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.

Additive effect of contractions and insulin on GLUT-4 translocation into the sarcolemma

1994 ◽  
Vol 77 (4) ◽  
pp. 1597-1601 ◽  
Author(s):  
J. Gao ◽  
J. Ren ◽  
E. A. Gulve ◽  
J. O. Holloszy
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Membrane Fraction ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Muscle Group ◽  
Intracellular Membrane ◽  
Glut 4 ◽  
Incremental Effect ◽  
Glut 4 Translocation

The maximal effects of insulin and muscle contractions on glucose transport are additive. GLUT-4 is the major glucose transporter isoform expressed in skeletal muscle. Muscle contraction and insulin each induce translocation of GLUT-4 from intracellular sites into the plasma membrane. The purpose of this study was to test the hypothesis that the incremental effect of contractions and insulin on glucose transport is mediated by additivity of the maximal effects of these stimuli on GLUT-4 translocation into the sarcolemma. Anesthetized rats were given insulin by intravenous infusion to raise plasma insulin to 2,635 +/- 638 microU/ml. The gastrocnemius-plantaris-soleus group was stimulated to contract via the sciatic nerve by using a protocol that maximally activates glucose transport. After treatment with insulin, contractions, or insulin plus contractions or no treatment, the gastrocnemius-plantaris-soleus muscle group was dissected out and was subjected to subcellular fractionation to separate the plasma membrane and intracellular membrane fractions. Insulin induced a 70% increase and contractions induced a 113% increase in the GLUT-4 content of the plasma membrane fraction. The effects of insulin and contractions were additive, as evidenced by a 185% increase in the GLUT-4 content of the sarcolemmal fraction. This finding provides evidence that the incremental effect of maximally effective insulin and contractile stimuli on glucose transport is mediated by additivity of their effects on GLUT-4 translocation into the sarcolemma.

scholarly journals Multiple endosomal recycling pathways in rat adipose cells

1998 ◽  
Vol 331 (3) ◽  
pp. 829-835 ◽  
Author(s):  
Konstantin V. KANDROR ◽  
Paul F. PILCH
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Sedimentation Coefficient ◽  
Membrane Vesicles ◽  
Average Diameter ◽  
Subcellular Fractionation ◽  
Adipose Cells

Adipose and skeletal-muscle cells can translocate several membrane proteins from intracellular compartment(s) to the cell surface in an insulin-dependent fashion. Among these proteins is Glut4, a physiologically important glucose transporter which mediates insulin's effect on blood glucose clearance. Under basal conditions, Glut4 is localized in uniform, intracellular membrane vesicles with an average diameter of 50–70 nm and a sedimentation coefficient of 100–120 S. The nature of this compartment and its trafficking pathway to the plasma membrane is still unresolved. We show here that, in addition to Glut4, the aminopeptidase gp160 or insulin-responsive aminopeptidase (‘IRAP’), sortilin, and an acutely recycling population of the insulin-like growth factor-II/mannose 6-phosphate receptor, this compartment includes 60% of the intracellular population of the transferrin receptor. We used subcellular fractionation, cell-surface biotinylation, and radioactive-ligand (125I-transferrin) uptake to demonstrate that the transferrin receptor recycles between this compartment and the plasma membrane in response to insulin along with Glut4 and other protein components of these vesicles. The co-localization of Glut4 and several endosomal markers in the terminally differentiated fat-cells during several stages of their cycling pathways suggests that the ‘Glut4 pathway ’ may derive from the hormone-insensitive endosomes of undifferentiated preadipocytes. The insulin receptor is excluded from Glut4-containing vesicles in both insulin-stimulated and unstimulated adipocytes, and thus it is likely to traffic independently from Glut4 through different intracellular compartments. Our data show that, in adipose cells, the ligand-dependent recycling pathway of the insulin receptor is structurally separated from the ligand-independent pathway of the transferrin receptor, and that Glut4 is specifically targetted to the latter.

scholarly journals Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes.

1983 ◽  
Vol 97 (2) ◽  
pp. 329-339 ◽  
Author(s):  
C Harding ◽  
J Heuser ◽  
P Stahl
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Intracellular Membrane ◽  
Coated Pits ◽  
Surface Receptors ◽  
Receptor Mediated Endocytosis ◽  
Freeze Dried ◽  
Inner Surface ◽  
Multivesicular Endosomes ◽  
Rat Reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin-resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.

scholarly journals Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells

1996 ◽  
Vol 313 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Jing YANG ◽  
James F. CLARKE ◽  
Catriona J. ESTER ◽  
Paul W. YOUNG ◽  
Masato KASUGA ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Receptor ◽  
Time Course ◽  
Glucose Transporter ◽  
Kinase Activity ◽  
Insulin Receptor Substrate ◽  
Low Density ◽  
Intracellular Membrane ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Glucose transporters (GLUTs) are continuously recycled in 3T3-L1 cells and so insulin, through its action on phosphatidylinositol 3-kinase (PI 3-kinase), could potentially alter the distribution of these transporters by enhancing retention in the plasma membrane or acting intracellularly to increase exocytosis, either by stimulating a budding or a docking and fusion process. To examine the site of involvement of PI 3-kinase in the glucose transporter recycling pathway, we have determined the kinetics of recycling under conditions in which the PI 3-kinase activity is inhibited by wortmannin. Wortmannin addition to fully insulin-stimulated cells induces a net reduction of glucose transport activity with a time course that is consistent with a major effect on the return of internalized transporters to the plasma membrane. The exocytosis of GLUT1 and GLUT4 is reduced to very low levels in wortmannin-treated cells (≈ 0.009 min-1), but the endocytosis of these isoforms is not markedly perturbed and the rate constants are approx. 10-fold higher than for exocytosis (0.099 and 0.165 min-1, respectively). The slow reduction in basal activity following treatment with wortmannin is consistent with a wortmannin effect on constitutive recycling as well as insulin-regulated exocytosis. PI 3-kinase activity that is precipitated by anti-phosphotyrosine, anti-[insulin receptor substrate 1 (IRS1)] and anti-α-p85 antibodies show the same level of insulin-stimulated activity, ≈ 0.5 pmol/20 min per dish of 3T3-L1 cells. Since the activities precipitated by all three antibodies are similar, it seems unlikely that a second insulin receptor substrate, IRS2, contributes significantly to the insulin signalling observed in 3T3-L1 cells. To examine whether insulin targets PI 3-kinase to intracellular membranes we have carried out subcellular fractionation studies. These suggest that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular, low-density, membranes. In addition, the association of PI 3-kinase with IRS1 appears to partially deplete the cytoplasm of α-p85-precipitatable activity, suggesting that IRS1 may redistribute PI 3-kinase from the cytoplasm to the low-density microsome membranes. Taken together, the trafficking kinetic and PI 3-kinase distribution studies suggest an intracellular membrane site of action of the enzyme in enhancing glucose transporter exocytosis.

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 Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation.

1987 ◽  
Vol 105 (3) ◽  
pp. 1241-1251 ◽  
Author(s):  
J R Bartles ◽  
H M Feracci ◽  
B Stieger ◽  
A L Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Subcellular Fractionation ◽  
Aminopeptidase N ◽  
Rat Hepatocyte ◽  
Dipeptidylpeptidase Iv ◽  
Plasma Membrane Fraction ◽  
Apical Domain ◽  
Basolateral Plasma Membrane

We have used pulse-chase metabolic radiolabeling with L-[35S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates. Approximate half-times for arrival are in the range of 90-120 min for aminopeptidase N and dipeptidylpeptidase IV whereas only 15-20% of the mature radiolabeled HA 4 associated with the hepatocyte plasma membrane fraction has become apical even after 150 min of chase. Our results suggest a mechanism for hepatocyte plasma membrane biogenesis in vivo in which all integral plasma membrane proteins are shipped first to the basolateral domain, followed by the specific retrieval and transport of apical proteins to the apical domain at distinct rates.

scholarly journals 5-lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes.

1993 ◽  
Vol 178 (6) ◽  
pp. 1935-1946 ◽  
Author(s):  
J W Woods ◽  
J F Evans ◽  
D Ethier ◽  
S Scott ◽  
P J Vickers ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Membrane Fraction ◽  
Subcellular Fractionation ◽  
Binding Activity ◽  
Resting Cells ◽  
Human Leukocytes ◽  
Ultrathin Frozen Sections

The intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-activated human leukocytes has been determined using immuno-electronmicroscopic labeling of ultrathin frozen sections and subcellular fractionation techniques. 5-LO is a 78-kD protein that catalyzes the conversion of arachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In response to ionophore stimulation, 5-LO translocates from a soluble to a sedimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Neither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was identical to that observed in activated cells. In addition, subcellular fractionation techniques showed > 83% of immunoblot-detectable FLAP protein and approximately 64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrated that a plasma membrane marker, detected by a monoclonal antibody PMN13F6, was not detectable in the nuclear membrane fraction. In contrast to FLAP, 5-LO in resting cells could not be visualized along the nuclear envelope. Except for weak labeling of the euchromatin region of the nucleus, 5-LO could not be readily detected in any other cellular compartment. These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and that ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope.

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.

scholarly journals Expression of the liver-type glucose transporter (GLUT2) in 3T3-L1 adipocytes: analysis of the effects of insulin on subcellular distribution

1994 ◽  
Vol 304 (1) ◽  
pp. 307-311 ◽  
Author(s):  
A M Brant ◽  
S Martin ◽  
G W Gould
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Glucose Transporter ◽  
Fold Increase ◽  
Subcellular Fractionation ◽  
Fibroblastic Cell ◽  
Clonal Cell Line ◽  
Cellular Environment ◽  
Cell Clones ◽  
Differentiation Protocol

We have expressed the liver-type facilitative glucose transporter, GLUT2, in the insulin-sensitive 3T3-L1 adipocyte clonal cell line in an effort to address the importance of transporter isoform and cellular environment on the ability of insulin to mediate glucose-transporter translocation. Analysis of non-differentiated fibroblastic cell clones transfected with the GLUT2 cDNA identified the presence of this isoform in several independent clones. These clones exhibited increased deoxyglucose and fructose transport rates compared with control cells. Upon differentiation, the fibroblastic clones selected for study achieved > 95% phenotypic conversion into adipocytes. Expression of the GLUT2 protein was maintained throughout the differentiation protocol. Subcellular fractionation revealed that in response to insulin, unlike the native GLUT4, GLUT2 protein did not undergo significant translocation to the plasma membrane; furthermore, the subcellular distribution of the expressed GLUT2 was quite distinct from that of the endogenous GLUT4. 3T3-L1 adipocytes expressing GLUT2 only exhibited a 2-fold increase in insulin-stimulated fructose uptake, further suggesting that GLUT2 does not undergo insulin-stimulated translocation.

Sign in / Sign up

Export Citation Format

Share Document