Insulin-regulated release from the endosomal recycling compartment is regulated by budding of specialized vesicles.

In several cell types, specific membrane proteins are retained intracellularly and rapidly redistributed to the surface in response to stimulation. In fat and muscle, the GLUT4 glucose transporter is dynamically retained because it is rapidly internalized and slowly recycled to the plasma membrane. Insulin increases the recycling of GLUT4, resulting in a net translocation to the surface. We have shown that fibroblasts also have an insulin-regulated recycling mechanism. Here we show that GLUT4 is retained within the transferrin receptor-containing general endosomal recycling compartment in Chinese hamster ovary (CHO) cells rather than being segregated to a specialized, GLUT4-recycling compartment. With the use of total internal reflection microscopy, we demonstrate that the TR and GLUT4 are transported from the pericentriolar recycling compartment in separate vesicles. These data provide the first functional evidence for the formation of distinct classes of vesicles from the recycling compartment. We propose that GLUT4 is dynamically retained within the endosomal recycling compartment in CHO cells because it is concentrated in vesicles that form more slowly than those that transport TR. In 3T3-L1 adipocytes, cells that naturally express GLUT4, we find that GLUT4 is partially segregated to a separate compartment that is inaccessible to the TR. We present a model for the formation of this specialized compartment in fat cells, based on the general mechanism described in CHO cells, which may explain the increased retention of GLUT4 and its insulin-induced translocation in fat cells.     

Sorting of GLUT4 into its insulin-sensitive store requires the Sec1/Munc18 protein mVps45

Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target–soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using sub­cellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment.     

Characterization of the insulin-regulated endocytic recycling mechanism in 3T3-L1 adipocytes using a novel reporter molecule

The endocytic trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) are regulated by insulin. We have used a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor (vpTR) to characterize IRAP-like trafficking in 3T3-L1 adipocytes. Our data demonstrate that the cytoplasmic domain of IRAP is sufficient to target vpTR to the insulin-regulated, slow recycling pathway in adipocytes and that the dynamic retention of vpTR is dependent on a di-leucine motif. Our kinetic analysis demonstrates that vpTR recycles as a single kinetic pool and that vpTR is very efficiently sorted from endosomes to the insulin-regulated recycling pathway. An implication of these findings is that the key step in the dynamic retention of vpTR occurs within the early endosomal system. We have previously shown that vpTR is trafficked by an insulin-regulated pathway in Chinese hamster ovary cells (Johnson, A. O., Subtil, A., Petrush, R., Kobylarz, K., Keller, S., and Mc Graw, T. E. (1998) J. Biol. Chem. 273, 17968-17977). The behavior of vpTR in Chinese hamster ovary cells is similar to its behavior in 3T3-L1 adipocytes. The main difference is that insulin has a larger effect on the trafficking of vpTR in the adipocytes. We concluded that the insulin-regulated slow recycling endocytic mechanism is expressed in many different cell types and therefore is not a unique characteristic of cells that express GLUT4.     

Rab11a and its binding partners regulate the recycling of the ß1-adrenergic receptor

ß1-adrenergic receptors (ß1-AR) are internalized in response to agonists and then recycle back for another round of signaling. The serine 312 to alanine mutant of the ß1-AR (S312A) is internalized but does not recycle. We determined that WT ß1-AR and S312A were internalized initially to an early sorting compartment because they colocalized by > 70% with the early endosomal markers rab5a and early endosomal antigen-1 (EEA1). Subsequently, the WT ß1-AR trafficked via rab4a-expressing sorting endosomes to recycling endosomes. In recycling endosomes WT ß1-AR were colocalized by > 70% with the rab11 GTPase. S312A did not colocalize with either rab4a or rab11, instead they exited from early endosomes to late endosomes/lysosomes in which they were degraded. Rab11a played a prominent role in recycling of the WT ß1-AR because dominant negative rab11a inhibited, while constitutively active rab11a accelerated the recycling of the ß1-AR. Next, we determined the effect of each of the rab11-interacting proteins on trafficking of the WT ß1-AR. The recycling of the ß1-AR was markedly inhibited when myosin Vb, FIP2, FIP3 and rabphillin were knocked down. These data indicate that rab11a and a select group of its binding partners play a prominent role in recycling of the human ß1-AR.     

GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins 6 and 16 but not TGN38: involvement of an acidic targeting motif

Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen 1 protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of the trans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process.     

Molecular mechanisms controlling GLUT4 intracellular retention

In basal adipocytes, glucose transporter 4 (GLUT4) is sequestered intracellularly by an insulin-reversible retention mechanism. Here, we analyze the roles of three GLUT4 trafficking motifs (FQQI, TELEY, and LL), providing molecular links between insulin signaling, cellular trafficking machinery, and the motifs in the specialized trafficking of GLUT4. Our results support a GLUT4 retention model that involves two linked intracellular cycles: one between endosomes and a retention compartment, and the other between endosomes and specialized GLUT4 transport vesicles. Targeting of GLUT4 to the former is dependent on the FQQI motif and its targeting to the latter is dependent on the TELEY motif. These two motifs act independently in retention, with the TELEY-dependent step being under the control of signaling downstream of the AS160 rab GTPase activating protein. Segregation of GLUT4 from endosomes, although positively correlated with the degree of basal retention, does not completely account for GLUT4 retention or insulin-responsiveness. Mutation of the LL motif slows return to basal intracellular retention after insulin withdrawal. Knockdown of clathrin adaptin protein complex-1 (AP-1) causes a delay in the return to intracellular retention after insulin withdrawal. The effects of mutating the LL motif and knockdown of AP-1 were not additive, establishing that AP-1 regulation of GLUT4 trafficking requires the LL motif.     

Transient changes in four GLUT4 compartments in rat adipocytes during the transition,insulin-stimulated to basal: implications for the GLUT4 trafficking pathway

In rat adipocytes, insulin-induced GLUT4 recruitment to the plasma membrane (PM) is associated with characteristic changes in the GLUT4 contents of three distinct endosomal fractions, T, H, and L. The organelle-specific marker distribution pattern suggests that these endosomal GLUT4 compartments are sorting endosomes (SR), GLUT4-storage endosomes (ST), and GLUT4 exocytotic vesicules (EV), respectively, prompting us to analyze GLUT4 recycling based upon a four-compartment kinetic model. Our analysis revealed that insulin modulates GLUT4 trafficking at multiple steps, including not only the endocytotic and exocytotic rates, but also the two rate coefficients coupling the three intracellular compartments. This analysis assumes that GLUT4 cycles through PM T, H,L, and back to PM, in that order, with transitions characterized by four first-order coefficients. Values assigned to these coefficients are based upon the four steady-state GLUT4 pool sizes assessed under both basal and insulin stimulated states and the transition time courses observed in the plasma membrane GLUT4 pool. Here we present the first reported experimental measurements of transient changes in each of the four GLUT4 compartments during the insulin-stimulated to basal transition in rat adipocytes and compare these experimental results with the corresponding model simulations. The close correlation of these results offers clear support for the general validity of the assumed model structure and the assignment of the T compartment to the sorting endosome GLUT4 pool. Variations in the recycling pathway from that of an unbranched cyclic topography are also considered in the light of these experimental observations. The possibility that H is a coupled GLUT4 storage compartment lying outside the direct cyclic pathway is contraindicated by the data. Okadaic acid-induced GLUT4 recruitment is accompanied by modulation of the rate coefficients linking individual endosomal GLUT4 compartments, further demonstrating a significant role of the endosomal pathways in GLUT4 exocytosis.     

Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit rab4 and perturb membrane recycling

In normal human skin fibroblasts (HSFs), fluorescent glycosphingolipid analogues are endocytosed and sorted into two pools, one that is recycled to the plasma membrane and one that is transported to the Golgi complex. Here, we investigated glycosphingolipid recycling in Niemann-Pick type A and C lipid storage disease fibroblasts (NPFs). Cells were incubated with a fluorescent analogue of lactosylceramide (LacCer) at 16 degrees C to label early endosomes (EEs), shifted to 37 degrees C, and lipid recycling was quantified. Using dominant negative rabs, we showed that, in normal HSFs, LacCer recycling was rapid (t1/2 approximately 8 min) and mainly rab4-dependent. In NPFs, LacCer recycling was delayed (t1/2 approximately 30-40 min), and rab4-dependent recycling was absent, whereas rab11-dependent recycling predominated. Transferrin recycling via the rab4 pathway was similarly perturbed in NPFs. Compared with normal HSFs, EEs in NPFs showed high cholesterol levels and an altered organization of rab4. In vitro extraction of rab4 (but not rab11) with GDP dissociation inhibitor was severely attenuated in NPF endosomal fractions. This impairment was reversed with cholesterol depletion of isolated endosomes or with high-salt treatment of endosomes. These data suggest that abnormal membrane recycling in NPFs results from specific inhibition of rab4 function by excess cholesterol in EEs.     

Insulin-regulated Aminopeptidase Is a Key Regulator of GLUT4 Trafficking by Controlling the Sorting of GLUT4 from Endosomes to Specialized Insulin-regulated Vesicles

Insulin stimulates glucose uptake by regulating translocation of the GLUT4 glucose transporter from intracellular compartments to the plasma membrane. In the absence of insulin GLUT4 is actively sequestered away from the general endosomes into GLUT4-specialized compartments, thereby controlling the amount of GLUT4 at the plasma membrane. Here, we investigated the role of the aminopeptidase IRAP in GLUT4 trafficking. In unstimulated IRAP knockdown adipocytes, plasma membrane GLUT4 levels are elevated because of increased exocytosis, demonstrating an essential role of IRAP in GLUT4 retention. Current evidence supports the model that AS160 RabGAP, which is required for basal GLUT4 retention, is recruited to GLUT4 compartments via an interaction with IRAP. However, here we show that AS160 recruitment to GLUT4 compartments and AS160 regulation of GLUT4 trafficking were unaffected by IRAP knockdown. These results demonstrate that AS160 is recruited to membranes by an IRAP-independent mechanism. Consistent with a role independent of AS160, we showed that IRAP functions in GLUT4 sorting from endosomes to GLUT4-specialized compartments. This is revealed by the relocalization of GLUT4 to endosomes in IRAP knockdown cells. Although IRAP knockdown has profound effects on GLUT4 traffic, GLUT4 knockdown does not affect IRAP trafficking, demonstrating that IRAP traffics independent of GLUT4. In sum, we show that IRAP is both cargo and a key regulator of the insulin-regulated pathway.     

Intracellular membrane trafficking pathways in bone-resorbing osteoclasts revealed by cloning and subcellular localization studies of small GTP-binding rab proteins

A variety of intracellular membrane trafficking pathways are involved in establishing the polarization of resorbing osteoclasts and regulating bone resorption activities. Small GTP-binding proteins of rab family have been implicated as key regulators of membrane trafficking in mammalian cells. Here we used a RT-PCR-based cloning method and confocal laser scanning microscopy to explore the expression array and subcellular localization of rab proteins in osteoclasts. Rab1B, rab4B, rab5C, rab7, rab9, rab11B, and rab35 were identified from rat osteoclasts in this study. Rab5C may be associated with early endosomes, while rab11B is localized at perinuclear recycling compartments and may function in the ruffled border membrane turnover and osteoclast motility. Interestingly, late endosomal rabs, rab7, and rab9, were found to localize at the ruffled border membrane indicating a late endosomal nature of this specialized plasma membrane domain in resorbing osteoclasts. This also suggests that late endocytotic pathways may play an important role in the secretion of lysosomal enzymes, such as cathepsin K, during bone resorption.     

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

Glucose transporter 4 (GLUT4) is the major insulin-regulated glucose transporter expressed mainly in muscle and adipose tissue. GLUT4 is stored in a poorly characterized intracellular vesicular compartment and translocates to the cell surface in response to insulin stimulation resulting in an increased glucose uptake. This process is essential for the maintenance of normal glucose homeostasis and involves a complex interplay of trafficking events and intracellular signaling cascades. Recent studies have identified sortilin as an essential element for the formation of GLUT4 storage vesicles during adipogenesis and Golgi-localized gamma-ear-containing Arf-binding protein (GGA) as a key coat adaptor for the entry of newly synthesized GLUT4 into the specialized compartment. Insulin-stimulated GLUT4 translocation from this compartment to the plasma membrane appears to require the Akt/protein kinase B substrate termed AS160 (Akt substrate of 160kDa). In addition, the VPS9 domain-containing protein Gapex-5 in complex with CIP4 appears to function as a Rab31 guanylnucleotide exchange factor that is necessary for insulin-stimulated GLUT4 translocation. Here, we attempt to summarize recent advances in GLUT4 vesicle biogenesis, intracellular trafficking and membrane fusion.     

Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis

BACKGROUND: Embryonic cleavage leads to the formation of an epithelial layer during development. In Drosophila, the process is specialized and called cellularization. The trafficking pathways that underlie this process and that are responsible for the mobilization of membrane pools, however, remain poorly understood. RESULTS: We provide functional evidence for the role of endocytic trafficking through Rab11 endosomes in remobilizing vesicular membrane pools to ensure lateral membrane growth. Part of the membrane stems from endocytosed apical material. Mutants in the endocytic regulators rab5 and shibire/dynamin inhibit basal-lateral membrane growth, and apical endocytosis is blocked in shibire mutants. In addition, shibire controls vesicular trafficking through Rab11-positive endosomes. In shibire mutants, the transmembrane protein Neurotactin follows the secretory pathway normally but is not properly inserted in the plasma membrane and accumulates instead in Rab11 subapical endosomes. Consistent with a direct role of shibire in vesicular trafficking through Rab11 endosomes, Shibire is enriched in this compartment. Moreover, we show by electron microscopy the large accumulation of intracellular coated pits on subapical endocytic structures in shibire mutants. Finally, we show that Rab11 is essential for membrane growth and invagination during cellularization. CONCLUSION: Together, the data show that endocytic trafficking is required for basal-lateral membrane growth during cellularization. We identify Rab11 endosomes as key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization. This pathway may be required in other morphogenetic processes characterized by the growth of a membrane domain.     

V-type ATPase is involved in biogenesis of GLUT4 vesicles

Proton pumps participate in several aspects of endocytic protein trafficking. However, their involvement specifically in the GLUT4 pathway has been a matter of great controversy. Here, we report that incubation of 3T3-L1 adipocytes with specific inhibitors of V-type ATPase, concanamycin A and bafilomycin A1, inhibits insulin-regulated glucose transport and results in accumulation of GLUT4 in heavy, rapidly sedimenting intracellular membranes. Correspondingly, the amount of small responsive GLUT4 vesicles in concanamycin A- and bafilomycin A1-treated cells is decreased. We conclude that these drugs block translocation of GLUT4 in adipose cells by inhibiting formation of small insulin-responsive vesicles on donor intracellular membranes. At the same time, proton pump inhibitors do not affect insulin-dependent translocation of preexisting vesicles or GLUT4 sorting in recycling endosomes. On the contrary, wortmannin acutely inhibits insulin-dependent translocation of the preexisting vesicles but has no effect on vesicle formation.     

A di-leucine sequence and a cluster of acidic amino acids are required for dynamic retention in the endosomal recycling compartment of fibroblasts

Insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase, is dynamically retained within the endosomal compartment of fibroblasts. The characteristics of this dynamic retention are rapid internalization from the plasma membrane and slow recycling back to the cell surface. These specialized trafficking kinetics result in <15% of IRAP on the cell surface at steady state, compared with 35% of the transferrin receptor, another transmembrane protein that traffics between endosomes and the cell surface. Here we demonstrate that a 29-amino acid region of IRAP's cytoplasmic domain (residues 56--84) is necessary and sufficient to promote trafficking characteristic of IRAP. A di-leucine sequence and a cluster of acidic amino acids within this region are essential elements of the motif that slows IRAP recycling. Rapid internalization requires any two of three distinct motifs: M(15,16), DED(64--66), and LL(76,77). The DED and LL sequences are part of the motif that regulates recycling, demonstrating that this motif is bifunctional. In this study we used horseradish peroxidase quenching of fluorescence to demonstrate that IRAP is dynamically retained within the transferrin receptor-containing general endosomal recycling compartment. Therefore, our data demonstrate that motifs similar to those that determine targeting among distinct membrane compartments can also regulate the rate of transport of proteins from endosomal compartments. We propose a model for dynamic retention in which IRAP is transported from the general endosomal recycling compartment in specialized, slowly budding recycling vesicles that are distinct from those that mediate rapid recycling back to the surface (e.g., transferrin receptor-containing transport vesicles). It is likely that the dynamic retention of IRAP is an example of a general mechanism for regulating the distribution of proteins between the surface and interior of cells.     

The insulin-sensitive GLUT4 storage compartment is a postendocytic and heterogeneous population recruited by acute exercise

Insulin and acute exercise stimulate glucose transport in skeletal muscle by translocating GLUT4 glucose transporters to the cell surface. GLUT4 is distributed in skeletal muscle in two intracellular membrane populations, an endosomal pool that remains unaltered after insulin treatment and an storage population that is markedly GLUT4 depleted in response to insulin. Here we have further characterized the storage GLUT4 compartment in regard to protein composition and sensitivity to acute exercise. This GLUT4 compartment contained IRAP (insulin-regulated aminopeptidase), transferrin receptors or mannose-6-phosphate/IGF-II receptors, indicating a postendocytic origin. Insulin administration caused a depletion of GLUT4 and IRAP but no changes in transferrin receptors, which suggests that this pool is heterogeneous. In addition, acute exercise caused a marked GLUT4 depletion in the storage compartment, whereas no changes were detected in the endosomal population. In all, our data indicate that the GLUT4 storage population represents a postendocytic and heterogeneous compartment; the storage compartment represents the recruitment site that triggers GLUT4 translocation to the cell surface in response to both insulin and acute exercise.     

Unconventional secretion of tissue transglutaminase involves phospholipid-dependent delivery into recycling endosomes

Although endosomal compartments have been suggested to play a role in unconventional protein secretion, there is scarce experimental evidence for such involvement. Here we report that recycling endosomes are essential for externalization of cytoplasmic secretory protein tissue transglutaminase (tTG). The de novo synthesized cytoplasmic tTG does not follow the classical ER/Golgi-dependent secretion pathway, but is targeted to perinuclear recycling endosomes, and is delivered inside these vesicles prior to externalization. On its route to the cell surface tTG interacts with internalized β1 integrins inside the recycling endosomes and is secreted as a complex with recycled β1 integrins. Inactivation of recycling endosomes, blocking endosome fusion with the plasma membrane, or downregulation of Rab11 GTPase that controls outbound trafficking of perinuclear recycling endosomes, all abrogate tTG secretion. The initial recruitment of cytoplasmic tTG to recycling endosomes and subsequent externalization depend on its binding to phosphoinositides on endosomal membranes. These findings begin to unravel the unconventional mechanism of tTG secretion which utilizes the long loop of endosomal recycling pathway and indicate involvement of endosomal trafficking in non-classical protein secretion.     

SNARE proteins underpin insulin-regulated GLUT4 traffic

Delivery of the glucose transporter type 4 (GLUT4) from an intracellular location to the cell surface in response to insulin represents a specialized form of membrane traffic, known to be impaired in the disease states of insulin resistance and type 2 diabetes. Like all membrane trafficking events, this translocation of GLUT4 requires members of the SNARE family of proteins. Here, we discuss two SNARE complexes that have been implicated in insulin-regulated GLUT4 traffic: one regulating the final delivery of GLUT4 to the cell surface in response to insulin and the other controlling GLUT4's intracellular trafficking.     

Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane

GLUT4 trafficking to the plasma membrane of muscle and fat cells is regulated by insulin. An important component of insulin-regulated GLUT4 distribution is the Akt substrate AS160 rab GTPase-activating protein. Here we show that Rab10 functions as a downstream target of AS160 in the insulin-signaling pathway that regulates GLUT4 translocation in adipocytes. Overexpression of a mutant of Rab10 defective for GTP hydrolysis increased GLUT4 on the surface of basal adipocytes. Rab10 knockdown resulted in an attenuation of insulin-induced GLUT4 redistribution to the plasma membrane and a concomitant 2-fold decrease in GLUT4 exocytosis rate. Re-expression of a wild-type Rab10 restored normal GLUT4 translocation. The basal increase in plasma-membrane GLUT4 due to AS160 knockdown was partially blocked by knocking down Rab10 in the same cells, further indicating that Rab10 is a target of AS160 and a positive regulator of GLUT4 trafficking to the cell surface upon insulin stimulation.     

C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression

Insulin regulates the uptake of glucose into skeletal muscle and adipocytes by redistributing the tissue-specific glucose transporter GLUT4 from intracellular vesicles to the cell surface. To date, GLUT4 is the only protein involved in insulin-regulated vesicular traffic that has this tissue distribution, thus raising the possibility that its expression alone may allow formation of an insulin-responsive vesicular compartment. We show here that treatment of differentiating C2C12 myoblasts with dexamethasone, acting via the glucocorticoid receptor, causes a >or=10-fold increase in GLUT4 expression but results in no significant change in insulin-stimulated glucose transport. Signaling from the insulin receptor to its target, Akt2, and expression of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor, or SNARE, proteins syntaxin 4 and vesicle-associated membrane protein are normal in dexamethasone-treated C2C12 cells. However, these cells show no insulin-dependent trafficking of the insulin-responsive aminopeptidase or the transferrin receptor, respective markers for intracellular GLUT4-rich compartments and endosomes that are insulin responsive in mature muscle and adipose cells. Therefore, these data support the hypothesis that GLUT4 expression by itself is insufficient to establish an insulin-sensitive vesicular compartment.