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 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.

scholarly journals Sphingomyelinase has an insulin-like effect on glucose transporter translocation in adipocytes

1998 ◽  
Vol 274 (5) ◽  
pp. R1446-R1453 ◽  
Author(s):  
T. S. David ◽  
P. A. Ortiz ◽  
T. R. Smith ◽  
J. Turinsky
Keyword(s):  
Plasma Membrane ◽  
Insulin Receptor ◽  
Glucose Uptake ◽  
Signaling Pathway ◽  
Insulin Signaling ◽  
Glucose Transporter ◽  
Insulin Signaling Pathway ◽  
Glut 1 ◽  
Glut 4 ◽  
Glut 4 Translocation

Rat epididymal adipocytes were incubated with 0, 0.1, and 1 mU sphingomyelinase/ml for 30 or 60 min, and glucose uptake and GLUT-1 and GLUT-4 translocation were assessed. Adipocytes exposed to 1 mU sphingomyelinase/ml exhibited a 173% increase in glucose uptake. Sphingomyelinase had no effect on the abundance of GLUT-1 in the plasma membrane of adipocytes. In contrast, 1 mU sphingomyelinase/ml increased plasma membrane content of GLUT-4 by 120% and produced a simultaneous decrease in GLUT-4 abundance in the low-density microsomal fraction. Sphingomyelinase had no effect on tyrosine phosphorylation of either the insulin receptor β-subunit or the insulin receptor substrate-1, a signaling molecule in the insulin signaling pathway. It is concluded that the incubation of adipocytes with sphingomyelinase results in insulin-like translocation of GLUT-4 to the plasma membrane and that this translocation does not occur via the activation of the initial components of the insulin signaling pathway.

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 Differential targeting of glucose transporter isoforms heterologously expressed in Xenopus oocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 707-715 ◽  
Author(s):  
H M Thomas ◽  
J Takeda ◽  
G W Gould
Keyword(s):  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Transport Activity ◽  
Glut 4 ◽  
Transporter Family ◽  
Native Cell

We have examined the subcellular distribution of three members of the human glucose transporter family expressed in oocytes from Xenopus laevis. Following injection of in vitro-transcribed mRNA encoding the transporter isoform to be studied, we have determined the subcellular localization of the expressed protein by immunofluorescence and by subcellular fractionation coupled with immunoblotting using specific anti-peptide antibodies. We have shown that both the liver-type (GLUT 2) and brain-type (GLUT 3) glucose transporters are expressed predominantly in the plasma membranes of oocytes, and in both cases high levels of glucose transport activity are exhibited. In contrast, the insulin-regulatable glucose transporter (GLUT 4) is localized predominantly to an intracellular membrane pool, and the levels of transport activity recorded in oocytes expressing GLUT 4 are correspondingly lower. The localization of the different transporter isoforms to distinct subcellular fractions mirrors the situation observed in their native cell type and thus demonstrates that oocytes may prove to be a useful system with which to study the targeting signals for this important class of membrane proteins. In addition, the determination of the amounts of the transporters expressed per oocyte together with a knowledge of their Km values has allowed us to estimate the turnover numbers of these transporters. Insulin was without effect on glucose transport in oocytes expressing any of these transporter isoforms. Microinjection of guanosine 5′-[gamma-thio]triphosphate into oocytes expressing GLUT 4 was also without effect on the transport rate.

Epinephrine translocates GLUT-4 but inhibits insulin-stimulated glucose transport in rat muscle

1998 ◽  
Vol 274 (4) ◽  
pp. E700-E707 ◽  
Author(s):  
Xiao-Xia Han ◽  
Arend Bonen
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Rat Muscle ◽  
Glut 4 ◽  
Glut 4 Translocation ◽  
Dose Dependent Decrease ◽  
Dose Dependent

We examined the effects of epinephrine (25, 50, and 150 nM) on 1) basal and insulin-stimulated 3- O-methylglucose (3-MG) transport in perfused rat muscles and 2) GLUT-4 in skeletal muscle plasma membranes. Insulin increased glucose transport 330–600% in three types of skeletal muscle [white (WG) and red (RG) gastrocnemius and soleus (SOL)]. Glucose transport was also increased by epinephrine (22–48%) in these muscles ( P < 0.05). In contrast, the insulin-stimulated 3-MG transport was reduced by epinephrine in all three types of muscles; maximal reductions were observed at 25 nM epinephrine in WG (−25%) and RG (−32.5%). A dose-dependent decrease occurred in SOL (−27% at 25 nM; −55% at 150 nM, P < 0.05). Insulin (20 mU/ml) and epinephrine (150 nM) each translocated GLUT-4 to the plasma membrane, and no differences in translocation were observed between insulin and epinephrine ( P > 0.05). In addition, epinephrine did not inhibit insulin-stimulated GLUT-4 translocation, and the combined epinephrine and insulin effects on GLUT-4 translocation were not additive. The increase in surface GLUT-4 was associated with increases in muscle cAMP concentrations, but only when epinephrine alone was present. No relationship was evident between muscle cAMP concentrations and surface GLUT-4 in the combined epinephrine and insulin-stimulated muscles. These studies indicate that epinephrine can translocate GLUT-4 while at the same time increasing glucose transport when insulin is absent, or can inhibit glucose transport when insulin is present.

Increased GLUT-4 translocation mediates enhanced insulin sensitivity of muscle glucose transport after exercise

1998 ◽  
Vol 85 (4) ◽  
pp. 1218-1222 ◽  
Author(s):  
Polly A. Hansen ◽  
Lorraine A. Nolte ◽  
May M. Chen ◽  
John O. Holloszy
Keyword(s):  
Insulin Sensitivity ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Glut 4 ◽  
Single Bout ◽  
Glut 4 Translocation ◽  
Muscle Glucose Transport

The purpose of this study was to determine whether the increase in insulin sensitivity of skeletal muscle glucose transport induced by a single bout of exercise is mediated by enhanced translocation of the GLUT-4 glucose transporter to the cell surface. The rate of 3- O-[3H]methyl-d-glucose transport stimulated by a submaximally effective concentration of insulin (30 μU/ml) was approximately twofold greater in the muscles studied 3.5 h after exercise than in those of the sedentary controls (0.89 ± 0.10 vs. 0.43 ± 0.05 μmol ⋅ ml−1 ⋅ 10 min−1; means ± SE for n = 6/group). GLUT-4 translocation was assessed by using the ATB-[2-3H]BMPA exofacial photolabeling technique. Prior exercise resulted in greater cell surface GLUT-4 labeling in response to submaximal insulin treatment (5.36 ± 0.45 dpm × 103/g in exercised vs. 3.00 ± 0.38 dpm × 103/g in sedentary group; n = 10/group) that closely mirrored the increase in glucose transport activity. The signal generated by the insulin receptor, as reflected in the extent of insulin receptor substrate-1 tyrosine phosphorylation, was unchanged after the exercise. We conclude that the increase in muscle insulin sensitivity of glucose transport after exercise is due to translocation of more GLUT-4 to the cell surface and that this effect is not due to potentiation of insulin-stimulated tyrosine phosphorylation.

scholarly journals Glucose regulates its transport in L8 myocytes by modulating cellular trafficking of the transporter GLUT-1

1992 ◽  
Vol 286 (1) ◽  
pp. 157-163 ◽  
Author(s):  
R Greco-Perotto ◽  
E Wertheimer ◽  
B Jeanrenaud ◽  
E Cerasi ◽  
S Sasson
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Glucose Concentration ◽  
Glucose Transporter ◽  
Polyclonal Antibodies ◽  
Culture Conditions ◽  
Intrinsic Activity ◽  
Western Blots ◽  
Glut 1 ◽  
Glut 4

The effect of culture conditions simulating hypo- and hyper-glycaemia on glucose transport and on the subcellular localization of the glucose transporter GLUT-1 was studied in L8 myocytes. Incubation of the cells with 20 mM-glucose for 25 h decreased the rate of 2-deoxy-D-[3H]glucose (dGlc) uptake to 0.106 +/- 0.016 nmol/min per 10(6) cells compared with 0.212 +/- 0.025 in cells maintained at 2 mM-glucose (final glucose concentrations at the end of the incubation period were 16-17 mM and 0.7-1.0 mM respectively). An additional 5 h incubation of these cells with medium containing the opposite glucose concentration (i.e. change from 17 mM to 1 mM and from 1 mM to 17 mM) increased the transport rate to 0.172 +/- 0.033 nmol/min per 10(6) cells in cultures initially conditioned at high glucose, and decreased the transport to 0.125 +/- 0.029 in those conditioned at low glucose. Plasma-membrane- and microsomal-membrane-enriched fractions were prepared from these cells for [3H]cytochalasin B (CB) binding and Western-blot analysis with antibodies against GLUT-1 and GLUT-4. A decrease in glucose concentration increased the number of D-glucose-displaceable CB-binding sites and GLUT-1 protein in the plasma-membrane fraction to the same extent as the increase in dGlc transport. Under downregulatory conditions, the lower dGlc-transport capacity could be accounted for by a decreased number of transporters in the plasma membrane of the cells. No apparent modification of the intrinsic activity of the glucose transporters was observed in up- or down-regulated cells. Under downregulatory conditions, the CB-binding data indicated a large increase in the number of transporters in the intracellular membranes of the myocytes. Western blots of the same membranes also indicated an increase in GLUT-1 content. However, the interaction of the intracellular GLUT-1 protein with the polyclonal antibodies was much weaker than that of the plasma-membrane-associated GLUT-1. The GLUT-4 concentration was too low to permit quantification in membrane fractions. Our findings suggest that autoregulation of glucose transport in L8 myocytes is accompanied by parallel changes in the number of GLUT-1 transporters in the plasma membrane, and that the rate of transporter degradation may be augmented in the upregulated myocytes. These glucose-induced changes are fully reversible.

scholarly journals Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.

1996 ◽  
Vol 7 (7) ◽  
pp. 1075-1082 ◽  
Author(s):  
A Volchuk ◽  
Q Wang ◽  
H S Ewart ◽  
Z Liu ◽  
L He ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Membrane Receptors ◽  
Low Density ◽  
Adipocyte Cell ◽  
Syntaxin 4 ◽  
Insulin Dependent ◽  
Plasma Membrane Receptors

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.

Exercise training increases GLUT-4 protein in rat adipose cells

1993 ◽  
Vol 264 (6) ◽  
pp. E882-E889 ◽  
Author(s):  
M. F. Hirshman ◽  
L. J. Goodyear ◽  
E. D. Horton ◽  
L. J. Wardzala ◽  
E. S. Horton
Keyword(s):  
Plasma Membrane ◽  
Exercise Training ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Low Density ◽  
Basal Cells ◽  
Glut 1 ◽  
Glut 4 ◽  
Cell Plasma ◽  
Adipose Cells

The relative abundance and subcellular distribution of the GLUT-1 and GLUT-4 glucose transporter isoforms were determined in basal and insulin-stimulated adipose cells from wheel cage exercise-trained rats and compared with both age-matched sedentary controls and young cell size-matched sedentary controls. Exercise training increased total estimated GLUT-4 by 67 and 54% compared with age-matched and young controls, respectively. Total estimated GLUT-1 per cell was not significantly different among the three groups. Expressed per cell, plasma membrane GLUT-4 protein in basal adipose cells from exercise-trained and age-matched control rats was 2.5-fold greater than in young controls (P < 0.05) and was associated with higher basal rates of glucose transport in these cells (P < 0.02). In insulin-stimulated cells, plasma membrane GLUT-4 was 67% greater in the exercise-trained animals than young controls (P < 0.01), and 31% greater than in age-matched controls. Rates of glucose transport were correspondingly higher. In basal cells, low-density microsomal GLUT-4 from exercise-trained rats was approximately twofold greater than from age-matched controls and young controls. With insulin stimulation, GLUT-4 in low-density microsomes decreased to similar levels in all groups. We conclude that the total amount of GLUT-4 protein, but not GLUT-1, is increased in adipose cells by exercise training and that this increase in GLUT-4 is due primarily to an increase in intracellular GLUT-4.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of endurance training on glucose transport capacity and glucose transporter expression in rat skeletal muscle

1990 ◽  
Vol 259 (6) ◽  
pp. E778-E786 ◽  
Author(s):  
T. Ploug ◽  
B. M. Stallknecht ◽  
O. Pedersen ◽  
B. B. Kahn ◽  
T. Ohkuwa ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Training Session ◽  
Residual Effect ◽  
Exercise Session ◽  
Transport Capacity ◽  
Glut 1 ◽  
Glut 4 ◽  
Fast Twitch

The effect of 10 wk endurance swim training on 3-O-methylglucose (3-MG) uptake (at 40 mM 3-MG) in skeletal muscle was studied in the perfused rat hindquarter. Training resulted in an increase of approximately 33% for maximum insulin-stimulated 3-MG transport in fast-twitch red fibers and an increase of approximately 33% for contraction-stimulated transport in slow-twitch red fibers compared with nonexercised sedentary muscle. A fully additive effect of insulin and contractions was observed both in trained and untrained muscle. Compared with transport in control rats subjected to an almost exhaustive single exercise session the day before experiment both maximum insulin- and contraction-stimulated transport rates were increased in all muscle types in trained rats. Accordingly, the increased glucose transport capacity in trained muscle was not due to a residual effect of the last training session. Half-times for reversal of contraction-induced glucose transport were similar in trained and untrained muscles. The concentrations of mRNA for GLUT-1 (the erythrocyte-brain-Hep G2 glucose transporter) and GLUT-4 (the adipocyte-muscle glucose transporter) were increased approximately twofold by training in fast-twitch red muscle fibers. In parallel to this, Western blot demonstrated a approximately 47% increase in GLUT-1 protein and a approximately 31% increase in GLUT-4 protein. This indicates that the increases in maximum velocity for 3-MG transport in trained muscle is due to an increased number of glucose transporters.

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