Isolation of vesicles containing insulin-responsive, intracellular glucose transporters from 3T3-L1 adipocytes

Insulin stimulation of glucose transport in fat and muscle cells occurs, at least in part, by the translocation of glucose transporters from intracellular membranes to the plasma membrane. In this report, we describe the isolation and partial characterization of vesicles containing translocatable intracellular transporters from 3T3-L1 adipocytes. The glucose transporter content of light microsomes in a 44,000 X g cell supernatant was found to decrease by 50% in response to insulin treatment of the adipocytes. A procedure was developed for the purification of transporter-containing vesicles from this supernatant by immunoadsorption onto Staphylococcus aureus cells coated with anti-transporter antibodies. The vesicles are about 50 nm in diameter and have a distinct polypeptide composition. After insulin treatment the number of transporter-containing vesicles decreased by about 50%, as determined both by microscopic analysis of vesicle number and by the relative abundance of vesicle polypeptides.     

Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes

The amounts of the brain type and muscle type glucose transporters (designated Glut 1 and 4, respectively) in 3T3-L1 adipocytes have been determined by quantitative immunoblotting with antibodies against their carboxyl-terminal peptides. There are about 950,000 and 280,000 copies of Glut 1 and 4, respectively, per cell. Insulin caused the translocation of both types of transporters from an intracellular location to the plasma membrane. The insulin-elicited increase in cell surface transporters was assessed by labeling the surface transporters with a newly developed, membrane-impermeant, photoaffinity labeling reagent for glucose transporters. The increases in Glut 1 and 4 averaged 6.5- and 17-fold, respectively, whereas there was a 21-fold in hexose transport. These results indicate that the translocation of Glut 4 could largely account for the insulin effect on transport rate, but only if the intrinsic activity of Glut 4 is much higher than that of Glut 1. The two transporters are colocalized intracellularly: vesicles (average diameter 72 nm) isolated from the intracellular membranes by immunoadsorption with antibodies against Glut 1 contained 95% of the Glut 4 and, conversely, vesicles isolated with antibodies against Glut 4 contained 85% of the Glut 1.     

Purification of insulin-dependent exocytic vesicles containing the glucose transporter

In muscle and fat, insulin causes the cellular redistribution of glucose transporters and insulin-like growth factor II receptors from an intracellular pool of membranes (low density microsomes) to the plasma membrane. This translocation is a major mechanism by which insulin stimulates cellular glucose uptake. Our aim was to purify and characterize the insulin-regulatable exocytic intracellular membranes that are enriched in glucose transporter. Low density microsome and plasma membrane fractions were isolated from basal and insulin-stimulated rat adipocytes by differential centrifugation. In cells exposed to insulin, glucose transporters were decreased in the low density microsomes and correspondingly increased in the plasma membranes as determined by immunoblotting and cytochalasin B binding. Low density microsomes were further fractionated by sucrose density gradient centrifugation. Membranes containing glucose transporters were separated from the major protein-containing peaks and from plasma membranes, Golgi, and endoplasmic reticulum. Further fractionation was achieved by agarose gel electrophoresis. Overall, the intracellular membranes enriched in transporter were purified 9-fold compared to low density microsomes. These purified membranes had the following characteristics: 1) uniformly sized vesicles, diameter 60-100 nm; 2) insulin-regulatable protein composition, one constituent being an Mr 43,000 protein that co-migrated with immunoblotted glucose transporters; 3) enrichment in insulin-like growth factor II receptors, but of a lesser degree than the enrichment in transporters. Thus, using a three-step procedure, insulin-sensitive translocatable vesicles from adipocytes have been highly purified. These are similar in size and density to endosomes, and the glucose transporter is a major constituent of this distinct vesicle population.     

Expression and compartmentalization of caveolin in adipose cells: coordinate regulation with and structural segregation from GLUT4

Native rat adipocytes and the mouse adipocyte cell line, 3T3-L1, possess transport vesicles of apparently uniform composition and size which translocate the tissue-specific glucose transporter isoform, GLUT4, from an intracellular pool to the cell surface in an insulin- sensitive fashion. Caveolin, the presumed structural protein of caveolae, has also been proposed to function in vesicular transport. Thus, we studied the expression and subcellular distribution of caveolin in adipocytes. We found that rat fat cells express the highest level of caveolin protein of any tissue studied, and caveolin is also expressed at high levels in cardiac muscle, another tissue possessing insulin responsive GLUT4 translocation. Both proteins are absent from 3T3-L1 fibroblasts and undergo a dramatic coordinate increase in expression upon differentiation of these cells into adipocytes. However, unlike GLUT4 in rat adipocytes not exposed to insulin, the majority of caveolin is present in the plasma membrane. In native rat adipocytes, intracellular GLUT4 and caveolin reside in vesicles practically indistinguishable by their size and buoyant density in sucrose gradients, and both proteins show insulin-dependent translocation to the cell surface. However, by immunoadsorption of GLUT4-containing vesicles with anti-GLUT4 antibody, we show that these vesicles have no detectable caveolin, and therefore, this protein is present in a distinct vesicle population. Thus, caveolin has no direct structural relation to the organization of the intracellular glucose transporting machinery in fat cells.     

Phenylarsine oxide stimulates hexose transport in 3T3-L1 adipocytes by a mechanism other than an increase in surface transporters

Phenylarsine oxide (PAO) has been shown to exert a biphasic effect on glucose transport in 3T3-L1 adipocytes. At 10 microM, PAO activates transport threefold, but at higher concentrations an inhibition of transport is observed. In this paper we report a procedure for the subcellular fractionation of these cells which we use to examine the distribution of glucose transporters following PAO challenge. Quantitative immunoblotting showed that the glucose transporter content of the plasma membrane fraction increased with increasing PAO concentrations; a parallel increase in another insulin-responsive protein, the transferrin receptor, also occurred. However, cell-surface labeling procedures for the glucose transporter and transferrin receptor showed that PAO actually decreased the cell-surface concentrations of these proteins; the basis of this discrepancy may be that in the presence of PAO, intracellular vesicles containing these proteins associate with the plasma membrane, but do not fuse with it. The possibility that PAO modulated transport by direct interaction with the glucose transporter was investigated by examining the effects of PAO on transport in both erythrocytes and a reconstituted system of purified erythrocyte transporter in lipid vesicles. PAO was without effect on the rate of transport in these systems. The hypothesis that the stimulatory effect of PAO on transport might be due to the activation of the insulin receptor kinase activity was examined by assessing the phosphotyrosine content of the receptor and other proteins using anti-phosphotyrosine antibodies. PAO alone caused no detectable increase in receptor phosphotyrosine content. However, the combination of PAO and insulin led to the tyrosine phosphorylation of two proteins of Mr 68,000 and 57,000 which were not detected in cells treated with either PAO or insulin, and an increased phosphotyrosine content of proteins of Mr 95,000 and 165,000 when compared to cells treated with insulin alone.     

Insulin stimulates the acute release of adipsin from 3T3-L1 adipocytes

The release of adipsin, a serine proteinase with complement factor D activity, from 3T3-L1 adipocytes was measured by quantitative immunoblotting. This protein is secreted constitutively from 3T3-L1 adipocytes, and there is a 2-fold increase in the amount of adipsin released from cells treated with insulin for 1 to 10 min. Longer exposure to insulin had no further effect on the rate of adipsin release. Adipsin does not appear to be anchored by a glycosylphosphatidylinositol moiety, since adipsin which was been released with Triton X-114 from an intracellular membrane fraction partitions into the aqueous phase. Using a previously described procedure for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters (GT vesicles), we show here that these GT vesicles contain an insulin-responsive pool of adipsin. Thus, insulin stimulates the secretion of a soluble protein, adipsin, as well as translocation to the plasma membrane of integral membrane proteins, including the glucose transporter, the transferrin receptors, and the insulin-like growth factor II receptor.     

Golgi-localized, gamma-ear-containing, Arf-binding protein adaptors mediate insulin-responsive trafficking of glucose transporter 4 in 3T3-L1 adipocytes

Small glucose transporter 4 (Glut4)-containing vesicles represent the major insulin-responsive compartment in fat and skeletal muscle cells. The molecular mechanism of their biogenesis is not yet elucidated. Here, we studied the role of the newly discovered family of monomeric adaptor proteins, GGA (Golgi-localized, gamma-ear-containing, Arf-binding proteins), in the formation of small Glut4 vesicles and acquisition of insulin responsiveness in 3T3-L1 adipocytes. In these cells, all three GGA isoforms are expressed throughout the differentiation process. In particular, GGA2 is primarily present in trans-Golgi network and endosomes where it demonstrates a significant colocalization with the recycling pool of Glut4. Using the techniques of immunoadsorption as well as glutathione-S-transferase pull-down assay we found that Glut4 vesicles (but not Glut4 per se) interact with GGA via the Vps-27, Hrs, and STAM (VHS) domain. Moreover, a dominant negative GGA mutant inhibits formation of Glut4 vesicles in vitro. To study a possible role of GGA in Glut4 traffic in the living cell, we stably expressed a dominant negative GGA mutant in 3T3-L1 adipocytes. Formation of small insulin-responsive Glut4-containing vesicles and insulin-stimulated glucose uptake in these cells were markedly impaired. Thus, GGA adaptors participate in the formation of the insulin-responsive vesicular compartment from the intracellular donor membranes both in vivo and in vitro.     

Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.     

Insulin-stimulated translocation of the HepG2/erythrocyte-type glucose transporter expressed in 3T3-L1 adipocytes

Insulin stimulates glucose transport into adipocytes, at least in part, via the translocation of intracellular transporters to the plasma membrane. The human HepG2-type transporter, which is not insulin-responsive in its native cell type, was expressed in 3T3-L1 adipocytes by infection with recombinant retrovirus harboring the HepG2 transporter cDNA in order to determine whether glucose transporter translocation in adipocytes is restricted to a distinct insulin-sensitive transporter species. The distributions of the endogenous murine and the HepG2 transporters were estimated by quantitative immunoblot analysis of subcellular fractions probed with either a monoclonal antibody that recognized only the human transporter or a polyclonal antibody that recognized both transporter species. In the basal state, the intracellular membrane fraction comprised approximately 50% of the total of each transporter type. Insulin decreased the content of both transporter species in the intracellular membranes by approximately 50% and increased the plasma membrane content of both species by approximately 1.5-2-fold. The similar insulin-mediated increase in the plasma membrane content of endogenous murine and HepG2 glucose transporters was verified by labeling of cell surface glycoproteins with [3H]NaBH4 followed by immunoprecipitation with glucose transporter antibodies. These data indicate that insulin-mediated translocation in 3T3-L1 adipocytes is not restricted to a tissue-specific insulin-responsive glucose transporter species and suggest that other tissue-specific factors regulate the translocation process.     

Restricted localization of the adipocyte/muscle glucose transporter species to a cell surface-derived vesicle fraction of 3T3-L1 adipocytes. Inhibited lateral mobility of integral plasma membrane proteins in newly inserted membrane areas of differentiated 3T3-L1 cells

The recent demonstration of a large cell surface-derived pool of insulin-sensitive glucose transporters, presumably concentrated in the microvilli of 3T3-L1 adipocytes, induced the assumption that in differentiated adipocytes, newly inserted plasma membrane areas may display restricted lateral mobility, thereby preventing diffusion of integral membrane proteins out of these areas into the adjoining plasma membrane. In order to test this assumption, the cell surface distributions of the two glucose transporter species expressed by 3T3-L1 cells were determined using specific antisera against the HepG2/erythrocyte transporter, GluT1, which is synthesized in both fibroblasts and adipocytes, and the adipocyte/muscle-specific transporter, GluT4, expressed for the first time 3-4 days after induction of adipose conversion. GluT1 was shown to be localized in the plasma membrane of both 3T3-L1 preadipocytes and adipocytes, whereas GluT4 was almost entirely restricted to the low density surface-derived vesicle (LDSV) fraction of 3T3-L1 adipocytes most likely consisting of microvilli-derived vesicles. In contrast to the minor portion of GluT4 found in the adipocyte plasma membrane fraction, equal amounts of the GluT1 protein were detected in both the plasma membrane and the LDSV fractions of adipocytes. Both transporter species were present in the microsomal and the LDSV fractions of adipocytes. The observed distribution of the two transporter species is in accordance with the postulated restriction of the lateral mobility in plasma membrane areas formed by newly inserted transgolgi vesicles of differentiated adipocytes.     

Photoaffinity labeling of insulin-sensitive hexose transporters in intact rat adipocytes. Direct evidence that latent transporters become exposed to the extracellular space in response to insulin

Irradiation of intact rat adipocytes with high intensity ultraviolet light in the presence of 0.5 microM [3H] cytochalasin B results in the labeling of Mr 43,000 and 46,000 proteins that reside in the plasma membrane fraction. In contrast to the Mr 46,000 protein, the Mr 43,000 component is not observed in the microsome fraction and exhibits lower affinity for [3H]cytochalasin B. Photolabeling of the Mr 43,000 protein is inhibited by cytochalasin D, indicating it is not a hexose transporter component. The Mr 46,000 protein exhibits characteristics expected for the glucose transporter such that D-glucose or 3-O-methylglucose but not cytochalasin D inhibits its photolabeling with [3H] cytochalasin B. Furthermore, insulin addition to intact cells either prior to or after photoaffinity labeling of the Mr 46,000 protein causes a redistribution of this component from the low density microsomes to the plasma membrane fraction, as expected for the hexose transporter. Photolabeling of transporters in both the low density microsome and plasma membrane fractions is inhibited when intact cells are equilibrated with 50 mM ethylidene glucose prior to irradiation with [3H]cytochalasin B. Incubation of intact cells with 50 mM ethylidene glucose for 1 min at 15 degrees C leads to an intracellular concentration of only 2 mM. Under these conditions, the photoaffinity labeling in intact cells of hexose transporters that fractionate with the low density microsomes is unaffected, indicating these transporters are not exposed to the extracellular medium. In contrast, photolabeling in intact insulin-treated cells of hexose transporters that fractionate with the plasma membrane is inhibited under these incubation conditions. The results demonstrate that insulin action results in the exposure to the extracellular medium of previously sequestered hexose transporters.     

Insulin-induced translocation of glucose transporters from post-Golgi compartments to the plasma membrane of 3T3-L1 adipocytes

A semiquantitative method using immunocytochemistry on ultrathin cryosections and the protein A-gold technique was performed to study the effect of insulin on the cellular distribution of the glucose transporters in cultured 3T3-L1 adipocytes. In basal cells a substantial portion of the label was present in a tubulovesicular structure at the trans side of the Golgi apparatus, likely to represent the trans-Golgi reticulum, and in small vesicles present in the cytoplasm. Treatment with insulin induced a rapid translocation of transporters from the tubulovesicular structure to the plasma membrane. The transporter labeling of the plasma membrane increased three-fold and that of the tubulovesicular structure decreased by half. There was no effect of insulin on the degree of label in the small cytoplasmic vesicles. Removal of insulin from stimulated cells rapidly reversed the distribution of transporters to that seen in basal cells. This study thus provides the first morphological evidence for the occurrence of transporter translocation in insulin action and identifies for the first time the intracellular location of the responsive transporters.     

Insulin-responsive glucose transporters are concentrated in a cell surface-derived membrane fraction of 3T3-L1 adipocytes

The recently proposed mechanistic concept of a receptor-regulated entrance compartment for hexose transport formed by microvilli on 3T3-L1 adipocytes predicted a preferential localization of glucose transporters in these structures. The cytochalasin B-binding technique was used to determine in basal and insulin-stimulated cells the distribution of glucose transporters between plasma membranes, low density microsomes (LDM) and two cell surface-derived membrane fractions prepared by a hydrodynamic shearing technique. The shearing procedure applied prior to homogenization yielded a low density surface-derived vesicle (LDSV) fraction which contained nearly 60% of the cellular glucose transporters and the total insulin-sensitive transporter pool. The rest of the glucose transporter population was localized within the plasma membrane (5%) and the LDM fraction (37%). Pretreatment of the cells with insulin (20 mU/ml for 10 min) reduced the transporter content of the LDSV fraction by 40% and increased that of the plasma membrane fraction 4-fold. The transporter containing LDSV fraction was clearly differentiated from the LDM fraction by its low specific galactosyltransferase activity and its insulin-sensitivity. Scanning electron microscopy revealed that the LDSV fraction contained a rather uniform population of spherical vesicles of 100-200 nm in diameter.     

Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations

We have recently described a monoclonal antibody (1F8) that recognizes a form of glucose transporter unique to fat and muscle (James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185), tissues that respond acutely to insulin by markedly increasing their glucose uptake. Here, we report that rat adipocytes possess two immunologically distinct glucose-transporters: one recognized by 1F8, and one reactive with antibodies raised against the human erythrocyte glucose transporter. Immunoadsorption experiments indicate that these glucose transporters reside in different vesicle populations and that both transporter isoforms translocate from intracellular sites to the plasma membrane in response to insulin. The insulin-regulatable transporter resides in a unique vesicle that comprises 3% or less of the low density microsomes of fat cells and has a limited protein composition that does not include the bulk of another translocatable protein, the insulin-like growth factor II receptor. Immunoprecipitation with 1F8 of microsomal glucose transporters photoaffinity labeled with [3H]cytochalasin B brings down 90% of the label. Similarly, immunoprecipitation with 1F8 of glucose transporters from insulin-stimulated plasma membranes photolabeled with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-f ors kolin, another transporter-selective reagent, results in 75% of the labeled transporter localized in the immunoprecipitate. Thus, insulin action involves the combined effect of translocation from at least two vesicle pools each containing different glucose transporters. The 1F8-reactive transporter comprises the majority of the total transporter pool that is responsible for the insulin-induced increase in glucose transporter number.     

Identification and partial purification of the insulin-responsive glucose transporter from 3T3-L1 adipocytes

The glucose transporter in 3T3-L1 adipocytes has been identified as a polypeptide of average Mr 51000 by means of its reaction with antibodies raised against the purified human erythrocyte glucose transporter and by photolabeling with [3H]cytochalasin B. The finding that the antibodies immunoprecipitated the photolabeled polypeptide demonstrated that both methods detected the same polypeptide. The 3T3-L1 adipocyte glucose transporter has been partially purified. The main steps in the purification procedure were the preparation of salt-washed cellular membranes, Triton X-100 solubilization, and immunoaffinity chromatography on affinity-purified antibodies against the human erythrocyte transporter. A simple method of affinity purification of these antibodies, which consists of adsorption from serum onto protein-depleted erythrocyte membranes and release with acid, and an assay for the 3T3-L1 adipocyte transporter polypeptide, which employs immunoblotting, have been developed.     

The formation of an insulin-responsive vesicular cargo compartment is an early event in 3T3-L1 adipocyte differentiation

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.     

Insulin-mediated translocation of glucose transporters from intracellular membranes to plasma membranes: sole mechanism of stimulation of glucose transport in L6 muscle cells

Plasma membranes and light microsomes were isolated from fused L6 muscle cells. Pre-treatment of cells with insulin did not affect marker enzyme or protein distribution in isolated membranes. The number of glucose transporters in the isolated membranes was calculated from the D-glucose-protectable binding of [3H]cytochalasin B. Glucose transporter number was higher in plasma membranes and lower in intracellular membranes derived from insulin-treated cells than in the corresponding fractions from untreated cells. The net increase in glucose transporters in plasma membranes was identical to the net decrease in glucose transporters in light microsomes (2 pmol/1.23 x 10(8) cells). The fold increase in glucose transporter number/mg protein in plasma membranes (2-fold) was similar to the fold increase in glucose transport caused by insulin. This suggests that recruitment of glucose transporters from intracellular membranes to the plasma membrane is the major mechanism of stimulation of hexose transport in L6 muscle cells. This is the first report of isolation of the two insulin-sensitive membrane elements from a cell line, and the results indicate that, in contrast to rat adipocytes, there is not change in the intrinsic activity of the transporters in response to insulin.     

Thrombin-stimulated elevation of human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production.

The nucleoside transporter has been purified by passage of a preparation of human erythrocyte-membrane band-4.5 proteins through a column of immobilized antibodies specific for the glucose transporter. This procedure removed greater than 99.8% of the glucose transporters and achieved an approx. 18-fold purification of the nucleoside transporter, constituting a 478-fold purification from erythrocyte membranes. The isolated protein migrated as a single broad band of average apparent Mr 55,000 on SDS/polyacrylamide gels and bound approx. 0.6 mol of nitrobenzylthioinosine/mol of polypeptide, with a Kd of 1.1 +/- 0.14 (S.E.M.) nM. Upon reconstitution into large unilamellar phospholipid vesicles it catalysed the uptake of uridine with an apparent specific activity 6-fold greater than that of the unfractionated band-4.5 proteins. Furthermore, the purified nucleoside transporter was not labelled on Western blots by monoclonal antibody raised against the glucose transporter. It is concluded that the nucleoside transporter has been purified to near homogeneity.     

The v-SNARE Vti1a regulates insulin-stimulated glucose transport and Acrp30 secretion in 3T3-L1 adipocytes

Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and trans-Golgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNA-based gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.     

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.