuously recycling dynamic even in the basal state (Martin et al., 2006). Even though it is not clear if the TGN serves as an insulin-sensitive sequestration compartment for GLUT4, the TGN does appear to function in GLUT4 transit to the GSV compartment (Figure 3). Golgi-localized g-ear-containing Arf binding proteins (GGAs) are a family of adaptors for Cell Metabolism 5, April 2007 ª2007 Elsevier Inc. 243

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Review clathrin coat assembly (Bonifacino, 2004) that define a subpopulation of transport vesicles involved in TGN-endosome trafficking. Importantly, GGA proteins apparently function in effective sorting of newly synthesized GLUT4 and IRAP into GSVs in adipocytes, which occurs before these molecules enter the GLUT4 recycling pathway (Figure 3; Watson et al., 2004b; Liu et al., 2005). Acquiring GSVs is an important component of adipocyte differentiation, and is assisted by sortilin, a resident membrane protein on GSVs (Morris et al., 1998; Shi and Kandror, 2005). Furthermore, GGAs may also be involved in regenerating GSVs following insulin stimulation (Li and Kandror, 2005). Cargo proteins recognized by GGAs have a dileucine sorting motif on their cytoplasmic domains (Bonifacino, 2004), consistent with the functional dileucine motif in IRAP (Johnson et al., 1998; Hou et al., 2006) and GLUT4 (Czech et al., 1993; Haney et al., 1995; Araki et al., 1996; Melvin et al., 1999; Shewan et al., 2000; Sandoval et al., 2000). Also, p115, a member of the Golgin family of proteins associated with the Golgi, colocalizes with GLUT4 in perinuclear structures through interaction with the N terminus of IRAP (Hosaka et al., 2005). Overexpression of the p115 N terminus disperses GLUT4 perinuclear localization and inhibits insulin-stimulated GLUT4 translocation. More recently, Golgin-160 has been shown to function at a step in the GLUT4 biosynthetic pathway prior to GGA-dependent sorting into GSVs (Williams et al., 2006). The Eps15-homology (EH) domain containing proteins are important components of the molecular machinery regulating intracellular membrane sorting and trafficking (Naslavsky and Caplan, 2005). Mammalian cells contain a subset of these proteins with the EH-domain located on the COOH terminus (EHD1-EHD4). EHD2 resides on GLUT4-containing membranes in primary adipocytes (Guilherme et al., 2004a), and EHD1 partially colocalizes with GLUT4 in perinuclear structures of 3T3-L1 adipocytes (Guilherme et al., 2004b). EHD2 functions in clathrin-mediated Tf-TfR and GLUT4 endocytosis (Guilherme et al., 2004a), while EHD1 is important for perinuclear sequestration of GLUT4 and may also play a role in generating GSVs (Guilherme et al., 2004b). These effects are in part mediated by EHBP1 (EH-domain binding protein 1), which binds to actin bundles through its CH domain and to EHD1/EHD2 through NPF motifs (Guilherme et al., 2004b). Importantly, RNAi knockdown of EHBP1 completely inhibits insulin-stimulated GLUT4 translocation in cultured adipocytes (Guilherme et al., 2004b), producing an inhibitory phenotype that is stronger than Akt knockdown in the same cells (Jiang et al., 2003). EHD1 is also required for exocytosis of major histocompartibility complex I and cystic fibrosis transmembrane conductance regulator (Caplan et al., 2002; Picciano et al., 2003). Interestingly, EHD1 is shown to interact with the Rab11 effector Rab11FIP2 (Naslavsky et al., 2006), and Rab11 is known to function in GLUT4 exocytosis (Zeigerer et al., 2002; Uhlig et al., 2005). Thus, EHD1, Rab11 and Rab11-FIP2 may coordinate intracellular sorting and trafficking of endocytosed GLUT4 in adipocytes. 244 Cell Metabolism 5, April 2007 ª2007 Elsevier Inc.

TIRF Imaging Applied to GLUT4 Recycling The concept that insulin signaling causes recruitment of GLUT4 to regions near the PM from the intracellular GSV and ERC (Figure 3) has recently been supported by total internal reflection fluorescence (TIRF) microscopy (Tengholm and Meyer, 2002; Lizunov et al., 2005; Gonzalez and McGraw, 2006; Bai et al., 2007, Huang et al., 2007). This technique images a region of about 100-250nm from the outside surface of the PM into the cytoplasm (Steyer and Almers, 2001), and reveals that insulin stimulates total GLUT4 levels in this region of the adipocyte by 2- to 3-fold (Tengholm and Meyer, 2002; Gonzalez and McGraw, 2006; Huang et al., 2007). This increased GLUT4 in the TIRF zone includes GLUT4 fused into the PM as well as GLUT4 in membrane vesicles underneath or docked to the PM. High resolution TIRF microscopy has now clearly consolidated the concept that insulin signaling directly modulates the PM membrane fusion mechanism (arrow 5 in Figure 3) that completes GLUT4 exocytosis in response to insulin (Huang et al., 2007, Bai et al., 2007). The insulin-mediated fusion step (arrow 5 in Figure 3) and insulin-stimulated recruitment/docking steps (arrows 1 and 2) are both dependent on PI 3-kinase activity. Akt inhibition greatly decreases GLUT4 in the TIRF zone, reflecting its presumed role in mediating the recruitment/docking of GLUT4 containing vesicles in response to insulin (Gonzalez and McGraw, 2006). Interestingly, there is continuing debate about the requirement of Akt for the fusion step of GLUT4 trafficking to the PM. The rate of membrane fusion of GLUT4-containing vesicles relative to the amount of GLUT4 in the TIRF zone appeared to be greater when Akt was inhibited than when PI 3-kinase was inhibited in the study by Gonzalez and McGraw, 2006. However, these results are based on the use of small molecule inhibitors and the analysis must be further confirmed by additional experiments. Indeed, reconstitution of GLUT4-containing vesicle fusion to the PM in a cell free system does seem to require Akt (Koumanov et al., 2005). It should also be noted that the mechanisms that may contribute to the increased GLUT4 in the TIRF zone in response to insulin include intracellular mobilization of GSVs (Bryant et al., 2002), movements on microtubules (Semiz et al., 2003) or actin (Tsakiridis et al., 1999; Kanzaki and Pessin, 2001; Jiang et al., 2002), motor proteins (Bose et al., 2002; Imamura et al., 2003), or even the formation of new GLUT4-containing membranes (Xu and Kandror, 2002). Another potential mechanism for intracellular retention of GLUT4 is insulinsensitive tethering through TUG protein (Bogan et al., 2003; Yu et al., 2007). These potential mechanisms all require further investigation to rigorously define their contributions to GLUT4 recruitment to the TIRF zone or docking prior to the fusion step. A particularly appealing approach to unraveling the mechanisms of GLUT4 recruitment into the TIRF zone (arrows 1 and 2 in Figure 3) is to identify additional putative Akt substrates that may mediate these effects. The mechanisms that regulate membrane fusion of GLUT4 containing vesicles with the PM remain elusive.

Cell Metabolism

Review The PM contains both the machinery for initiation of insulin signaling and the sites for exocytic GLUT4 vesicle fusion and clathrin-dependent and cholesterol-dependent GLUT4 endocytosis (Figure 3). The SNARE core proteins (i.e., VAMP2, syntaxin 4 and SNAP23) mediating exocytic GLUT4 vesicle fusion with the PM have been well characterized, and a few SNARE-associated proteins potentially regulating SNARE core formation have been identified (i.e., Munc18c, synip, tomosyn). These results are summarized in recent reviews (Bryant et al., 2002; Watson et al., 2004a; Ishiki and Klip, 2005; James, 2005). In addition, the exocyst complex has been proposed to function in exocytic GLUT4 vesicle tethering (Inoue et al., 2003, 2006; Ewart et al., 2005). Recently, a two-color TIRF microscope that is capable of imaging membrane trafficking events at 10 frames per second (fps) and at a spatial resolution approaching the optical limitation (i.e., 259 nm at 488 nm laser excitation) has been used to image GLUT4 docking and fusion events (Huang et al., 2007). Novel image-analysis tools have been developed to process thousands of TIRF images obtained from a single experiment (Huang et al., 2007). Analysis of hundreds of single-vesicle fusion events showed that insulin stimulates GLUT4 vesicle fusion frequency by 4 fold while significantly shortening vesicle tethering/docking durations prior to membrane fusion (arrow 5 in Figure 3). These conclusions have also been recently reported by Bai et al., 2007 using a different type of TIRF-based analysis. Insulin action is also associated with immobilization of GLUT4containing structures in the TIRF evanescence field (Lizunov et al., 2005; Huang et al., 2007), which is largely due to endocytic GLUT4 accumulation in static clathrin coats on the PM (Bellve et al., 2006; Huang et al., 2007). Taken together, the data available to date strongly support multiple direct effects of insulin on the movement of GLUT4 into proximity of the PM as well as direct effects of insulin on one or more steps of the docking and fusion process at the PM (Figure 3). Conclusions The role of GLUT4 as a dominant regulator of whole-body glucose homeostasis is now well established, based on many genetically engineered mouse models of GLUT4 overexpression or deficiency. These data reinforce the concept that either acute or long-term changes in the abundance of GLUT4 on the cell surface of adipose or muscle cells could provoke systemic changes in glucose disposal in vivo. Such changes include decreased GLUT4 expression in adipocytes in obesity and increased GLUT4 expression in adipocytes and muscle cells in response to exercise. However, we are still at an early stage of understanding the molecular mechanisms that underlie GLUT4 expression in these tissues. Some transcription factors involved in GLUT4 mRNA regulation are identified, but their exact roles within the various physiological states that alter GLUT4 expression need to be further clarified within transgenic mouse models. Other transcriptional regulators also likely play important roles and remain to be discovered.

Significant progress in understanding the membrane recycling pathway of GLUT4 has been made, including the identification of a RabGAP substrate (AS160) of the insulin-sensitive protein kinase Akt that may help restrain GLUT4 exocytosis in the basal state. Other unknown substrates also likely play a role, and their identification is crucial to further progress. Application of new imaging techniques to GLUT4 recycling has also yielded important new insights, including the hypothesis that both Akt-dependent and Akt-independent steps downstream of PI 3-kinase may be involved. Importantly, it is now clear that multiple steps in the GLUT4 trafficking pathway are regulated by insulin, and that the overall response of GLUT4 to insulin is combinatorial. For example, TIRF microscopy has demonstrated direct effects of insulin signaling on both the movement of GLUT4 from intracellular sites to distances within about 200nm of the PM as well as on the kinetics of GLUT4-containing vesicle docking and fusion. The ability to visualize GLUT4 recycling and docking/fusion steps by such imaging technology reveals the exciting complexity we face as each of the many trafficking steps are dissected and analyzed in future studies. ACKNOWLEDGMENTS The authors thank former and current laboratory members for insights, discussion and data that have contributed to this topic. The authors are grateful for the funding of our studies by the National Institutes of Health (DK030648, DK030898, DK063023, and Program Project grant DK06056) and to the University of Massachusetts Medical School Diabetes and Endocrinology Center (DK325220) for support of several core facilities that made our studies possible. M.P.C. is a member of the Scientific Advisory Board of CytRx Corporation. REFERENCES Abel, E.D., Peroni, O., Kim, J.K., Kim, Y.B., Boss, O., Hadro, E., Minnemann, T., Shulman, G.I., and Kahn, B.B. (2001). Adiposeselective targeting of the GLUT4 gene impairs insulin action in muscle and liver. Nature 409, 729–733. Aledo, J.C., Lavoie, L., Volchuk, A., Keller, S.R., Klip, A., and Hundal, H.S. (1997). Identification and characterization of two distinct intracellular GLUT4 pools in rat skeletal muscle: evidence for an endosomal and an insulin-sensitive GLUT4 compartment. Biochem. J. 325, 727– 732. Al-Hasani, H., Kunamneni, R.K., Dawson, K., Hinck, C.S., MullerWieland, D., and Cushman, S.W. (2002). Roles of the N- and C-termini of GLUT4 in endocytosis. J. Cell Sci. 115, 131–140. Antonin, W., Riedel, D., and von Mollard, G.F. (2000). The SNARE Vti1a-beta is localized to small synaptic vesicles and participates in a novel SNARE complex. J. Neurosci. 20, 5724–5732. Araki, S., Yang, J., Hashiramoto, M., Tamori, Y., Kasuga, M., and Holman, G.D. (1996). Subcellular trafficking kinetics of GLU4 mutated at the N- and C-terminal. Biochem. J. 315, 153–159. Bae, S.S., Cho, H., Mu, J., and Birnbaum, M.J. (2003). Isoform-specific regulation of insulin-dependent glucose uptake by Akt/protein kinase B. J. Biol. Chem. 278, 49530–49536. Bai, L., Wang, Y., Fan, J., Chen, Y., Ji, W., Qu, A., Xu, P., James, D.E., and Xu, T. (2007). Dissecting multiple steps of GLUT4 trafficking and identifying the sites of insulin action. Cell Metab. 5, 47–57. Becker, C., Sevilla, L., Tomas, E., Palacin, M., Zorzano, A., and Fischer, Y. (2001). The endosomal compartment is an insulin-sensitive

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