scholarly journals Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8751 ◽  
Author(s):  
Silke Morris ◽  
Niall D. Geoghegan ◽  
Jessica B.A. Sadler ◽  
Anna M. Koester ◽  
Hannah L. Black ◽  
...  
Keyword(s):  
Cell Surface ◽  
Hela Cells ◽  
Glucose Transporter ◽  
Characteristic Property ◽  
Cell Types ◽  
Model System ◽  
Human Model ◽  
Small Scale ◽  
Glucose Transporter Glut4 ◽  
Surface Fusion

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA–GLUT4–GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA–GLUT4–GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells.

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 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.

scholarly journals Adipsin and the Glucose Transporter GLUT4 Traffic to the Cell Surface via Independent Pathways in Adipocytes

Traffic ◽  
2000 ◽  
Vol 1 (2) ◽  
pp. 141-151 ◽  
Author(s):  
Caroline A. Millar ◽  
Timo Meerloo ◽  
Sally Martin ◽  
Gilles R.X. Hickson ◽  
Neil J. Shimwell ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Glucose Transporter Glut4

scholarly journals Characterization of Insulin-Responsive GLUT4 Storage Vesicles Isolated from 3T3-L1 Adipocytes

2000 ◽  
Vol 20 (1) ◽  
pp. 416-427 ◽  
Author(s):  
Mitsuru Hashiramoto ◽  
David E. James
Keyword(s):  
Glucose Transporter ◽  
Significant Proportion ◽  
Protein Composition ◽  
Cell Types ◽  
Storage Compartment ◽  
Storage Vesicles ◽  
Equilibrium Sedimentation ◽  
Insulin Responsiveness ◽  
Glucose Transporter Glut4 ◽  
Golgi Network

ABSTRACT 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.

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

1996 ◽  
Vol 16 (12) ◽  
pp. 6879-6886 ◽  
Author(s):  
M Cormont ◽  
M N Bortoluzzi ◽  
N Gautier ◽  
M Mari ◽  
E van Obberghen ◽  
...  
Keyword(s):  
Cell Surface ◽  
Subcellular Distribution ◽  
Potential Role ◽  
Glucose Transporter ◽  
Small Gtpase ◽  
Wild Type ◽  
Transfected Cells ◽  
Isolated Adipocytes ◽  
Glucose Transporter Glut4

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

scholarly journals Identification of the carboxy terminus as important for the isoform-specific subcellular targeting of glucose transporter proteins.

1993 ◽  
Vol 123 (1) ◽  
pp. 137-147 ◽  
Author(s):  
K J Verhey ◽  
S F Hausdorff ◽  
M J Birnbaum
Keyword(s):  
Amino Acids ◽  
Gene Transfer ◽  
Cell Surface ◽  
Laser Scanning ◽  
Glucose Transporter ◽  
Cell Types ◽  
Laser Scanning Confocal Microscopy ◽  
Additional Contribution ◽  
Subcellular Targeting ◽  
Amino Terminal

Differential trafficking of glucose transporters contributes significantly to the establishment of a cell's capacity for hormone-regulatable hexose uptake. In the true insulin-sensitive peripheral target tissues, muscle and adipose, the transporter isoform GLUT1 residues on the cell surface and interior of the cell whereas the highly homologous isoform GLUT4 displays virtually exclusive intracellular sequestration, allowing the latter to redistribute to the cell surface in response to hormone. These patterns are equally pronounced in cells into which the transporters have been introduced by DNA-mediated gene transfer, suggesting that signals for isoform-specific sorting are recognized in diverse cell types. To determine the primary sequences responsible for the characteristic distributions, chimeric transporters were constructed in which reciprocal domains were exchanged between GLUT1 and GLUT4. In addition, a non-disruptive, species-specific epitope "tag" was introduced into a neutral region of the transporter to allow analysis of reciprocal chimeras using a single antibody. These recombinant transporters were stably expressed in HIH 3T3 and PC12 cells by retrovirus-mediated gene transfer, and were localized by indirect immunofluorescence and laser scanning confocal microscopy, as well as by staining of plasma membrane sheets prepared from these cells. The results indicate that the carboxy-terminal 30 amino acids are primarily responsible for the differential targeting of the glucose transporter isoforms GLUT1 and GLUT4, though there is a lesser additional contribution by the amino-terminal 183 amino acids.

A dual role of the N-terminal FQQI motif in GLUT4 trafficking

2009 ◽  
Vol 390 (9) ◽  
Author(s):  
Ulrike Bernhardt ◽  
Françoise Carlotti ◽  
Rob C. Hoeben ◽  
Hans-Georg Joost ◽  
Hadi Al-Hasani
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Dual Role ◽  
Endosomal Trafficking ◽  
Endosomal Sorting ◽  
Storage Compartment ◽  
Storage Vesicles ◽  
Intracellular Storage ◽  
Glucose Transporter Glut4

AbstractIn adipocytes, the glucose transporter GLUT4 recycles between intracellular storage vesicles and the plasma membrane. GLUT4 is internalized by a clathrin- and dynamin-dependent mechanism, and sorted into an insulin-sensitive storage compartment. Insulin stimulation leads to GLUT4 accumulation on the cell surface. The N-terminal F5QQI motif in GLUT4 has been shown previously to be required for sorting of the protein in the basal state. Here, we show that the FQQI motif is a binding site for the medium chain adaptin μ1, a subunit of the AP-1 adaptor complex that plays a role in post-Golgi/endosomal trafficking events. In order to investigate the role of AP-1 and AP-2 in GLUT4 trafficking, we generated 3T3-L1 adipocytes expressing HA-GLUT4-GFP and knocked down the AP-1 and AP-2 complex by RNAi, respectively. In AP-1 and AP-2 knockdown adipocytes, GLUT4 accumulates at the cell surface in the basal state, consistent with a role of AP-1 in post-endosomal sorting of GLUT4 to the insulin-sensitive storage compartment, and of AP-2 in clathrin-mediated endocytosis. Our data demonstrate a dual role of the F5QQI motif and support the conclusion that the AP complexes direct GLUT4 trafficking and endocytosis.

scholarly journals Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization.

1993 ◽  
Vol 123 (1) ◽  
pp. 127-135 ◽  
Author(s):  
M P Czech ◽  
A Chawla ◽  
C W Woon ◽  
J Buxton ◽  
M Armoni ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cho Cells ◽  
Cellular Localization ◽  
Glucose Transporter ◽  
Intracellular Localization ◽  
Similar Data ◽  
Amino Acid Residues ◽  
Structural Elements ◽  
Residue Substitution ◽  
Glucose Transporter Glut4

The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti-HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH-terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transporters as important determinants of cellular localization in COS-7 and CHO cells.

scholarly journals A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal.

1994 ◽  
Vol 126 (4) ◽  
pp. 979-989 ◽  
Author(s):  
S Corvera ◽  
A Chawla ◽  
R Chakrabarti ◽  
M Joly ◽  
J Buxton ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Surface Display ◽  
Cell Surface Display ◽  
Underlying Mechanism ◽  
Terminal Domain ◽  
Amino Acid Region ◽  
Intracellular Compartments ◽  
A Cell ◽  
Glucose Transporter Glut4

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.

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