Immunocytochemical and biochemical studies of GLUT4 in rat skeletal muscle.

In muscle and adipocytes, glucose transport is regulated by the translocation of insulin regulatable glucose transporters (GLUT4) between an intracellular compartment and the cell surface. In these studies we have characterized the cellular compartments containing GLUT4 in rat skeletal muscle. Immunocytochemical studies showed that in unstimulated muscle, GLUT4 was not present in surface membranes. Tubulo-vesicular structures clustered in the trans Golgi reticulum were enriched in GLUT4. GLUT4 underwent translocation to the sarcolemma in response to combined stimulation with insulin and exercise. Using immunoisolation, the intracellular GLUT4 vesicles (IRGTV) were purified 300-fold over the cell homogenate. IRGTV from unstimulated muscle were not enriched in markers specific for the sarcolemma, transverse tubules, sarcoplasmic reticulum or mitochondria; this was confirmed using gel filtration chromatography. Insulin resulted in a 40% decrease in GLUT4 levels in IRGTV confirming that this represents the intracellular compartment of GLUT4. GLUT4 is a major component of the IRGTV, constituting at least 5% of total vesicle protein. A subset of polypeptides are also markedly enriched in the muscle IRGTV. In conclusion, these data suggest that translocation of GLUT4 from intracellular tubulo-vesicular structures is the major mechanism by which insulin and exercise regulate muscle glucose transport.     

Subcellular localization and trafficking of the GLUT4 glucose transporter isoform in insulin-responsive cells

The rate-limiting step in the uptake and metabolism of Dglucose by insulin target cells is thought to be glucose transport mediated by glucose transporters (primarily the GLUT4 isoform) localized to the plasma membrane. However, subcellular fractionation, photolabelling and immunocytochemical studies have shown that the pool of GLUT4 present in the plasma membrane is only one of many subcellular pools of this protein. GLUT4 has been found in occluded vesicles at the plasma membrane, clathrin-coated pits and vesicles, early endosomes, and tubulo-vesicular structures; the latter are analogous to known specialized secretory compartments. Tracking the movement of GLUT4 through these compartments, and defining the mechanism and site of action of insulin in stimulating this subcellular trafficking, are major topics of current investigation. Recent evidence focuses attention on the exocytosis of GLUT4 as the major site of insulin action. Increased exocytosis may be due to decreased retention of glucose transporters in an intracellular pool, or possibly to increased assembly of a vesicle docking and fusion complex. Although details are unknown, the presence in GLUT4 vesicles of a synaptobrevin homologue leads us to propose that a process analogous to that occurring in synaptic vesicle trafficking is involved in the assembly of GLUT4 vesicles into a form suitable for fusion with the plasma membrane. Evidence that the pathways of signalling from the insulin receptor and of GLUT4 vesicle exocytosis may converge at the level of the key signalling enzyme, phosphatidylinositol 3-kinase, is discussed.     

Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type

Insulin stimulates glucose transport in adipocytes via the rapid redistribution of the GLUT1 and GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. Insulin sensitivity is dependent on the proper intracellular trafficking of the glucose transporters in the basal state. The bulk of insulin-sensitive transport in adipocytes appears to be due to the translocation of GLUT4, which is more efficiently sequestered inside the cell and is present in much greater abundance than GLUT1. The cell type and isoform specificity of GLUT4 intracellular targeting were investigated by examining the subcellular distribution of GLUT1 and GLUT4 in cell types that are refractory to the effect of insulin on glucose transport. Rat GLUT4 was expressed in 3T3-L1 fibroblasts and HepG2 hepatoma cells by DNA-mediated transfection. Transfected 3T3-L1 fibroblasts over-expressing human GLUT1 exhibited increased glucose transport, and laser confocal immunofluorescent imaging of GLUT1 in these cells indicated that the protein was concentrated in the plasma membrane. In contrast, 3T3-L1 fibroblasts expressing GLUT4 exhibited no increase in transport activity, and confocal imaging demonstrated that this protein was targeted almost exclusively to cytoplasmic compartments. 3T3-L1 fibroblasts expressing GLUT4 were unresponsive to insulin with respect to transport activity, and no change was observed in the subcellular distribution of the protein after insulin administration. Immunogold labeling of frozen ultrathin sections revealed that GLUT4 was concentrated in tubulo-vesicular elements of the trans-Golgi reticulum in these cells. Sucrose density gradient analysis of 3T3-L1 homogenates was consistent with the presence of GLUT1 and GLUT4 in discrete cytoplasmic compartments. Immunogold labeling of frozen thin sections of HepG2 cells indicated that endogenous GLUT1 was heavily concentrated in the plasma membrane. Sucrose density gradient analysis of homogenates of HepG2 cells expressing rat GLUT4 suggested that GLUT4 is targeted to an intracellular location in these cells. The density of the putative GLUT4-containing cytoplasmic membrane vesicles was very similar in HepG2 cells, 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and rat adipocytes. These data indicate that the intracellular trafficking of GLUT4 is isoform specific. Additionally, these observations support the notion that GLUT4 is targeted to its proper intracellular locale even in cell types that do not exhibit insulin-responsive glucose transport, and suggest that the machinery that regulates the intracellular targeting of GLUT4 is distinct from the factors that regulate insulin-dependent recruitment to the cell surface.     

Insulin recruits GLUT4 from specialized VAMP2-carrying vesicles as well as from the dynamic endosomal/trans-Golgi network in rat adipocytes

Insulin treatment of fat cells results in the translocation of the insulin-responsive glucose transporter type 4, GLUT4, from intracellular compartments to the plasma membrane. However, the precise nature of these intracellular GLUT4-carrying compartments is debated. To resolve the nature of these compartments, we have performed an extensive morphological analysis of GLUT4-containing compartments, using a novel immunocytochemical technique enabling high labeling efficiency and 3-D resolution of cytoplasmic rims isolated from rat epididymal adipocytes. In basal cells, GLUT4 was localized to three morphologically distinct intracellular structures: small vesicles, tubules, and vacuoles. In response to insulin the increase of GLUT4 at the cell surface was compensated by a decrease in small vesicles, whereas the amount in tubules and vacuoles was unchanged. Under basal conditions, many small GLUT4 positive vesicles also contained IRAP (88%) and the v-SNARE, VAMP2 (57%) but not markers of sorting endosomes (EEA1), late endosomes, or lysosomes (lgp120). A largely distinct population of GLUT4 vesicles (56%) contained the cation-dependent mannose 6-phosphate receptor (CD-MPR), a marker protein that shuttles between endosomes and the trans-Golgi network (TGN). In response to insulin, GLUT4 was recruited both from VAMP2 and CD-MPR positive vesicles. However, while the concentration of GLUT4 in the remaining VAMP2-positive vesicles was unchanged, the concentration of GLUT4 in CD-MPR-positive vesicles decreased. Taken together, we provide morphological evidence indicating that, in response to insulin, GLUT4 is recruited to the plasma membrane by fusion of preexisting VAMP2-carrying vesicles as well as by sorting from the dynamic endosomal-TGN system.     

Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: effects of ATP insulin, and GTP gamma S and localization of GLUT4 to clathrin lattices

Insulin stimulates the movement of two glucose transporter isoforms (GLUT1 and GLUT4) to the plasma membrane (PM) in adipocytes. To study this process we have prepared highly purified PM fragments by gently sonicating 3T3-L1 adipocytes grown on glass coverslips. Using confocal laser immunofluorescence microscopy we observed increased PM labeling for GLUT1 (2.3-fold) and GLUT4 (eightfold) after insulin treatment in intact cells. EM immunolabeling of PM fragments indicated that in the nonstimulated state GLUT4 was mainly localized to flat clathrin lattices. Whereas GLUT4 labeling of clathrin lattices was only slightly increased after insulin treatment, labeling of uncoated PM regions was markedly increased with insulin. These data suggest that GLUT4 recycles from the cell surface both in the presence and absence of insulin. In streptolysin-O permeabilized adipocytes, insulin, and GTP gamma S increased PM levels of GLUT4 to a similar extent as observed with insulin in intact cells. In the absence of an exogenous ATP source the magnitude of these effects was considerably reduced. Removal of ATP per se caused a significant increase in cell surface levels of GLUT4 suggesting that ATP may be required for intracellular sequestration of these transporters. When insulin and GTP gamma S were added together, in the presence of ATP, PM GLUT4 levels were similar to levels observed when either insulin or GTP gamma S was added individually. Addition of GTP gamma S was able to overcome this ATP dependence of insulin-stimulated GLUT4 movement. GTP gamma S had no effect on constitutive secretion of adipsin in permeabilized cells. In addition, there was no effect of insulin or GTP gamma S on GLUT4 movement to the PM in noninsulin sensitive streptolysin-O-permeabilized 3T3-L1 fibroblasts overexpressing GLUT4. We conclude that the insulin-stimulated movement of GLUT4 to the cell surface in adipocytes may require ATP early in the insulin signaling pathway and a GTP-binding protein(s) at a later step(s). We propose that the association of GLUT4 with clathrin lattices may be important in maintaining the exclusive intracellular location of this transporter in the absence of insulin.     

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.     

Understanding the distinct subcellular trafficking of CD36 and GLUT4 during the development of myocardial insulin resistance

CD36 and GLUT4 are the main cardiac trans-sarcolemmal transporters for long-chain fatty acids and glucose, respectively. Together they secure the majority of cardiac energy demands. Moreover, these transporters each represent key governing kinetic steps in cardiac fatty acid and glucose fluxes, thereby offering major sites of regulation. The underlying mechanism of this regulation involves a perpetual vesicle-mediated trafficking (recycling) of both transporters between intracellular stores (endosomes) and the cell surface. In the healthy heart, CD36 and GLUT4 translocation to the cell surface is under short-term control of the same physiological stimuli, most notably increased contraction and insulin secretion. However, under chronic lipid overload, a condition that accompanies a Western lifestyle, CD36 and GLUT4 recycling are affected distinctly, with CD36 being expelled to the sarcolemma while GLUT4 is imprisoned within the endosomes. Moreover, the increased CD36 translocation towards the cell surface is a key early step, setting the heart on a route towards insulin resistance and subsequent contractile dysfunction. Therefore, the proteins making up the trafficking machinery of CD36 need to be identified with special focus to the differences with the protein composition of the GLUT4 trafficking machinery. These proteins that are uniquely dedicated to either CD36 or GLUT4 traffic may offer targets to rectify aberrant substrate uptake seen in the lipid-overloaded heart. Specifically, CD36-dedicated trafficking regulators should be inhibited, whereas such GLUT4-dedicated proteins would need to be activated. Recent advances in the identification of CD36-dedicated trafficking proteins have disclosed the involvement of vacuolar-type H⁺-ATPase and of specific vesicle-associated membrane proteins (VAMPs). In this review, we summarize these recent findings and sketch a roadmap of CD36 and GLUT4 trafficking compatible with experimental findings.     

GLUT4 is retained by an intracellular cycle of vesicle formation and fusion with endosomes

The intracellularly stored GLUT4 glucose transporter is rapidly translocated to the cell surface upon insulin stimulation. Regulation of GLUT4 distribution is key for the maintenance of whole body glucose homeostasis. We find that GLUT4 is excluded from the plasma membrane of adipocytes by a dynamic retention/retrieval mechanism. Our kinetic studies indicate that GLUT4-containing vesicles continually bud and fuse with endosomes in the absence of insulin and that these GLUT4 vesicles are 5 times as likely to fuse with an endosome as with the plasma membrane. We hypothesize that this intracellular cycle of vesicle budding and fusion is an element of the active mechanism by which GLUT4 is retained. The GLUT4 trafficking pathway does not extensively overlap with that of furin, indicating that the trans-Golgi network, a compartment in which furin accumulates, is not a significant storage reservoir of GLUT4. An intact microtubule cytoskeleton is required for insulin-stimulated recruitment to the cell surface, although it is not required for the basal budding/fusion cycle. Nocodazole disruption of the microtubule cytoskeleton reduces the insulin-stimulated exocytosis of GLUT4, accounting for the reduced insulin-stimulated translocation of GLUT4 to the cell surface.     

A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal [published erratum appears in J Cell Biol 1994 Sep;126(6):1625]

The unique COOH-terminal 30-amino acid region of the adipocyte/skeletal muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test whether an underlying mechanism of this domain's function involves glucose transporter endocytosis rates, transfected cells were generated expressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUT1) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incubation of COS-7 or CHO cells expressing the HA-tagged chimera with anti-HA antibody at 37 degrees resulted in an increased rate of antibody internalization compared to cells expressing similar levels of HA-tagged GLUT1, which displays a cell surface disposition. Colocalization of the internalized anti-HA antibody in vesicular structures with internalized transferrin and with total transporters was established by digital imaging microscopy, suggesting the total cellular pool of transporters are continuously recycling through the coated pit endocytosis pathway. Mutation of the unique double leucines 489 and 490 in the rat GLUT4 COOH-terminal domain to alanines caused the HA-tagged chimera to revert to the slow endocytosis rate and steady- state cell surface display characteristic of GLUT1. These results support the hypothesis that the double leucine motif in the GLUT4 COOH terminus operates as a rapid endocytosis and retention signal in the GLUT4 transporter, causing its localization to intracellular compartments in the absence of insulin.     

Detection of insulin-regulated GLUT4-translocation by the insertion of a protein C epitope in L6 myoblasts

Insulin stimulates glucose transport by translocation of the membrane glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. GLUT4 is highly expressed in adipose tissue and skeletal muscle. We have constructed a cDNA containing the human GLUT4 inserted by a 12 amino acid protein C epitope in the first extracellular (exofacial) domain of the human GLUT4 (GLUT4-PC). Stable expression of GLUT4-PC in L6 myoblasts (L6-GLUT4-PC) was confirmed in immunofluorescence using monoclonal antibodies against protein C. The protein C staining yielded labeling in perinuclear vesicles strongly co-localizing with GLUT4 detected with antibodies directed against the endofacial part of GLUT4. The L6-GLUT4-PC cells were further characterized in a direct cell-based enzyme-linked immunosorbent assay by the use of beta-galactosidase. Cell surface binding of monoclonal protein C antibodies was detected with beta-galactosidase-conjugated secondary antibodies and chlorophenolred-beta-D-galactopyranoside (CPRG) as substrate in 2% paraformaldehyde fixed cells. In this assay, stimulation with insulin created a rapidly detectable recruitment of GLUT4-PC to the cell surface. This cell-based enzyme-linked immunosorbent GLUT4 assay was shown to be comparable with that of previously reported radioactive assays.     

Insulin increases cell surface GLUT4 levels by dose dependently discharging GLUT4 into a cell surface recycling pathway

The insulin-responsive glucose transporter GLUT4 plays an essential role in glucose homeostasis. A novel assay was used to study GLUT4 trafficking in 3T3-L1 fibroblasts/preadipocytes and adipocytes. Whereas insulin stimulated GLUT4 translocation to the plasma membrane in both cell types, in nonstimulated fibroblasts GLUT4 readily cycled between endosomes and the plasma membrane, while this was not the case in adipocytes. This efficient retention in basal adipocytes was mediated in part by a C-terminal targeting motif in GLUT4. Insulin caused a sevenfold increase in the amount of GLUT4 molecules present in a trafficking cycle that included the plasma membrane. Strikingly, the magnitude of this increase correlated with the insulin dose, indicating that the insulin-induced appearance of GLUT4 at the plasma membrane cannot be explained solely by a kinetic change in the recycling of a fixed intracellular GLUT4 pool. These data are consistent with a model in which GLUT4 is present in a storage compartment, from where it is released in a graded or quantal manner upon insulin stimulation and in which released GLUT4 continuously cycles between intracellular compartments and the cell surface independently of the nonreleased pool.     

The glucose transporter 4-regulating protein TUG is essential for highly insulin-responsive glucose uptake in 3T3-L1 adipocytes

Insulin stimulates glucose uptake in fat and muscle by redistributing GLUT4 glucose transporters from intracellular membranes to the cell surface. We previously proposed that, in 3T3-L1 adipocytes, TUG retains GLUT4 within unstimulated cells and insulin mobilizes this retained GLUT4 by stimulating its dissociation from TUG. Yet the relative importance of this action in the overall control of glucose uptake remains uncertain. Here we report that transient, small interfering RNA-mediated depletion of TUG causes GLUT4 translocation and enhances glucose uptake in unstimulated 3T3-L1 adipocytes, similar to insulin. Stable TUG depletion or expression of a dominant negative fragment likewise stimulates GLUT4 redistribution and glucose uptake, and insulin causes a 2-fold further increase. Microscopy shows that TUG governs the accumulation of GLUT4 in perinuclear membranes distinct from endosomes and indicates that it is this pool of GLUT4 that is mobilized by TUG disruption. Interestingly, in addition to translocating GLUT4 and enhancing glucose uptake, TUG disruption appears to accelerate the degradation of GLUT4 in lysosomes. Finally, we find that TUG binds directly and specifically to a large intracellular loop in GLUT4. Together, these findings demonstrate that TUG is required to retain GLUT4 intracellularly in 3T3-L1 adipocytes in the absence of insulin and further implicate the insulin-stimulated dissociation of TUG and GLUT4 as an important action by which insulin stimulates glucose uptake.     

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.     

Visualization of TGN to endosome trafficking through fluorescently labeled MPR and AP-1 in living cells

We have stably expressed in HeLa cells a chimeric protein made of the green fluorescent protein (GFP) fused to the transmembrane and cytoplasmic domains of the mannose 6-phosphate/insulin like growth factor II receptor in order to study its dynamics in living cells. At steady state, the bulk of this chimeric protein (GFP-CI-MPR) localizes to the trans-Golgi network (TGN), but significant amounts are also detected in peripheral, tubulo-vesicular structures and early endosomes as well as at the plasma membrane. Time-lapse videomicroscopy shows that the GFP-CI-MPR is ubiquitously detected in tubular elements that detach from the TGN and move toward the cell periphery, sometimes breaking into smaller tubular fragments. The formation of the TGN-derived tubules is temperature dependent, requires the presence of intact microtubule and actin networks, and is regulated by the ARF-1 GTPase. The TGN-derived tubules fuse with peripheral, tubulo-vesicular structures also containing the GFP-CI-MPR. These structures are highly dynamic, fusing with each other as well as with early endosomes. Time-lapse videomicroscopy performed on HeLa cells coexpressing the CFP-CI-MPR and the AP-1 complex whose gamma-subunit was fused to YFP shows that AP-1 is present not only on the TGN and peripheral CFP-CI-MPR containing structures but also on TGN-derived tubules containing the CFP-CI-MPR. The data support the notion that tubular elements can mediate MPR transport from the TGN to a peripheral, tubulo-vesicular network dynamically connected with the endocytic pathway and that the AP-1 coat may facilitate MPR sorting in the TGN and endosomes.     

Infusion of a biotinylated bis-glucose photolabel: a new method to quantify cell surface GLUT4 in the intact mouse heart

Glucose uptake in the heart is mediated by specific glucose transporters (GLUTs) present on cardiomyocyte cell surface membranes. Metabolic stress and insulin both increase glucose transport by stimulating the translocation of glucose transporters from intracellular storage vesicles to the cell surface. Isolated perfused transgenic mouse hearts are commonly used to investigate the molecular regulation of heart metabolism; however, current methods to quantify cell surface glucose transporter content in intact mouse hearts are limited. Therefore, we developed a novel technique to directly assess the cell surface content of the cardiomyocyte glucose transporter GLUT4 in perfused mouse hearts, using a cell surface impermeant biotinylated bis-glucose photolabeling reagent (bio-LC-ATB-BGPA). Bio-LC-ATB-BGPA was infused through the aorta and cross-linked to cell surface GLUTs. Bio-LC-ATB-BGPA-labeled GLUT4 was recovered from cardiac membranes by streptavidin isolation and quantified by immunoblotting. Bio-LC-ATB-BGPA-labeling of GLUT4 was saturable and competitively inhibited by d-glucose. Stimulation of glucose uptake by insulin in the perfused heart was associated with parallel increases in bio-LC-ATB-BGPA-labeling of cell surface GLUT4. Bio-LC-ATB-BGPA also labeled cell surface GLUT1 in the perfused heart. Thus, photolabeling provides a novel approach to assess cell surface glucose transporter content in the isolated perfused mouse heart and may prove useful to investigate the mechanisms through which insulin, ischemia, and other stimuli regulate glucose metabolism in the heart and other perfused organs.     

Immunohistochemical localization of glucose transporters and insulin receptors in human fetal membranes at term

The localization has been investigated of the isoforms GLUT1, GLUT3 and GLUT4 of glucose transporter proteins as well as of insulin receptors. Fetal membranes (n=10) were examined by immunohistochemical methods at the light and electron microscopic levels using mono- and polyclonal antibodies. In all amnion epithelial cells, GLUT1 and GLUT3 antibodies were bound to the apical membrane. Very rarely the GLUT1 antibody also immunostained the basolateral membrane and reacted weakly with the endomembrane system and membranes of the lateral cell protrusions. Fibroblasts reacted with the antibodies against GLUT1, GLUT4 and insulin receptor, whereas they were labelled only in one case with GLUT3 antibody. Cytotrophoblast cells were only stained with antibodies against GLUT1 and GLUT3. Antibodies against GLUT4 only reacted with fibroblasts in the membranes. On amnion epithelial cells, weak immunoreactivity with insulin receptor antibodies was detected only at the electron microscopic level. The data indicate: (1) GLUT1 is located on all cells of the amnion, whereas GLUT3 is present in detectable amounts only on amnion epithelial cells and cytotrophoblast; (2) GLUT1 and GLUT3 on amnion epithelial cells are predominantly located on the apical surface; (3) GLUT4 and insulin receptors are not regularly expressed. We suggest that amnion epithelial cells cover their basal glucose requirements from the amniotic fluid and not from the maternal circulation.     

Receptor-mediated transcytosis of follicle-stimulating hormone through the rat testicular microvasculature

Accumulated data suggest that endothelial cells express specific receptors for several peptide and (glyco)protein hormones that may transport hormones across the cell to be delivered to the interstitial fluid and tissue target cells. Surprisingly, very little information is available on the actual endothelial organelles involved in this cellular process. In the present study the transfer of follicle-stimulating hormone (FSH) through the endothelial barrier of rat testes was examined by analysing the binding and transport of gold-tagged recombinant human (rh)FSH under various conditions using electron microscopy. At 4 degrees C the probe bound specifically to the luminal surface of the endothelial cells without internalization. The use of 125I-rhFSH, which allows precise quantitation of the binding, confirmed the specificity of hormone interaction with the testicular microvasculature. At 37 degrees C the hormone was internalized via coated pits and vesicles into an extensive subluminal tubulo-vesicular compartment and was transported across the endothelium via a system of tubules and vesicles. Moreover, monoclonal antibodies against the FSH receptor ectodomain coupled to colloidal gold followed the same route. In contrast, a non specific, fluid-phase uptake via caveolae was observed for a major plasma protein - rat serum albumin and a fluid-phase tracer - peroxidase. These results suggest that FSH transcytosis across the testicular endothelial barrier is receptor-mediated and involves luminal uptake via coated pits/vesicles, sorting at the level of luminal early endosomes, and transcellular transport through transcytotic tubulo-vesicular organelles. Similar receptor-mediated pathways are likely to be involved in the physiological functioning of a number of other protein and peptide hormones that must translocate specifically from blood to the target cells.     

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.     

Biogenesis of insulin-responsive GLUT4 vesicles is independent of brefeldin A-sensitive trafficking

Insulin stimulates translocation of GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. As a significant amount of GLUT4 is localised to the TGN, independently of the biosynthetic pathway, one possibility is that trafficking via the TGN is important in either intracellular sequestration or insulin-dependent movement to the cell surface. In this study we have used immuno-electron microscopy to show that GLUT4 is localised to AP-1 vesicles in the TGN region in 3T3-L1 adipocytes. To dissect the role of this trafficking pathway we used brefeldin A (BFA) to disrupt AP-1 association with membranes. Despite a reorganisation of GLUT4 compartments following BFA treatment, the intracellular sequestration of GLUT4, and its insulin-dependent movement to the cell surface, was unaffected. BFA increased the half time of reversal of insulin-stimulated glucose transport from 17 to 30 min but did not prevent complete reversal. Furthermore, following reversal re-stimulation of glucose transport activity by insulin was not compromised. We conclude that under basal conditions GLUT4 cycles between the TGN and endosomes via the AP-1 pathway. However, neither this pathway, nor any other BFA-sensitive pathway, appears to play a major role in insulin-dependent recruitment of GLUT4 to the cell surface.     

Development of an intracellular pool of glucose transporters in 3T3-L1 cells.

The membrane-impermeant bis-mannose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) has been used to study the development of an intracellular pool of glucose transporters in 3T3-L1 cells. The subcellular distributions of the transporter isoforms GLUT1 and GLUT4 were determined by comparing the labeling obtained in cells in which the impermeant reagent only had access to the cell surface and the labeling obtained in digitonin-permeabilized cells. ATB-BMPA labeling showed that only GLUT1 was present in preconfluent fibroblasts and that most of the transporters were distributed to the cell surface. In preconfluent fibroblasts, the 2-deoxy-D-glucose transport activity was approximately 5 times higher than in confluent fibroblasts. ATB-BMPA labeling showed that the decrease in transport as cells reached confluence was associated with a decrease in the proportion of GLUT1 distributed to the cell surface. The sequestration of these transporters was associated with the development of an insulin-responsive transport activity which increased by approximately 2.5-fold compared with unstimulated confluent cells. ATB-BMPA labeling showed that insulin stimulation resulted in an approximately 2-fold increase in surface GLUT1 so that about one-half of the available transporters became recruited to the cell surface. Measurements of the changes in the distribution of both GLUT1 and GLUT4 throughout the differentiation of confluent fibroblasts into adipocytes showed that both transporters were sequestered in parallel. Basal levels of transport and photolabeling remained low throughout the differentiation period when the total pool of transporters (GLUT1 plus GLUT4) was increased by approximately 5-fold. These results suggest that the sequestration process was present before new transporters were synthesized. Thus, the sequestration mechanism develops in confluent growth-arrested fibroblasts although the capacity to sequester additional transporters may increase as differentiation proceeds.