scholarly journals EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes.

2021 ◽  
Author(s):  
Anna M Koester ◽  
Kamilla M Laidlaw ◽  
Silke Morris ◽  
Marie F.A. Cutiongco ◽  
Laura Stirrat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Recent Work ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Vesicle Fusion ◽  
Scaffold Protein ◽  
Glucose Transporter Glut4 ◽  
Phosphatidylinositol 4 ◽  
Intracellular Glucose

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localisation of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

scholarly journals The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus

1992 ◽  
Vol 117 (4) ◽  
pp. 729-743 ◽  
Author(s):  
RC Piper ◽  
C Tai ◽  
JW Slot ◽  
CS Hahn ◽  
CM Rice ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Cho Cells ◽  
Sindbis Virus ◽  
Glucose Transporter ◽  
Intracellular Compartment ◽  
Glut 1 ◽  
Glut 4 ◽  
Intracellular Sequestration

GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.

scholarly journals Effects of noradrenaline on the cell-surface glucose transporters in cultured brown adipocytes: novel mechanism for selective activation of GLUT1 glucose transporters

1998 ◽  
Vol 330 (1) ◽  
pp. 397-403 ◽  
Author(s):  
Yasutake SHIMIZU ◽  
Shinobu SATOH ◽  
Hajime YANO ◽  
Yasuhiko MINOKOSHI ◽  
W. Samuel CUSHMAN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Brown Adipocytes ◽  
Photoaffinity Labelling ◽  
Glucose Binding ◽  
Glut1 Protein ◽  
Dependent Mechanism

Glucose transport into rat brown adipocytes has been shown to be stimulated directly by the sympathetic neurotransmitter, noradrenaline, without a significant increase in the protein content of either GLUT1 or GLUT4 glucose transporter in the plasma membrane [Shimizu, Kielar, Minokoshi and Shimazu (1996) Biochem. J.314, 485-490]. In the present study, we labelled the exofacial glucose-binding sites of GLUT1 and GLUT4 with a membrane-impermeant photoaffinity reagent, 2-N-[4-(1-azitrifluoroethyl)benzoyl]-[2-3H]1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-[3H]BMPA), to determine which isoform is responsible for the noradrenaline-induced increase in glucose transport into intact brown adipocytes in culture. Insulin stimulated the rate of hexose transport by increasing ATB-[3H]BMPA-labelled cell-surface GLUT4. In contrast, the noradrenaline-induced increase in glucose transport was not accompanied by an increased ATB-[3H]BMPA labelling of GLUT4, nor with an increased amount of GLUT4 in the plasma membrane fraction as assessed by Western blotting, indicating that noradrenaline does not promote the translocation of GLUT4. However, noradrenaline induced an increase in photoaffinity labelling of cell-surface GLUT1 without an apparent increase in the immunoreactive GLUT1 protein in the plasma membrane. This is suggestive of an increased affinity of GLUT1 for the ligand. In fact, the Ki value of non-radioactive ATB-BMPA for 2-deoxy-d-glucose uptake was significantly decreased after treatment of the cells with noradrenaline. The increased photoaffinity labelling of GLUT1 and increased glucose transport caused by noradrenaline were inhibited by a cAMP antagonist, cAMP-S Rp-isomer. These results demonstrate that noradrenaline stimulates glucose transport in brown adipocytes by enhancing the functional activity of GLUT1 through a cAMP-dependent mechanism.

Thyroid hormone increases the partitioning of glucose transporters to the plasma membrane in ARL 15 cells

1995 ◽  
Vol 269 (3) ◽  
pp. E605-E610
Author(s):  
R. S. Haber ◽  
C. M. Wilson ◽  
S. P. Weinstein ◽  
A. Pritsker ◽  
S. W. Cushman
Keyword(s):  
Gene Expression ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Transporter Gene ◽  
Glut 1 ◽  
Surface Partitioning ◽  
Enhanced Surface ◽  
Stimulation Of

The stimulation of glucose transport by 3,5,3'-triiodo-L-thyronine (T3) in the liver-derived ARL 15 cell line is only partly attributable to increased GLUT-1 glucose transporter gene expression. To test the hypothesis that T3 increases the partitioning of GLUT-1 to the cell surface, we quantitated surface GLUT-1 using the photolabel ATB-[3H]BMPA. In control cells only approximately 20% of total cellular GLUT-1 was present at the cell surface. T3 treatment (100 nM) for 6 h increased the rate of 2-deoxy-[3H]glucose (2-DG) uptake by 30, 92, and 95% in three experiments and increased surface GLUT-1 photolabeling by 17, 81, and 72%, respectively, with no increase in total cellular GLUT-1. T3 treatment for 48 h increased 2-DG uptake by 143, 172, and 216% in three experiments and increased cell surface GLUT-1 photolabeling by 88, 161, and 184%, respectively, with smaller increases in total cellular GLUT-1. T3 treatment for 48 h thus increased the fraction of cellular GLUT-1 at the plasma membrane from 21 +/- 2 to 35 +/- 3% (SE). We conclude that most of the early (6-h) stimulation of glucose transport by T3 in ARL 15 cells is mediated by an increase in the partitioning of GLUT-1 to the plasma membrane. With more chronic T3 treatment (48 h), the enhanced surface partitioning of GLUT-1 is persistent and is superimposed on an increase in total cellular GLUT-1, accounting for a further increase in glucose transport.

scholarly journals Regulation of cell surface GLUT4 in skeletal muscle of transgenic mice

1997 ◽  
Vol 321 (1) ◽  
pp. 75-81 ◽  
Author(s):  
Joseph T. BROZINICK ◽  
Scott C. McCOID ◽  
Thomas H. REYNOLDS ◽  
Cindy M. WILSON ◽  
Ralph W. STEVENSON ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Muscle Membrane ◽  
Transport Activity ◽  
Hindlimb Muscles ◽  
Subcellular Membrane ◽  
Glucose Transporter Glut4

Marked overexpression of the glucose transporter GLUT4 in skeletal muscle membrane fractions of GLUT4 transgenic (TG) mice is accompanied by disproportionately small increases in basal and insulin-stimulated glucose transport activity. Thus we have assessed cell surface GLUT4 by photolabelling with the membrane-impermeant reagent 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(d-mannos-4-yloxy)-2-propylamine (ATB-BMPA) and measured the corresponding glucose transport activity using 2-deoxyglucose in isolated extensor digitorum longus (EDL) muscles from non-transgenic (NTG) and GLUT4 TG mice in the absence and presence of 13.3 nM (2000 µ-units/ml) insulin, without or with hypoxia as a model of muscle contraction. TG mice displayed elevated rates of glucose transport activity under basal and insulin-stimulated conditions, and in the presence of insulin plus hypoxia, compared with NTG mice. Photoaffinity labelling of cell surface GLUT4 indicated corresponding elevations in plasma membrane GLUT4 in the basal and insulin-stimulated states, and with insulin plus hypoxia, but no difference in cell surface GLUT4 during hypoxia stimulation. Subcellular fractionation of hindlimb muscles confirmed the previously observed 3-fold overexpression of GLUT4 in the TG compared with the NTG mice. These results suggest that: (1) alterations in glucose transport activity which occur with GLUT4 overexpression in EDL muscles are directly related to cell surface GLUT4 content, regardless of the levels observed in the corresponding subcellular membrane fractions, (2) while overexpression of GLUT4 influences both basal and insulin-stimulated glucose transport activity, the response to hypoxia/contraction-stimulated glucose transport is unchanged, and (3) subcellular fractionation provides little insight into the subcellular trafficking of GLUT4, and whatever relationship is demonstrated in EDL muscles from NTG mice is disrupted on GLUT4 overexpression.

scholarly journals Insulin Increases Cell Surface GLUT4 Levels by Dose Dependently Discharging GLUT4 into a Cell Surface Recycling Pathway

2004 ◽  
Vol 24 (14) ◽  
pp. 6456-6466 ◽  
Author(s):  
Roland Govers ◽  
Adelle C. F. Coster ◽  
David E. James
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transporter ◽  
Insulin Dose ◽  
Cell Types ◽  
Insulin Stimulation ◽  
Intracellular Compartments ◽  
A Cell ◽  
Recycling Pathway ◽  
Glucose Transporter Glut4

ABSTRACT 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.

scholarly journals Knockout of Syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion

2021 ◽  
Author(s):  
Hannah L. Black ◽  
Rachel Livingstone ◽  
Cynthia C. Mastick ◽  
Mohammed Al Tobi ◽  
Holly Taylor ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Metabolic Regulation ◽  
Glucose Transporter ◽  
Ectopic Expression ◽  
Glut4 Translocation ◽  
Syntaxin 4 ◽  
Rate Limiting ◽  
Insight Into

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport which is underpinned by the insulin-stimulated delivery of glucose transporter-4 (GLUT4)- containing vesicles to the plasma membrane where they dock and fuse increasing cell surface GLUT4 levels. Adipocytokines such as adiponectin are secreted via a similar mechanism. We used genome editing to knockout Syntaxin-4 a protein reported to mediate GLUT4-vesicle fusion with the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of HA-GLUT4-GFP showed that Syntaxin-4 knockout cells retain significant GLUT4 translocation capacity demonstrating that Syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that Syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that Syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells.

Functional characterization of an insulin-responsive glucose transporter (GLUT4) from fish adipose tissue

2004 ◽  
Vol 287 (2) ◽  
pp. E348-E357 ◽  
Author(s):  
Encarnación Capilla ◽  
Mònica Díaz ◽  
Amaya Albalat ◽  
Isabel Navarro ◽  
Jeffrey E. Pessin ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Plasma Membrane ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Intracellular Localization ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Native Form ◽  
Protein Motifs ◽  
Glucose Transporter Glut4

Glucose transport across the plasma membrane is mediated by a family of glucose transporter proteins (GLUTs), several of which have been identified in mammalian, avian, and, more recently, in fish species. Here, we report on the cloning of a salmon GLUT from adipose tissue with a high sequence homology to mammalian GLUT4 that has been named okGLUT4. Kinetic analysis of glucose transport following expression in Xenopus laevis oocytes demonstrated a 7.6 ± 1.4 mM Km for 2-deoxyglucose (2-DG) transport measured under zero- trans conditions and 14.4 ± 1.5 mM by equilibrium exchange of 3- O-methylglucose. Transport of 2-DG by okGLUT4-injected oocytes was stereospecific and was competed by d-glucose, d-mannose, and, to a lesser extent, d-galactose and d-fructose. In addition, 2-DG uptake was inhibited by cytochalasin B and ethylidene glucose. Moreover, insulin stimulated glucose uptake in Xenopus oocytes expressing okGLUT4 and in isolated trout adipocytes, which contain the native form of okGLUT4. Despite differences in protein motifs important for insulin-stimulated translocation of mammalian GLUT4, okGLUT4 was able to translocate to the plasma membrane from intracellular localization sites in response to insulin when expressed in 3T3-L1 adipocytes. These data demonstrate that okGLUT4 is a structural and functional fish homolog of mammalian GLUT4 but with a lower affinity for glucose, which could in part explain the lower ability of fish to clear a glucose load.

scholarly journals Cell surface accessibility of GLUT4 glucose transporters in insulin-stimulated rat adipose cells. Modulation by isoprenaline and adenosine

1992 ◽  
Vol 288 (1) ◽  
pp. 325-330 ◽  
Author(s):  
S J Vannucci ◽  
H Nishimura ◽  
S Satoh ◽  
S W Cushman ◽  
G D Holman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Transport Activity ◽  
Surface Accessibility ◽  
Adipose Cells ◽  
Membrane Concentration

Insulin-stimulated glucose transport activity in rat adipocytes is inhibited by isoprenaline and enhanced by adenosine. Both of these effects occur without corresponding changes in the subcellular distribution of the GLUT4 glucose transporter isoform. In this paper, we have utilized the impermeant, exofacial bis-mannose glucose transporter-specific photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine (ATB-BMPA) [Clark & Holman (1990) Biochem. J. 269, 615-622], to examine the cell surface accessibility of GLUT4 glucose transporters under these conditions. Compared with cells treated with insulin alone, adenosine in the presence of insulin increased the accessibility of GLUT4 to the extracellular photolabel by approximately 25%, consistent with its enhancement of insulin-stimulated glucose transport activity; the plasma membrane concentration of GLUT4 as assessed by Western blotting was unchanged. Conversely, isoprenaline, in the absence of adenosine, promoted a time-dependent (t1/2 approximately 2 min) decrease in the accessibility of insulin-stimulated cell surface GLUT4 of > 50%, which directly correlated with the observed inhibition of transport activity; the plasma membrane concentration of GLUT4 decreased by 0-15%. Photolabelling the corresponding plasma membranes revealed that these alterations in the ability of the photolabel to bind to GLUT4 are transient, as the levels of both photolabel incorporation and plasma membrane glucose transport activity were consistent with the observed GLUT4 concentration. These data suggest that insulin-stimulated GLUT4 glucose transporters can exist in two distinct states within the adipocyte plasma membrane, one which is functional and accessible to extracellular substrate, and one which is non-functional and unable to bind extracellular substrate. These effects are only observed in the intact adipocyte and are not retained in plasma membranes isolated from these cells when analysed for their ability to transport glucose or bind photolabel.

scholarly journals Increased intracellular sequestration of the insulin-regulated aminopeptidase upon differentiation of 3T3-L1 cells

1998 ◽  
Vol 330 (2) ◽  
pp. 1003-1008 ◽  
Author(s):  
A. Stuart ROSS ◽  
R. Susanna KELLER ◽  
E. Gustav LIENHARD
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transporter ◽  
Fold Increase ◽  
Membrane Receptors ◽  
Cell Surface Biotinylation ◽  
A Cell ◽  
Plasma Membrane Receptors ◽  
Glucose Transporter Glut4 ◽  
Intracellular Sequestration

In fat and muscle cells, the glucose transporter GLUT4 is sequestered in an intracellular compartment under basal conditions and redistributes markedly to the plasma membrane in response to insulin. Recently, we characterized a membrane aminopeptidase, designated IRAP (insulin-regulated aminopeptidase), that colocalizes with intracellular GLUT4 and similarly redistributes markedly to the plasma membrane in response to insulin in adipocytes. In contrast to GLUT4, IRAP is also expressed in 3T3-L1 fibroblasts, and this finding provided an opportunity to compare its subcellular distribution in fibroblasts and adipocytes. The relative amount of IRAP at the cell surface was measured by a cell surface biotinylation method. The portion of total IRAP at the cell surface in unstimulated adipocytes was 30% of that in unstimulated fibroblasts. Upon insulin treatment the portion of IRAP at the cell surface was the same in fibroblasts and adipocytes, and was increased 1.8-fold in fibroblasts and 8-fold in adipocytes. A similar analysis of the distribution of the transferrin receptor (TfR), the paradigm for recycling plasma membrane receptors, revealed that the portions of the TfR at the cell surface in both the basal and insulin-treated states were almost unchanged upon differentiation, and that insulin caused an increase of about 1.6-fold in the amount of TfR at the cell surface. These results show that enhanced intracellular sequestration of IRAP occurs during adipogenesis, and that this effect underlies the larger insulin-elicited fold increase of IRAP at the cell surface in adipocytes.

scholarly journals GLUT4 Recycles via a trans-Golgi Network (TGN) Subdomain Enriched in Syntaxins 6 and 16 But Not TGN38: Involvement of an Acidic Targeting Motif

2003 ◽  
Vol 14 (3) ◽  
pp. 973-986 ◽  
Author(s):  
Annette M. Shewan ◽  
Ellen M. van Dam ◽  
Sally Martin ◽  
Tang Bor Luen ◽  
Wanjin Hong ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Adipocyte Differentiation ◽  
Attachment Protein ◽  
Trans Golgi Network ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Glucose Transporter Glut4 ◽  
Golgi Network ◽  
Endosomal System

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)-solubleN-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 thetrans-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.

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