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.

scholarly journals Insulin-sensitive regulation of glucose transport and GLUT4 translocation in skeletal muscle of GLUT1 transgenic mice

1998 ◽  
Vol 337 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Garret J. ETGEN ◽  
William J. ZAVADOSKI ◽  
Geoffrey D. HOLMAN ◽  
E. Michael GIBBS
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Transgenic Mice ◽  
Glucose Transport ◽  
Hind Limb ◽  
Glucose Transporter ◽  
Glut4 Translocation ◽  
Transport Activity ◽  
Fast Twitch Muscle ◽  
Fast Twitch

Skeletal muscle glucose transport was examined in transgenic mice overexpressing the glucose transporter GLUT1 using both the isolated incubated-muscle preparation and the hind-limb perfusion technique. In the absence of insulin, 2-deoxy-d-glucose uptake was increased ∼ 3–8-fold in isolated fast-twitch muscles of GLUT1 transgenic mice compared with non-transgenic siblings. Similarly, basal glucose transport activity was increased ∼ 4–14-fold in perfused fast-twitch muscles of transgenic mice. In non-transgenic mice insulin accelerated glucose transport activity ∼ 2–3-fold in isolated muscles and to a much greater extent (∼ 7–20-fold) in perfused hind-limb preparations. The observed effect of insulin on glucose transport in transgenic muscle was similarly dependent upon the technique used for measurement, as insulin had no effect on isolated fast-twitch muscle from transgenic mice, but significantly enhanced glucose transport in perfused fast-twitch muscle from transgenic mice to ∼ 50–75% of the magnitude of the increase observed in non-transgenic mice. Cell-surface glucose transporter content was assessed via 2-N-4-(l-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(d -mannos-4-yloxy)-2-propylamine photolabelling methodology in both isolated and perfused extensor digitorum longus (EDL). Cell-surface GLUT1 was enhanced by as much as 70-fold in both isolated and perfused EDL of transgenic mice. Insulin did not alter cell-surface GLUT1 in either transgenic or non-transgenic mice. Basal levels of cell-surface GLUT4, measured in either isolated or perfused EDL, were similar in transgenic and non-transgenic mice. Interestingly, insulin enhanced cell-surface GLUT4 ∼ 2-fold in isolated EDL and ∼ 6-fold in perfused EDL of both transgenic and non-transgenic mice. In summary, these results reveal differences between isolated muscle and perfused hind-limb techniques, with the latter method showing a more robust responsiveness to insulin. Furthermore, the results demonstrate that muscle overexpressing GLUT1 has normal insulin-induced GLUT4 translocation and the ability to augment glucose-transport activity above the elevated basal rates.

scholarly journals Proteolytic cleavage of cellubrevin and vesicle-associated membrane protein (VAMP) by tetanus toxin does not impair insulin-stimulated glucose transport or GLUT4 translocation in rat adipocytes

1997 ◽  
Vol 321 (1) ◽  
pp. 233-238 ◽  
Author(s):  
Eric HAJDUCH ◽  
J. Carlos ALEDO ◽  
Colin WATTS ◽  
Harinder S. HUNDAL
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Tetanus Toxin ◽  
Glucose Transporter ◽  
Detectable Effect ◽  
Glut4 Translocation ◽  
Rat Adipocytes ◽  
Isolated Rat

Acute insulin stimulation of glucose transport in fat and skeletal muscle occurs principally as a result of the hormonal induced translocation of the GLUT4 glucose transporter from intracellular vesicular stores to the plasma membrane. The precise mechanisms governing the fusion of GLUT4 vesicles with the plasma membrane are very poorly understood at present but may share some similarities with synaptic vesicle fusion, as vesicle-associated membrane protein (VAMP) and cellubrevin, two proteins implicated in the process of membrane fusion, are resident in GLUT4-containing vesicles isolated from rat and murine 3T3-L1 adipocytes respectively. In this study we show that proteolysis of both cellubrevin and VAMP, induced by electroporation of isolated rat adipocytes with tetanus toxin, does not impair insulin-stimulated glucose transport or GLUT4 translocation. The hormone was found to stimulate glucose uptake by approx. 16-fold in freshly isolated rat adipocytes. After a single electroporating pulse, the ability of insulin to activate glucose uptake was lowered, but the observed stimulation was nevertheless nearly 5-fold higher than the basal rate of glucose uptake. Electroporation of adipocytes with 600 nM tetanus toxin resulted in a complete loss of both cellubrevin and VAMP expression within 60 min. However, toxin-mediated proteolysis of both these proteins had no effect on the ability of insulin to stimulate glucose transport which was elevated approx. 5-fold, an activation of comparable magnitude to that observed in cells electroporated without tetanus toxin. The lack of any significant change in insulin-stimulated glucose transport was consistent with the finding that toxin-mediated proteolysis of both cellubrevin and VAMP had no detectable effect on insulin-induced translocation of GLUT4 in adipocytes. Our findings indicate that, although cellubrevin and VAMP are resident proteins in adipocyte GLUT4-containing vesicles, they are not required for the acute insulin-induced delivery of GLUT4 to 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 Substrate specificity and effect on GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1

2007 ◽  
Vol 403 (2) ◽  
pp. 353-358 ◽  
Author(s):  
William G. Roach ◽  
Jose A. Chavez ◽  
Cristinel P. Mîinea ◽  
Gustav E. Lienhard
Keyword(s):  
Plasma Membrane ◽  
Glucose Transporter ◽  
Insulin Treatment ◽  
Ectopic Expression ◽  
Rab Gtpase ◽  
Glut4 Translocation ◽  
Gtpase Activating Protein ◽  
Insulin Stimulation ◽  
Glucose Transporter Glut4 ◽  
Stimulation Of

Insulin stimulation of the trafficking of the glucose transporter GLUT4 to the plasma membrane is controlled in part by the phosphorylation of the Rab GAP (GTPase-activating protein) AS160 (also known as Tbc1d4). Considerable evidence indicates that the phosphorylation of this protein by Akt (protein kinase B) leads to suppression of its GAP activity and results in the elevation of the GTP form of a critical Rab. The present study examines a similar Rab GAP, Tbc1d1, about which very little is known. We found that the Rab specificity of the Tbc1d1 GAP domain is identical with that of AS160. Ectopic expression of Tbc1d1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect. Insulin treatment led to the phosphorylation of Tbc1d1 on an Akt site that is conserved between Tbc1d1 and AS160. These results show that Tbc1d1 regulates GLUT4 translocation through its GAP activity, and is a likely Akt substrate. An allele of Tbc1d1 in which Arg125 is replaced by tryptophan has very recently been implicated in susceptibility to obesity by genetic analysis. We found that this form of Tbc1d1 also inhibited GLUT4 translocation and that this effect also required a functional GAP domain.

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 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 Activation of Protein Kinase Cζ Induces Serine Phosphorylation of VAMP2 in the GLUT4 Compartment and Increases Glucose Transport in Skeletal Muscle

2001 ◽  
Vol 21 (22) ◽  
pp. 7852-7861 ◽  
Author(s):  
Liora Braiman ◽  
Addy Alt ◽  
Toshio Kuroki ◽  
Motoi Ohba ◽  
Asia Bak ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Primary Cultures ◽  
Dominant Negative ◽  
Serine Phosphorylation ◽  
Glut4 Translocation

ABSTRACT Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKCζ in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKCζ to associate specifically with the GLUT4 compartments and that PKCζ together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKCζ and GLUT4 recycled independently of one another. To further establish the importance of PKCζ in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKCζ (DNPKCζ) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKCζ was associated with a marked increase in the activity of this isoform. The overexpressed, active PKCζ coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKCζ caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKCζ induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKCζ disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKCζ regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.

scholarly journals Role of Insulin-dependent Cortical Fodrin/Spectrin Remodeling in Glucose Transporter 4 Translocation in Rat Adipocytes

2006 ◽  
Vol 17 (10) ◽  
pp. 4249-4256 ◽  
Author(s):  
Libin Liu ◽  
Mark P. Jedrychowski ◽  
Steven P. Gygi ◽  
Paul F. Pilch
Keyword(s):  
Plasma Membrane ◽  
Glucose Transporter ◽  
Cytochalasin D ◽  
Resting Cells ◽  
Glut4 Translocation ◽  
Glucose Transporter 4 ◽  
Rat Adipocytes ◽  
Protein Receptor ◽  
Latrunculin A ◽  
Syntaxin 4

Fodrin or nonerythroid spectrin is an abundant component of the cortical cytoskeletal network in rat adipocytes. Fodrin has a highly punctate distribution in resting cells, and insulin causes a dramatic remodeling of fodrin to a more diffuse pattern. Insulin-mediated remodeling of actin occurs to a lesser extent than does that of fodrin. We show that fodrin interacts with the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 4, and this interaction is increased by insulin stimulation and decreased by prior latrunculin A treatment. Latrunculin A disrupts all actin filaments, inhibits glucose transporter 4 (GLUT4) translocation, and causes fodrin to partially redistribute from the plasma membrane to the cytosol. In contrast, cytochalasin D disrupts only the short actin filament signal, and cytochalasin D neither inhibits GLUT4 translocation nor fodrin redistribution in adipocytes. Together, our data suggest that insulin induces remodeling of the fodrin–actin network, which is required for the fusion of GLUT4 storage vesicles with the plasma membrane by permitting their access to the t-SNARE syntaxin 4.

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