Insulin regulation of protein traffic in rat adipose cells.

Rat adipocytes were biotinylated with cell-impermeable reagents, sulfo-N-hydroxysuccinimide-biotin and sulfo-N-hydroxysuccinimide-S-S-biotin in the absence and presence of insulin. Biotinylated and nonbiotinylated populations of the insulin-like growth factor-II/mannose 6-phosphate receptor, the transferrin receptor, and insulin-responsive aminopeptidase were separated by adsorption to streptavidin-agarose to determine the percentage of the biotinylated protein molecules versus their total amount in different subcellular compartments. Results indicate that adipose cells possess at least two distinct cell surface recycling pathways for insulin-like growth factor-II/mannose 6-phosphate receptor (MPR) and transferrin receptor (TfR): one which is mediated by glucose transporter isoform 4(Glut4)-vesicles and another that bypasses this compartment. Under basal conditions, the first pathway is not active, and cell surface recycling of TfR and, to a lesser extent, MPR proceeds via the second pathway. Insulin dramatically stimulates recycling through the first pathway and has little effect on the second. Within the Glut4-containing compartment, insulin has profoundly different effects on intracellular trafficking of insulin-responsive aminopeptidase on one hand and MPR and TfR on the other. After insulin administration, insulin-responsive aminopeptidase is redistributed from Glut4-containing vesicles to the plasma membrane and stays there for at least 30 min with minimal detectable internalization and recycling, whereas MPR and TfR rapidly shuttle between Glut4 vesicles and the plasma membrane in such a way that after 30 min of insulin treatment, virtually every receptor molecule in this compartment completes at least one trafficking cycle to the cell surface. Thus, different recycling proteins, which compose Glut4-containing vesicles, are internalized into this compartment at their own distinctive rates.     

Regulation of insulin-responsive aminopeptidase expression and targeting in the insulin-responsive vesicle compartment of glucose transporter isoform 4-deficient cardiomyocytes

In adipocytes and cardiac or skeletal muscle, glucose transporter isoform 4 (GLUT4) is targeted to insulin-responsive intracellular membrane vesicles (IRVs) that contain several membrane proteins, including insulin-responsive aminopeptidase (IRAP) that completely colocalizes with GLUT4 in basal and insulin-treated cells. Cardiac GLUT4 content is reduced by 65-85% in IRAP knockout mice, suggesting that IRAP may regulate the targeting or degradation of GLUT4. To determine whether GLUT4 is required for maintenance of IRAP content within IRVs, we studied the expression and cellular localization of IRAP and other GLUT4 vesicle-associated proteins, in hearts of mice with cardiac-specific deletion of GLUT4 (G4H-/-). In G4H-/- hearts, IRAP content was reduced by 60%, but the expression of other vesicle-associated proteins, namely cellugyrin, IGF-II/mannose-6-phosphate, and transferrin receptors, secretory carrier-associated membrane proteins and vesicle-associated membrane protein were unchanged. Using sucrose gradient centrifugation and cell surface biotinylation, we found that IRAP content in 50-80S vesicles where GLUT4 vesicles normally sediment was markedly depleted in G4H-/- hearts, and the remaining IRAP was found in the heavy membrane fraction. Although insulin caused a discernible increase in cell surface IRAP content of G4H-/- cardiomyocytes, cell surface IRAP remained 70% lower than insulin-stimulated controls. Immunoabsorption of intracellular vesicles with anticellugyrin antibodies revealed that IRAP content was reduced by 70% in both cellugyrin-positive and cellugyrin-negative vesicles. Endosomal recycling, as measured by transferrin receptor recycling was normal. Thus, GLUT4 and IRAP content of early endosome-derived sorting vesicles and of IRVs are coordinately regulated, and both proteins are required for maintenance of key constituents of these compartments in cardiac muscle cells in vivo.     

Phosphatidylinositol 4-kinase type IIalpha is targeted specifically to cellugyrin-positive glucose transporter 4 vesicles

Phosphoinositides now emerge as important regulators of membrane traffic. In particular, phosphatidylinositol 4-phosphate may serve as a precursor for polyphosphorylated derivatives of phosphatidylinositol and, also, may regulate vesicular traffic by recruiting specific proteins to the membrane. Early results have demonstrated the presence of phosphatidylinositol 4-kinase (PI4K) activity in glucose transporter 4 (Glut4) vesicles from fat and skeletal muscle cells. However, the molecular identity of phosphatidylinositol 4-kinase(s) associated with Glut4 vesicles has not been characterized. It has also been determined that Glut4 vesicles are not homogeneous and represent a mixture of at least two vesicular populations: ubiquitous cellugyrin-positive transport vesicles and specialized cellugyrin-negative insulin-responsive Glut4 storage vesicles, which are different in size, protein composition, and functional properties. Using sequential immunoadsorption, subcellular fractionation, and immunofluorescence staining, we show that virtually all PI4K activity in Glut4 vesicles is represented by PI4K type IIalpha, which is associated with cellugyrin-positive vesicles and is not detectable in the Glut4 storage vesicles. The unique N terminus of PI4K type IIalpha is required for the targeting of the enzyme to cellugyrin-positive vesicles. Knockdown of PI4K type IIalpha with the help of short hairpin RNA does not decrease the amount of cellugyrin-positive vesicles in human embryonic kidney 293 cells.     

Insulin-regulated aminopeptidase marks an antigen-stimulated recycling compartment in mast cells

Insulin-regulated aminopeptidase (IRAP) is a marker for insulin-sensitive recycling compartments of fat and muscle cells that contain the glucose transporter isoform GLUT4. Unlike GLUT4, IRAP is expressed in many other cell types. Thus, it is a potential marker for regulated recycling compartments that are analogous to GLUT4 vesicles. In bone marrow-derived mast cells, IRAP is highly expressed and localizes to an intracellular compartment different from secretory granules. Using cell-surface biotinylation, we determined that IRAP underwent rapid redistribution to the plasma membrane on antigen/immunoglobulin E (IgE) stimulation and was re-internalized within 30 min. When granule exocytosis was inhibited, by removing extracellular calcium, adding the protein kinase C inhibitor bisindolylmaleimide or the phosphatidylinositol 3-kinase inhibitor wortmannin, IRAP redistribution was still detected in stimulated cells. However, the redistribution of IRAP required intracellular calcium. By immunofluorescence, IRAP significantly co-localized with the transferrin receptor (TfR), a marker for constitutively recycling endosomes. However, antigen/IgE stimulation did not increase TfR on the cell surface, indicating that IRAP and TfR may follow different pathways to the plasma membrane. In rat peritoneal mast cells, the distributions of IRAP and TfR overlapped to only a limited extent, indicating that overlap may decrease with cell differentiation. We propose that IRAP vesicles represent a second IgE-sensitive exocytotic compartment in mast cells, which is regulated differently from secretory granules, and that these vesicles may be similar to GLUT4 vesicles.     

Self-assembly of Glut4 storage vesicles during differentiation of 3T3-L1 adipocytes

Glut4 storage vesicles (GSVs) represent translocation-competent vesicular carriers in fat and skeletal muscle cells that deliver Glut4 to the plasma membrane in response to insulin stimulation. GSVs include three major cargo proteins: Glut4, insulin-responsive aminopeptidase (IRAP), and sortilin. Previous work has suggested that the lumenal interaction between Glut4 and sortilin and the cytoplasmic interaction between sortilin and GGA adaptors play an important role in recruitment of Glut4 into the GSVs. However, the mechanism of IRAP targeting to this compartment remains unknown. To address this question, we show that in differentiating adipocytes IRAP enters the GSVs from the "donor" membranes on day 3 of differentiation. Forced expression of sortilin in undifferentiated cells does not recruit IRAP into the vesicles. However, double expression of sortilin and Glut4 reconstitutes functional GSVs that incorporate endogenous IRAP. To explain this process, we show by a yeast two-hybrid system and chemical cross-linking that the lumenal domain of IRAP can interact with the lumenal loop of Glut4. IRAP without the lumenal domain is faithfully targeted to the donor membranes but has significantly lower insulin responsiveness than full-length IRAP. We suggest that lumenal interactions between Glut4 and IRAP play an important role in the assembly of the GSVs.     

A Glut4-vesicle marker protein, insulin-responsive aminopeptidase, is localized in a novel vesicular compartment in PC12 cells

Glut4-containing vesicles represent a regulated recycling compartment in insulin-sensitive fat and skeletal muscle cells, the nature and origin of which are not fully understood. In addition to Glut4 itself, these vesicles compartmentalize a number of proteins, at least one of which, insulin-responsive aminopeptidase, or IRAP, is completely colocalized with Glut4 in insulin-sensitive tissues. However, unlike Glut4, IRAP is expressed in a variety of other tissues and cell lines. Here, we explored the intracellular localization of IRAP in the rat pheochromocytoma cell line PC12. We found that this protein is present in a distinct population of slowly recycling light vesicles. By gradient centrifugations, immunoadsorption and double immunofluorescent staining, these vesicles are different from transferrin-containing endosomes, small synaptic vesicles and secretory granules and may thus represent a novel compartment in PC12 cells. Glut4-GFP chimera transiently expressed in PC12 cells is targeted to IRAP-containing vesicles indicating that cotargeting of Glut4 and IRAP is not specific for adipocytes and myocytes, but is faithful in a foreign cell type. We suggest that PC12 cells may possess a novel type of a vesicular carrier that may represent the homolog of Glut4-vesicles.     

Cellugyrin is a marker for a distinct population of intracellular Glut4-containing vesicles

Although Glut4 traffic is routinely described as translocation from an "intracellular storage pool" to the plasma membrane, it has been long realized that Glut4 travels through at least two functionally distinct intracellular membrane compartments on the way to and from the cell surface. Biochemical separation and systematic studies of the individual Glut4-containing compartments have been limited by the lack of appropriate reagents. We have prepared a monoclonal antibody against a novel component protein of Glut4 vesicles and have identified this protein as cellugyrin, a ubiquitously expressed homologue of a major synaptic vesicle protein, synaptogyrin. By means of sucrose gradient centrifugation, immunoadsorption, and confocal microscopy, we have shown that virtually all cellugyrin is co-localized with Glut4 in the same vesicles. However, unlike Glut4, cellugyrin is not re-distributed to the plasma membrane in response to insulin stimulation, and at least 40-50% of the total population of Glut4 vesicles do not contain this protein. We suggest that cellugyrin represents a specific marker of a functionally distinct population of Glut4 vesicles that permanently maintains its intracellular localization and is not recruited to the plasma membrane by insulin.     

The vesicle- and target-SNARE proteins that mediate Glut4 vesicle fusion are localized in detergent-insoluble lipid rafts present on distinct intracellular membranes

Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 ( approximately 35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-beta-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.     

Single Point Mutations Result in the Miss-Sorting of Glut4 to a Novel Membrane Compartment Associated with Stress Granule Proteins

Insulin increases cellular glucose uptake and metabolism in the postprandial state by acutely stimulating the translocation of the Glut4 glucose transporter from intracellular membrane compartments to the cell surface in muscle and fat cells. The intracellular targeting of Glut4 is dictated by specific structural motifs within cytoplasmic domains of the transporter. We demonstrate that two leucine residues at the extreme C-terminus of Glut4 are critical components of a motif (IRM, insulin responsive motif) involved in the sorting of the transporter to insulin responsive vesicles in 3T3L1 adipocytes. Light microscopy, immunogold electron microscopy, subcellular fractionation, and sedimentation analysis indicate that mutations in the IRM cause the aberrant targeting of Glut4 to large dispersed membrane vesicles that are not insulin responsive. Proteomic characterization of rapidly and slowly sedimenting membrane vesicles (RSVs and SSVs) that were highly enriched by immunoadsorption for either wild-type Glut4 or an IRM mutant revealed that the major vesicle fraction containing the mutant transporter (IRM-RSVs) possessed a relatively small and highly distinct protein population that was enriched for proteins associated with stress granules. We suggest that the IRM is critical for an early step in the sorting of Glut4 to insulin-responsive subcellular membrane compartments and that IRM mutants are miss-targeted to relatively large, amorphous membrane vesicles that may be involved in a degradation pathway for miss-targeted or miss-folded proteins or represent a transitional membrane compartment that Glut4 traverses en route to insulin responsive storage compartments.     

p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation

Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1-109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site.     

Translocation of small preformed vesicles is responsible for the insulin activation of glucose transport in adipose cells. Evidence from the in vitro reconstitution assay

Insulin stimulates translocation of the glucose transporter isoform 4 (Glut4) from an intracellular storage compartment to the plasma membrane in fat and skeletal muscle cells. At present, the nature of the Glut4 storage compartment is unclear. According to one model, this compartment represents a population of preformed small vesicles that fuse with the plasma membrane in response to insulin stimulation. Alternatively, Glut4 may be retained in large donor membranes, and insulin stimulates the formation of transport vesicles that deliver Glut4 to the cell surface. Finally, insulin can induce plasma membrane fusion of the preformed vesicles and, also, stimulate the formation of new vesicles. In extracts of fat and skeletal muscle cells, Glut4 is predominantly found in small insulin-sensitive 60-70 S membrane vesicles that may or may not artificially derive from large donor membranes during cell homogenization. Here, we use a cell-free reconstitution assay to demonstrate that small Glut4-containing vesicles are formed from large rapidly sedimenting donor membranes in a cytosol-, ATP-, time-, and temperature-dependent fashion and, therefore, do not represent an artifact of homogenization. Thus, small insulin-responsive vesicles represent the major form of Glut4 storage in the living adipose cell. Fusion of these vesicles with the plasma membrane may be largely responsible for the primary effect of insulin on glucose transport in fat tissue. In addition, our results suggest that insulin may also stimulate the formation of Glut4 vesicles and accelerate Glut4 recycling to the plasma membrane.     

The role of small G-proteins in the regulation of glucose transport (review).

Insulin increases the rate of glucose transport into fat and muscle cells by stimulating the translocation of intracellular Glut 4-containing vesicles to the plasma membrane. This results in a marked increase in the amount of the facilitative glucose transporter Glut 4 at the cell surface, allowing for an enhanced glucose uptake. This process requires a continuous cycling through the early endosomes, a Glut 4 specific storage compartment and the plasma membrane. The main effect of insulin is to increase the rate of Glut 4 trafficking from its specific storage compartment to the plasma membrane. The whole phenomenon involves signal transduction from the insulin receptor, vesicle trafficking (sorting and fusion processes) and actin cytoskeleton modifications, which are all supposed to require small GTPases. This review describes the potential role of the various members of the Ras, Rad, Rho, Arf and Rab families in the traffic of the Glut 4-containing vesicles.     

The role of small G-proteins in the regulation of glucose transport

Insulin increases the rate of glucose transport into fat and muscle cells by stimulating the translocation of intracellular Glut 4-containing vesicles to the plasma membrane. This results in a marked increase in the amount of the facilitative glucose transporter Glut 4 at the cell surface, allowing for an enhanced glucose uptake. This process requires a continuous cycling through the early endosomes, a Glut 4 specific storage compartment and the plasma membrane. The main effect of insulin is to increase the rate of Glut 4 trafficking from its specific storage compartment to the plasma membrane. The whole phenomenon involves signal transduction from the insulin receptor, vesicle trafficking (sorting and fusion processes) and actin cytoskeleton modifications, which are all supposed to require small GTPases. This review describes the potential role of the various members of the Ras, Rad, Rho, Arf and Rab families in the traffic of the Glut 4-containing vesicles.     

GLUT4 overexpression or deficiency in adipocytes of transgenic mice alters the composition of GLUT4 vesicles and the subcellular localization of GLUT4 and insulin-responsive aminopeptidase

The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2-GLUT4-Tg) mice were studied. GLUT4 amount was reduced by 80-95% in aP2-GLUT4-/- adipocytes and increased approximately 10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase (IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2-GLUT4-Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.     

Characterization of insulin-responsive GLUT4 storage vesicles isolated from 3T3-L1 adipocytes

Insulin regulates glucose transport in muscle and adipose tissue by triggering the translocation of a facilitative glucose transporter, GLUT4, from an intracellular compartment to the cell surface. It has previously been suggested that GLUT4 is segregated between endosomes, the trans-Golgi network (TGN), and a postendosomal storage compartment. The aim of the present study was to isolate the GLUT4 storage compartment in order to determine the relationship of this compartment to other organelles, its components, and its presence in different cell types. A crude intracellular membrane fraction was prepared from 3T3-L1 adipocytes and subjected to iodixanol equilibrium sedimentation analysis. Two distinct GLUT4-containing vesicle peaks were resolved by this procedure. The lighter of the two peaks (peak 2) was comprised of two overlapping peaks: peak 2b contained recycling endosomal markers such as the transferrin receptor (TfR), cellubrevin, and Rab4, and peak 2a was enriched in TGN markers (syntaxin 6, the cation-dependent mannose 6-phosphate receptor, sortilin, and sialyltransferase). Peak 1 contained a significant proportion of GLUT4 with a smaller but significant amount of cellubrevin and relatively little TfR. In agreement with these data, internalized transferrin (Tf) accumulated in peak 2 but not peak 1. There was a quantitatively greater loss of GLUT4 from peak 1 than from peak 2 in response to insulin stimulation. These data, combined with the observation that GLUT4 became more sensitive to ablation with Tf-horseradish peroxidase following insulin treatment, suggest that the vesicles enriched in peak 1 are highly insulin responsive. Iodixanol gradient analysis of membranes isolated from other cell types indicated that a substantial proportion of GLUT4 was targeted to peak 1 in skeletal muscle, whereas in CHO cells most of the GLUT4 was targeted to peak 2. These results indicate that in insulin-sensitive cells GLUT4 is targeted to a subpopulation of vesicles that appear, based on their protein composition, to be a derivative of the endosome. We suggest that the biogenesis of this compartment may mediate withdrawal of GLUT4 from the recycling system and provide the basis for the marked insulin responsiveness of GLUT4 that is unique to muscle and adipocytes.     

Identification of amino acid residues within the C terminus of the Glut4 glucose transporter that are essential for insulin-stimulated redistribution to the plasma membrane

The Glut4 glucose transporter undergoes complex insulin-regulated subcellular trafficking in adipocytes. Much effort has been expended in an attempt to identify targeting motifs within Glut4 that direct its subcellular trafficking, but an amino acid motif responsible for the targeting of the transporter to insulin-responsive intracellular compartments in the basal state or that is directly responsible for its insulin-stimulated redistribution to the plasma membrane has not yet been delineated. In this study we define amino acid residues within the C-terminal cytoplasmic tail of Glut4 that are essential for its insulin-stimulated translocation to the plasma membrane. The residues were identified based on sequence similarity (LXXLXPDEXD) between cytoplasmic domains of Glut4 and the insulin-responsive aminopeptidase (IRAP). Alteration of this putative targeting motif (IRM, insulin-responsive motif) resulted in the targeting of the bulk of the mutant Glut4 molecules to dispersed membrane vesicles that lacked detectable levels of wild-type Glut4 in either the basal or insulin-stimulated states and completely abolished the insulin-stimulated translocation of the mutant Glut4 to the plasma membrane in 3T3L1 adipocytes. The bulk of the dispersed membrane vesicles containing the IRM mutant did not contain detectable levels of any subcellular marker tested. A fraction of the total IRM mutant was also detected in a wild-type Glut4/Syntaxin 6-containing perinuclear compartment. Interestingly, mutation of the IRM sequence did not appreciably alter the subcellular trafficking of IRAP. We conclude that residues within the IRM are critical for the targeting of Glut4, but not of IRAP, to insulin-responsive intracellular membrane compartments in 3T3-L1 adipocytes.     

The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin- sensitive cells

Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle- associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP- 2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.     

Potential role of Rab4 in the regulation of subcellular localization of Glut4 in adipocytes.

A role for Rab4 in the translocation of the glucose transporter Glut4 induced by insulin has been recently proposed. To study more directly the role of this small GTPase, freshly isolated adipocytes were transiently transfected with the cDNAs of both an epitope-tagged Glut4-myc and Rab4, a system which allows direct measurement of the concentration of Glut4 molecules at the cell surface. When cells were cotransfected with Glut4-myc and Rab4, the concentration of Glut4-myc at the cell surface decreased in parallel with the increased expression of Rab4, suggesting that Rab4 participates in the intracellular retention of Glut4. In parallel, the amount of Rab4 associated with the Glut4-containing vesicles increased. When Rab4 was moderately overexpressed, the number of Glut4-myc molecules recruited to the cell surface in response to insulin was similar to that observed in mock-transfected cells, and thus the insulin efficiency was increased. When Rab4 was expressed at a higher level, the amount of Glut4-myc present at the cell surface in response to insulin decreased. Since the overexpressed protein was predominantly cytosolic, this suggests that the cytosolic Rab4 might complex some factor(s) necessary for insulin action. This hypothesis was strengthened by the fact that Rab4 deltaCT, a Rab4 mutant lacking the geranylgeranylation sites, inhibited insulin-induced recruitement of Glut4-myc to the cell surface, even when moderately overexpressed. Rab3D was without effect on Glut4-myc subcellular distribution in basal or insulin-stimulated conditions. While two mutated proteins unable to bind GTP did not decrease the number of Glut4-myc molecules in basal or insulin-stimulated conditions at the plasma membrane, the behavior of a mutated Rab4 protein without GTPase activity was similar to that of the wild-type Rab4 protein, indicating that GTP binding but not its hydrolysis was required for the observed effects. Altogether, our results suggest that Rab4, but not Rab3D, participates in the molecular mechanism involved in the subcellular distribution of the Glut4 molecules both in basal and in insulin-stimulated conditions in adipocytes.     

Loss of AS160 Akt substrate causes Glut4 protein to accumulate in compartments that are primed for fusion in basal adipocytes

The Akt substrate AS160 (TCB1D4) regulates Glut4 exocytosis; shRNA knockdown of AS160 increases surface Glut4 in basal adipocytes. AS160 knockdown is only partially insulin-mimetic; insulin further stimulates Glut4 translocation in these cells. Insulin regulates translocation as follows: 1) by releasing Glut4 from retention in a slowly cycling/noncycling storage pool, increasing the actively cycling Glut4 pool, and 2) by increasing the intrinsic rate constant for exocytosis of the actively cycling pool (k(ex)). Kinetic studies were performed in 3T3-L1 adipocytes to measure the effects of AS160 knockdown on the rate constants of exocytosis (k(ex)), endocytosis (k(en)), and release from retention into the cycling pool. AS160 knockdown released Glut4 into the actively cycling pool without affecting k(ex) or k(en). Insulin increased k(ex) in the knockdown cells, further increasing cell surface Glut4. Inhibition of phosphatidylinositol 3-kinase or Akt affected both k(ex) and release from retention in control cells but only k(ex) in AS160 knockdown cells. Glut4 vesicles accumulate in a primed pre-fusion pool in basal AS160 knockdown cells. Akt regulates the rate of exocytosis of the primed vesicles through an AS160-independent mechanism. Therefore, there is an additional Akt substrate that regulates the fusion of Glut4 vesicles that remain to be identified. Mathematical modeling was used to test the hypothesis that this substrate regulates vesicle priming (release from retention), whereas AS160 regulates the reverse step by stimulating GTP turnover of a Rab protein required for vesicle tethering/docking/fusion. Our analysis indicates that fusion of the primed vesicles with the plasma membrane is an additional non-Akt-dependent insulin-regulated step.     

Coordinated Regulation of Vasopressin Inactivation and Glucose Uptake by Action of TUG Protein in Muscle

In adipose and muscle cells, insulin stimulates the exocytic translocation of vesicles containing GLUT4, a glucose transporter, and insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase. A substrate of IRAP is vasopressin, which controls water homeostasis. The physiological importance of IRAP translocation to inactivate vasopressin remains uncertain. We previously showed that in skeletal muscle, insulin stimulates proteolytic processing of the GLUT4 retention protein, TUG, to promote GLUT4 translocation and glucose uptake. Here we show that TUG proteolysis also controls IRAP targeting and regulates vasopressin action in vivo. Transgenic mice with constitutive TUG proteolysis in muscle consumed much more water than wild-type control mice. The transgenic mice lost more body weight during water restriction, and the abundance of renal AQP2 water channels was reduced, implying that vasopressin activity is decreased. To compensate for accelerated vasopressin degradation, vasopressin secretion was increased, as assessed by the cosecreted protein copeptin. IRAP abundance was increased in T-tubule fractions of fasting transgenic mice, when compared with controls. Recombinant IRAP bound to TUG, and this interaction was mapped to a short peptide in IRAP that was previously shown to be critical for GLUT4 intracellular retention. In cultured 3T3-L1 adipocytes, IRAP was present in TUG-bound membranes and was released by insulin stimulation. Together with previous results, these data support a model in which TUG controls vesicle translocation by interacting with IRAP as well as GLUT4. Furthermore, the effect of IRAP to reduce vasopressin activity is a physiologically important consequence of vesicle translocation, which is coordinated with the stimulation of glucose uptake.