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.

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 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 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 Golgin-160 Is Required for the Golgi Membrane Sorting of the Insulin-responsive Glucose Transporter GLUT4 in Adipocytes

2006 ◽  
Vol 17 (12) ◽  
pp. 5346-5355 ◽  
Author(s):  
Dumaine Williams ◽  
Stuart W. Hicks ◽  
Carolyn E. Machamer ◽  
Jeffrey E. Pessin
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Glucose Transporter ◽  
General Distribution ◽  
Amino Terminal ◽  
Vesicular Stomatitis ◽  
Glucose Transporter Glut4 ◽  
Golgi Network ◽  
Interfering Rna ◽  
Regulated Trafficking

The peripheral Golgi protein golgin-160 is induced during 3T3L1 adipogenesis and is primarily localized to the Golgi cisternae distinct from the trans-Golgi network (TGN) in a general distribution similar to p115. Small interfering RNA (siRNA)-mediated reduction in golgin-160 protein resulted in an increase accumulation of the insulin-responsive amino peptidase (IRAP) and the insulin-regulated glucose transporter (GLUT4) at the plasma membrane concomitant with enhanced glucose uptake in the basal state. The redistribution of GLUT4 was rescued by expression of a siRNA-resistant golgin-160 cDNA. The basal state accumulation of plasma membrane GLUT4 occurred due to an increased rate of exocytosis without any significant effect on the rate of endocytosis. This GLUT4 trafficking to the plasma membrane in the absence of golgin-160 was independent of TGN/Golgi sorting, because it was no longer inhibited by the expression of a dominant-interfering Golgi-localized, γ-ear–containing ARF-binding protein mutant and displayed reduced binding to the lectin wheat germ agglutinin. Moreover, expression of the amino terminal head domain (amino acids 1–393) had no significant effect on the distribution or insulin-regulated trafficking of GLUT4 or IRAP. In contrast, expression of carboxyl α helical region (393–1498) inhibited insulin-stimulated GLUT4 and IRAP translocation, but it had no effect on the sorting of constitutive membrane trafficking proteins, the transferrin receptor, or vesicular stomatitis virus G protein. Together, these data demonstrate that golgin-160 plays an important role in directing insulin-regulated trafficking proteins toward the insulin-responsive compartment in adipocytes.

scholarly journals Akt Activation Is Required at a Late Stage of Insulin-Induced GLUT4 Translocation to the Plasma Membrane

2005 ◽  
Vol 19 (4) ◽  
pp. 1067-1077 ◽  
Author(s):  
Ellen M. van Dam ◽  
Roland Govers ◽  
David E. James
Keyword(s):  
Plasma Membrane ◽  
Glucose Transporter ◽  
Cell Cortex ◽  
Glut4 Translocation ◽  
Akt Activation ◽  
Multistep Process ◽  
Intracellular Compartments ◽  
Insulin Dependent ◽  
Glucose Transporter Glut4 ◽  
Golgi Network

Abstract Insulin stimulates the translocation of glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (PM). This involves multiple steps as well as multiple intracellular compartments. The Ser/Thr kinase Akt has been implicated in this process, but its precise role is ill defined. To begin to dissect the role of Akt in these different steps, we employed a low-temperature block. Upon incubation of 3T3-L1 adipocytes at 19 C, GLUT4 accumulated in small peripheral vesicles with a slight increase in PM labeling concomitant with reduced trans-Golgi network labeling. Although insulin-dependent translocation of GLUT4 to the PM was impaired at 19 C, we still observed movement of vesicles toward the surface. Strikingly, insulin-stimulated Akt activity, but not phosphatidylinositol 3 kinase activity, was blocked at 19 C. Consistent with a multistep process in GLUT4 trafficking, insulin-stimulated GLUT4 translocation could be primed by treating cells with insulin at 19 C, whereas this was not the case for Akt activation. These data implicate two insulin-regulated steps in GLUT4 translocation: 1) redistribution of GLUT4 vesicles toward the cell cortex—this process is Akt-independent and is not blocked at 19 C; and 2) docking and/or fusion of GLUT4 vesicles with the PM—this process may be the major Akt-dependent step in the insulin regulation of glucose transport.

scholarly journals Insulin-responsive amino peptidase follows the Glut4 pathway but is dispensable for the formation and translocation of insulin-responsive vesicles

2019 ◽  
Vol 30 (12) ◽  
pp. 1536-1543 ◽  
Author(s):  
Xiang Pan ◽  
Anatoli Meriin ◽  
Guanrong Huang ◽  
Konstantin V. Kandror
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transporter ◽  
Muscle Cells ◽  
Retrograde Transport ◽  
Skeletal Muscle Cells ◽  
Glucose Transporter 4 ◽  
Trans Golgi Network ◽  
Insulin Responsiveness ◽  
Golgi Network ◽  
Component Protein

In fat and skeletal muscle cells, insulin-responsive amino peptidase (IRAP) along with glucose transporter 4 (Glut4) and sortilin, represents a major component protein of the insulin-responsive vesicles (IRVs). Here, we show that IRAP, similar to Glut4 and sortilin, is retrieved from endosomes to the trans-Golgi network by retromer. Unlike Glut4, retrograde transport of IRAP does not require sortilin, as retromer can directly bind to the cytoplasmic tail of IRAP. Ablation of IRAP in 3T3-L1 adipocytes shifts the endosomal pool of Glut4 to more acidic endosomes, but does not affect IRV targeting, stability, and insulin responsiveness of Glut4.

GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with TGN

1996 ◽  
Vol 109 (13) ◽  
pp. 2967-2978 ◽  
Author(s):  
E. Ralston ◽  
T. Ploug
Keyword(s):  
Immunoelectron Microscopy ◽  
Golgi Complex ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Brefeldin A ◽  
Immunogold Electron Microscopy ◽  
Trans Golgi Network ◽  
Storage Vesicles ◽  
Skeletal Myotubes ◽  
Golgi Network

There is little consensus on the nature of the storage compartment of the glucose transporter GLUT4, in non-stimulated cells of muscle and fat. More specifically, it is not known whether GLUT4 is localized to unique, specialized intracellular storage vesicles, or to vesicles that are part of the constitutive endosomal-lysosomal pathway. To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by immunofluorescence and immunoelectron microscopy. We have used a panel of antibodies to markers of the Golgi complex (alpha mannosidase II and giantin), of the trans-Golgi network (TGN38), of lysosomes (lgp110), and of early and late endosomes (transferrin receptor and mannose-6-phosphate receptor, respectively), to define the position of their subcellular compartments. By immunofluorescence, GLUT4 appears concentrated in the core of the myotubes. It is primarily found around the nuclei, in a pattern suggesting an association with the Golgi complex, which is further supported by colocalization with giantin and by immunogold electron microscopy. GLUT4 appears to be in the trans-most cisternae of the Golgi complex and in vesicles just beyond, i.e. in the structures that constitute the trans-Golgi network (TGN). In myotubes treated with brefeldin A, the immunofluorescence pattern of GLUT4 is modified, but it differs from both Golgi complex markers and TGN38. Instead, it resembles the pattern of the transferrin receptor, which forms long tubules. In untreated cells, double staining for GLUT4 and transferrin receptor by immunofluorescence shows similar but distinct patterns. Immunoelectron microscopy localizes transferrin receptor, detected by immunoperoxidase, to large vesicles, presumably endosomes, very close to the GLUT4-containing tubulo-vesicular elements. In brefeldin A-treated cells, a network of tubules of approximately 70 nm diameter, studded with varicosities, stains for both GLUT4 and transferrin receptor, suggesting that brefeldin A has caused fusion of the transferrin receptor and GLUT4-containing compartments. The results suggest that GLUT4 storage vesicles constitute a specialized compartment that is either a subset of the TGN, or is very closely linked to it. The link between GLUT4 vesicles and transferrin receptor containing endosomes, as revealed by brefeldin A, may be important for GLUT4 translocation in response to muscle stimulation.

scholarly journals Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase

2007 ◽  
Vol 402 (2) ◽  
pp. 279-290 ◽  
Author(s):  
Tsung-Yin J. Yeh ◽  
Juan I. Sbodio ◽  
Zhi-Yang Tsun ◽  
Biao Luo ◽  
Nai-Wen Chi
Keyword(s):  
Protein Modification ◽  
Glucose Transporter ◽  
Glut4 Translocation ◽  
Small Interfering Rnas ◽  
Insulin Stimulation ◽  
Storage Vesicles ◽  
Cargo Proteins ◽  
Polymerase Inhibitor ◽  
Parp Activity ◽  
Glucose Transporter Glut4

The glucose transporter GLUT4 and the aminopeptidase IRAP (insulin-responsive aminopeptidase) are the major cargo proteins of GSVs (GLUT4 storage vesicles) in adipocytes and myocytes. In the basal state, most GSVs are sequestered in perinuclear and other cytosolic compartments. Following insulin stimulation, GSVs undergo exocytic translocation to insert GLUT4 and IRAP into the plasma membrane. The mechanisms regulating GSV trafficking are not fully defined. In the present study, using 3T3-L1 adipocytes transfected with siRNAs (small interfering RNAs), we show that insulin-stimulated IRAP translocation remained intact despite substantial GLUT4 knockdown. By contrast, insulin-stimulated GLUT4 translocation was impaired upon IRAP knockdown, indicating that IRAP plays a role in GSV trafficking. We also show that knockdown of tankyrase, a Golgi-associated IRAP-binding protein that co-localizes with perinuclear GSVs, attenuated insulin-stimulated GSV translocation and glucose uptake without disrupting insulin-induced phosphorylation cascades. Moreover, iodixanol density gradient analyses revealed that tankyrase knockdown altered the basal-state partitioning of GLUT4 and IRAP within endosomal compartments, apparently by shifting both proteins toward less buoyant compartments. Importantly, the afore-mentioned effects of tankyrase knockdown were reproduced by treating adipocytes with PJ34, a general PARP (poly-ADP-ribose polymerase) inhibitor that abrogated tankyrase-mediated protein modification known as poly-ADP-ribosylation. Collectively, these findings suggest that physiological GSV trafficking depends in part on the presence of IRAP in these vesicles, and that this process is regulated by tankyrase and probably its PARP activity.

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.

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