scholarly journals GLUT4 Retention in Adipocytes Requires Two Intracellular Insulin-regulated Transport Steps

2002 ◽  
Vol 13 (7) ◽  
pp. 2421-2435 ◽  
Author(s):  
Anja Zeigerer ◽  
Michael A. Lampson ◽  
Ola Karylowski ◽  
David D. Sabatini ◽  
Milton Adesnik ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Trafficking Pathway ◽  
Endosomal Recycling ◽  
Step Model ◽  
Recycling Pathway ◽  
Endosomal Pathway ◽  
Endosomal System ◽  
Regulated Trafficking

Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level.

Demonstration of insulin-responsive trafficking of GLUT4 and vpTR in fibroblasts

2000 ◽  
Vol 113 (22) ◽  
pp. 4065-4076 ◽  
Author(s):  
M.A. Lampson ◽  
A. Racz ◽  
S.W. Cushman ◽  
T.E. McGraw
Keyword(s):  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Insulin Response ◽  
Physiological Role ◽  
Muscle Cells ◽  
Cell Types ◽  
Whole Body ◽  
Trafficking Pathway ◽  
Undifferentiated Cells ◽  
Regulated Trafficking

Insulin-responsive trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) in adipose and muscle cells is well established. Insulin regulation of GLUT4 trafficking in these cells underlies the role that adipose tissue and muscle play in the maintenance of whole body glucose homeostasis. GLUT4 is expressed in a very limited number of tissues, most highly in adipose and muscle, while IRAP is expressed in many tissues. IRAP's physiological role in any of the tissues in which it is expressed, however, is unknown. The fact that IRAP, which traffics by the same insulin-regulated pathway as GLUT4, is expressed in ‘non-insulin responsive’ tissues raises the question of whether these other cell types also have a specialized insulin-regulated trafficking pathway. The existence of an insulin-responsive pathway in other cell types would allow regulation of IRAP activity at the plasma membrane as a potentially important physiological function of insulin. To address this question we use reporter molecules for both GLUT4 and IRAP trafficking to measure insulin-stimulated translocation in undifferentiated cells by quantitative fluorescence microscopy. One reporter (vpTR), a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor, has been previously characterized. The other is a GLUT4 construct with an exofacial HA epitope and a C-terminal GFP. By comparing these reporters to the transferrin receptor, a marker for general endocytic trafficking, we demonstrate the existence of a specialized, insulin-regulated trafficking pathway in two undifferentiated cell types, neither of which normally express GLUT4. The magnitude of translocation in these undifferentiated cells (approximately threefold) is similar to that reported for the translocation of GLUT4 in muscle cells. Thus, undifferentiated cells have the necessary retention and translocation machinery for an insulin response that is large enough to be physiologically important.

scholarly journals Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation.

1988 ◽  
Vol 106 (4) ◽  
pp. 1061-1066 ◽  
Author(s):  
T E McGraw ◽  
K W Dunn ◽  
F R Maxfield
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Cho Cells ◽  
Transferrin Receptor ◽  
Phosphorylation Site ◽  
Major Protein ◽  
Kinase C ◽  
Recycling Pathway

In Chinese hamster ovary (CHO) fibroblast cells the protein kinase C activating phorbol ester, phorbol myristate acetate (PMA), stimulates an increase in cell surface transferrin receptor (TR) expression by increasing the exocytic rate of the recycling pathway. The human TR expressed in CHO cells is similarly affected by PMA treatment. A mutant human TR in which the major protein kinase C phosphorylation site, serine 24, has been replaced with the non-phosphorylatable amino acid glycine has been constructed to investigate the role of receptor phosphorylation in the PMA induced up-regulation. The Gly-24-substituted receptor binds, internalizes, and recycles Tf. Furthermore, the altered receptor mediates cellular Fe accumulation from diferric-Tf, thereby fulfilling the receptor's major biological role. The Gly-24 TR behaves identically to the wild-type TR when cells are treated with PMA. Therefore, Ser-24 phosphorylation is not required for the PMA-induced redistribution of the human TR expressed in CHO cells. The increased TR expression on the cell surface after PMA treatment results from an increase in the rate of exocytosis of the recycling receptors. No change in the endocytic rate or the size of the recycling receptor pool was observed. These results indicate that the PMA effect on the TR surface expression may result from a more general perturbation of membrane trafficking rather than a specific modulation of the TR.

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 The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells.

1996 ◽  
Vol 134 (3) ◽  
pp. 625-635 ◽  
Author(s):  
S Martin ◽  
J Tellam ◽  
C Livingstone ◽  
J W Slot ◽  
G W Gould ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Intracellular Distribution ◽  
Expression Library ◽  
Recycling Endosomes ◽  
Glut 4 ◽  
Intracellular Compartments ◽  
Intracellular Vesicles

Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle-associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP-2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.

scholarly journals Rapid endocytosis of the transferrin receptor in the absence of bound transferrin.

1985 ◽  
Vol 100 (2) ◽  
pp. 633-637 ◽  
Author(s):  
C Watts
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Cell Line ◽  
Transferrin Receptor ◽  
Protein A ◽  
Transferrin Receptors ◽  
Coated Pits ◽  
Prolonged Incubation ◽  
Surface Receptors ◽  
Recycling Pathway

The rate of endocytosis of transferrin receptors, occupied or unoccupied with transferrin, was measured on the cell line K562. At 37 degrees C, receptors, radioiodinated on the cell surface at 4 degrees C, were internalized equally rapidly in the presence or absence of transferrin. In both cases, 50% of the labeled receptors became resistant to externally added trypsin in 5 min. An antitransferrin antibody was used to show directly that the receptors had entered the cells without bound transferrin. The distribution of the receptors on the cell surface was revealed by antibody and protein A-gold staining after prolonged incubation in the presence or absence of transferrin. The receptors were concentrated in coated pits under both conditions. The data suggest that endocytosis of transferrin receptors is not "triggered" by ligand binding and raise the possibility that ligand-induced down-regulation of surface receptors may not occur by this mechanism. Instead receptors may be recognized as being ligand-occupied, not at the cell surface, but at some other site in the recycling pathway such as the endosome.

In Vivo Internalization of the Somatostatin sst2A Receptor in Rat Brain: Evidence for Translocation of Cell-Surface Receptors into the Endosomal Recycling Pathway

2001 ◽  
Vol 17 (4) ◽  
pp. 646-661 ◽  
Author(s):  
Zsolt Csaba ◽  
Véronique Bernard ◽  
Lone Helboe ◽  
Marie-Thérèse Bluet-Pajot ◽  
Bertrand Bloch ◽  
...  
Keyword(s):  
Cell Surface ◽  
Rat Brain ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Endosomal Recycling ◽  
Recycling Pathway

scholarly journals Isoform-specific subcellular targeting of glucose transporters in mouse fibroblasts.

1992 ◽  
Vol 116 (3) ◽  
pp. 785-797 ◽  
Author(s):  
A W Hudson ◽  
M Ruiz ◽  
M J Birnbaum
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Amino Acid Sequences ◽  
3T3 Cells ◽  
Subcellular Targeting ◽  
Cellular Factors ◽  
Endosomal Pathway ◽  
Hexose Uptake ◽  
Cellular Compartments ◽  
Nih 3T3

GLUT1, the erythrocyte glucose transporter, and GLUT4, the adipose/muscle transporter, were each expressed in NIH-3T3 cells by retrovirus-mediated gene transfer. In fibroblasts overexpressing GLUT1, basal as well as insulin-stimulated deoxyglucose uptake was increased. Expression of GLUT4 was without affect on either basal or hormone stimulated hexose uptake. Localization of each of the transporters by indirect immunofluorescence revealed that, whereas GLUT1 was found primarily on the cell surface, GLUT4 was directed to vesicles in a perinuclear distribution and throughout the cytoplasm. The GLUT4-containing compartment represented neither Golgi complex nor lysosomes, as evidenced by the failure of lgp110 or Golgi mannosidase to co-localize. However, there was substantial overlap between the distribution of GLUT4 and the transferrin receptor, and some colocalization of the transporter isoform with the manose-6-phosphate receptor. In addition, when FITC-wheat germ agglutinin bound to the cell surface was allowed to internalize at 37 degrees C, it concentrated in vesicular structures coincident with GLUT4 immunoreactivity. These data establish that GLUT1 and GLUT4 contain within their amino acid sequences information which dictates targeting to distinct cellular compartments. Moreover, GLUT4 can be recognized by those cellular factors which direct membrane proteins to the endosomal pathway.

scholarly journals Genetic dissection of early endosomal recycling highlights a TORC1-independent role for Rag GTPases

2017 ◽  
Vol 216 (10) ◽  
pp. 3275-3290 ◽  
Author(s):  
Chris MacDonald ◽  
Robert C. Piper
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Membrane ◽  
Physiological Role ◽  
Amino Acid Transporters ◽  
Genetic Dissection ◽  
Cellular Processes ◽  
Rag Gtpases ◽  
Endosomal Recycling ◽  
Recycling Pathway

Endocytosed cell surface membrane proteins rely on recycling pathways for their return to the plasma membrane. Although endosome-to-plasma membrane recycling is critical for many cellular processes, much of the required machinery is unknown. We discovered that yeast has a recycling route from endosomes to the cell surface that functions efficiently after inactivation of the sec7-1 allele of Sec7, which controls transit through the Golgi. A genetic screen based on an engineered synthetic reporter that exclusively follows this pathway revealed that recycling was subject to metabolic control through the Rag GTPases Gtr1 and Gtr2, which work downstream of the exchange factor Vam6. Gtr1 and Gtr2 control the recycling pathway independently of TORC1 regulation through the Gtr1 interactor Ltv1. We further show that the early-endosome recycling route and its control though the Vam6>Gtr1/Gtr2>Ltv1 pathway plays a physiological role in regulating the abundance of amino acid transporters at the cell surface.

scholarly journals GLUT4 Is Retained by an Intracellular Cycle of Vesicle Formation and Fusion with Endosomes

2004 ◽  
Vol 15 (2) ◽  
pp. 870-882 ◽  
Author(s):  
Ola Karylowski ◽  
Anja Zeigerer ◽  
Alona Cohen ◽  
Timothy E. McGraw
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transporter ◽  
Kinetic Studies ◽  
Whole Body ◽  
Microtubule Cytoskeleton ◽  
Vesicle Budding ◽  
Trafficking Pathway ◽  
Retrieval Mechanism ◽  
Golgi Network

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.

scholarly journals Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes

1996 ◽  
Vol 315 (2) ◽  
pp. 487-495 ◽  
Author(s):  
Callum LIVINGSTONE ◽  
David E. JAMES ◽  
Jacqueline E. RICE ◽  
David HANPETER ◽  
Gwyn W. GOULD
Keyword(s):  
Plasma Membrane ◽  
Okadaic Acid ◽  
Transferrin Receptor ◽  
Membrane Fraction ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Type I ◽  
Intracellular Membrane ◽  
Intracellular Compartment ◽  
Endosomal System

The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the co-localization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained > 90% of the immunoreactive GLUT4 found in this fraction, but only 40% of the transferrin receptor (TfR). These results suggest only a limited degree of co-localization of these proteins. Using a technique to cross-link and render insoluble (‘ablate’) intracellular compartments containing the TfR by means of a transferrin–horseradish peroxidase conjugate (Tf–HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf–HRP for 3 h at 37 °C resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40% of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant ablation of either TGN38 or lgp120, which are markers for the trans Golgi reticulum and lysosomes respectively. Subcellular fractionation analysis revealed that most of the ablated pools of GLUT4 and TfR were found in the intracellular membrane fraction. The extent of ablation of GLUT4 from the intracellular fraction was unchanged in cells which were insulin-stimulated prior to ablation, whereas GLUT1 exhibited increased ablation in insulin-stimulated cells. Pretreatment of adipocytes with okadaic acid, an inhibitor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the internalization of GLUT4 from the plasma membrane under these conditions. Using a combination of subcellular fractionation, vesicle immunoadsorption and compartment ablation using the Tf–HRP conjugate we have been able to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative, TfR-negative/GLUT4-positive, and TfR-positive/GLUT4-positive, as defined by the relative abundance of these two markers. We propose that the TfR-negative/GLUT4-positive compartment, which contains approximately 60% of the intracellular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteristics of this compartment may be fundamental to the unique insulin regulation of GLUT4.

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