scholarly journals Insulin-dependent protein trafficking in skeletal muscle cells

1998 ◽  
Vol 275 (2) ◽  
pp. E187-E196 ◽  
Author(s):  
Min Zhou ◽  
Lidia Sevilla ◽  
Gino Vallega ◽  
Peng Chen ◽  
Manuel Palacin ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Protein Trafficking ◽  
Glucose Transporter ◽  
Simple Procedure ◽  
Sedimentation Coefficient ◽  
Intracellular Compartment ◽  
Glut 4 ◽  
Fractionation Procedure

We have established a simple procedure for the separation of intracellular pool(s) of glucose transporter isoform GLUT-4-containing vesicles from the surface sarcolemma and T tubule membranes of rat skeletal myocytes. This procedure enabled us to immunopurify intracellular GLUT-4-containing vesicles and to demonstrate that 20–30% of the receptors for insulin-like growth factor II/mannose 6-phosphate and transferrin are colocalized with GLUT-4 in the same vesicles. Using our new fractionation procedure as well as cell surface biotinylation, we have shown that these receptors are translocated from their intracellular compartment(s) to the cell surface along with GLUT-4 after insulin stimulation in vivo. Denervation causes a considerable downregulation of GLUT-4 protein in skeletal muscle but does not affect the level of expression of other known component proteins of the corresponding vesicles. Moreover, the sedimentation coefficient of these vesicles remains unchanged by denervation. We suggest that the normal level of GLUT-4 expression is not necessary for the structural organization and insulin-sensitive translocation of its cognate intracellular compartment.

Regulation of glucose transporter GLUT-4 and hexokinase II gene transcription by insulin and epinephrine

1997 ◽  
Vol 273 (4) ◽  
pp. E682-E687 ◽  
Author(s):  
Jared P. Jones ◽  
G. Lynis Dohm
Keyword(s):  
Skeletal Muscle ◽  
Gene Transcription ◽  
Glucose Transporter ◽  
Male Rats ◽  
Rat Skeletal Muscle ◽  
Hexokinase Ii ◽  
Epinephrine Infusion ◽  
Glut 4 ◽  
Gastrocnemius Muscles

Transport of glucose across the plasma membrane by GLUT-4 and subsequent phosphorylation of glucose by hexokinase II (HKII) constitute the first two steps of glucose utilization in skeletal muscle. This study was undertaken to determine whether epinephrine and/or insulin regulates in vivo GLUT-4 and HKII gene transcription in rat skeletal muscle. In the first experiment, adrenodemedullated male rats were fasted 24 h and killed in the control condition or after being infused for 1.5 h with epinephrine (30 μg/ml at 1.68 ml/h). In the second experiment, male rats were fasted 24 h and killed after being infused for 2.5 h at 1.68 ml/h with saline or glucose (625 mg/ml) or insulin (39.9 μg/ml) plus glucose (625 mg/ml). Nuclei were isolated from pooled quadriceps, tibialis anterior, and gastrocnemius muscles. Transcriptional run-on analysis indicated that epinephrine infusion decreased GLUT-4 and increased HKII transcription compared with fasted controls. Both glucose and insulin plus glucose infusion induced increases in GLUT-4 and HKII transcription of twofold and three- to fourfold, respectively, compared with saline-infused rats. In conclusion, epinephrine and insulin may regulate GLUT-4 and HKII genes at the level of transcription in rat skeletal muscle.

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 Fish Glucose Transporter (GLUT)-4 Differs from Rat GLUT4 in Its Traffic Characteristics but Can Translocate to the Cell Surface in Response to Insulin in Skeletal Muscle Cells

Endocrinology ◽  
2007 ◽  
Vol 148 (11) ◽  
pp. 5248-5257 ◽  
Author(s):  
Mònica Díaz ◽  
Costin N. Antonescu ◽  
Encarnación Capilla ◽  
Amira Klip ◽  
Josep V. Planas
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Uptake ◽  
Brown Trout ◽  
Glucose Transporter ◽  
Intracellular Localization ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Glut 4

In mammals, glucose transporter (GLUT)-4 plays an important role in glucose homeostasis mediating insulin action to increase glucose uptake in insulin-responsive tissues. In the basal state, GLUT4 is located in intracellular compartments and upon insulin stimulation is recruited to the plasma membrane, allowing glucose entry into the cell. Compared with mammals, fish are less efficient restoring plasma glucose after dietary or exogenous glucose administration. Recently our group cloned a GLUT4-homolog in skeletal muscle from brown trout (btGLUT4) that differs in protein motifs believed to be important for endocytosis and sorting of mammalian GLUT4. To study the traffic of btGLUT4, we generated a stable L6 muscle cell line overexpressing myc-tagged btGLUT4 (btGLUT4myc). Insulin stimulated btGLUT4myc recruitment to the cell surface, although to a lesser extent than rat-GLUT4myc, and enhanced glucose uptake. Interestingly, btGLUT4myc showed a higher steady-state level at the cell surface under basal conditions than rat-GLUT4myc due to a higher rate of recycling of btGLUT4myc and not to a slower endocytic rate, compared with rat-GLUT4myc. Furthermore, unlike rat-GLUT4myc, btGLUT4myc had a diffuse distribution throughout the cytoplasm of L6 myoblasts. In primary brown trout skeletal muscle cells, insulin also promoted the translocation of endogenous btGLUT4 to the plasma membrane and enhanced glucose transport. Moreover, btGLUT4 exhibited a diffuse intracellular localization in unstimulated trout myocytes. Our data suggest that btGLUT4 is subjected to a different intracellular traffic from rat-GLUT4 and may explain the relative glucose intolerance observed in fish.

scholarly journals Comparison of glucose-transporter-containing vesicles from rat fat and muscle tissues: evidence for a unique endosomal compartment

1995 ◽  
Vol 307 (2) ◽  
pp. 383-390 ◽  
Author(s):  
K V Kandror ◽  
L Coderre ◽  
A V Pushkin ◽  
P F Pilch
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Glucose Transporter ◽  
Simple Procedure ◽  
Protein Composition ◽  
Muscle Cells ◽  
Membrane Structures ◽  
Rat Adipocytes ◽  
Endosomal Compartment ◽  
Biochemical Comparison

Insulin-sensitive tissues (fat and muscle) express a specific isoform of glucose-transporter protein, GLUT4, which normally resides in intracellular vesicular structures and is translocated to the cell surface in response to insulin. Here we provide a biochemical comparison of GLUT4-containing structures from fat and muscle cells. We demonstrate that, in spite of totally different protocols for cell homogenization and fractionation used for adipocytes as compared with skeletal-muscle tissue, GLUT4-containing vesicles from both sources have identical buoyant densities, sedimentation coefficients, and a very similar, if not identical, protein composition. The individual proteins first identified in GLUT4-containing vesicles from adipocytes (GTV3/SCAMPs proteins and aminopeptidase gp160) are also present in the analogous vesicles from muscle. Intracellular microsomes from rat adipocytes also contain GLUT1, a ubiquitously expressed glucose-transporter isoform. GLUT1 has not been detected in intracellular vesicular pool(s) from skeletal-muscle cells, probably because of its low abundance there. GLUT1 in adipocytes is excluded from GLUT4-containing vesicles, but is found in membrane structures which are indistinguishable from the former by all methods tested and demonstrate the same type of regulation by insulin. That is, the GLUT1- and GLUT4-containing vesicles have identical densities and sedimentation coefficients in sucrose gradients, and translocate to the cell surface in response to hormonal exposure. Also, we describe a simple procedure for the purification of native glucose-transporter vesicles from rat adipocytes. Overall, our data suggest the existence of a unique endosomal compartment in fat and muscle cells which is functionally and compositionally different from other microsomal vesicles and which is responsible for insulin-sensitive glucose transport in these tissues.

Skeletal muscle glucose transporter (GLUT-4) protein is decreased in lactating goats

1997 ◽  
Vol 65 (2) ◽  
pp. 257-265 ◽  
Author(s):  
M. Balage ◽  
J. F. Hocquette ◽  
B. Graulet ◽  
P. Ferre ◽  
J. Grizard
Keyword(s):  
Skeletal Muscle ◽  
Protein Content ◽  
Skeletal Muscles ◽  
Glucose Transporter ◽  
Glucose Utilization ◽  
Glut 4 ◽  
Lactating Goats ◽  
Insulin Responsiveness ◽  
Triton X

AbstractLactation in goats is associated with an insulin resistance manifested by an impairment of the ability of insulin maximally to stimulate skeletal muscle glucose utilization. The mechanism responsible for this modification is unknown. Therefore an investigation was made of the insulin-sensitive glucose transporter (GLUT-4) in three skeletal muscles from six lactating (peak of lactation) and six non-lactating goats. GLUT-4 protein content was assessed in crude membrane preparations and Triton X-100 extracts by Western-blot analysis. Lactation resulted in a decrease in GLUT-4 protein content. This decrease was more pronounced in oxidoglycolytic muscles (proportionately -0·40 to -0·60 in m. tensor fasciae latae and longissimus dorsi) than in oxidative muscles (-0·20 in masseter). Down-regulation of the insulin-sensitive glucose transporter (GLUT-4) expression in skeletal muscles from lactating goats may be responsible for the decrease in insulin responsiveness of glucose utilization previously observed in vivo.

A new procedure for the isolation of plasma membranes, T tubules, and internal membranes from skeletal muscle

1996 ◽  
Vol 270 (4) ◽  
pp. E667-E676 ◽  
Author(s):  
L. Dombrowski ◽  
D. Roy ◽  
B. Marcotte ◽  
A. Marette
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Subcellular Fractionation ◽  
Intracellular Fraction ◽  
Transporter Protein ◽  
Glut 4 ◽  
T Tubules ◽  
Surface Domains

A new subcellular fractionation procedure for the simultaneous isolation of plasma membranes and transverse (T) tubule membranes from a rat skeletal muscle was developed. This new technique allows the isolation and separation of plasma membranes and T tubules in distinct subcellular fractions, as revealed by the membrane distribution of enzymatic and immunologic markers of both cell surface compartments. The procedure also yields a novel membrane fraction that is devoid of markers of both surface domains but is markedly enriched with GLUT-4 glucose transporters, thus strongly suggesting that it represents an intracellular pool of GLUT-4. Using this new procedure, we found that acute in vivo insulin administration (30 min) increased GLUT-4 protein content in the plasma membrane and a T tubule fraction (by approximately 80%), whereas a smaller elevation (35%) was observed in another fraction enriched with T tubules. Insulin induced a concomitant reduction (approximately 40%) in GLUT-4 abundance in the intracellular fraction. These results further support the hypothesis that T tubules are involved in the regulation of glucose transport in skeletal muscle. This novel fractionation method will be useful in investigating the regulation of muscle GLUT-4 transporters in other physiological and disease states such as diabetes, where defective translocation of the transporter protein to either one or both cell surface domains is suspected to occur.

Insulin-mediated translocation of GLUT-4-containing vesicles is preserved in denervated muscles

2000 ◽  
Vol 278 (6) ◽  
pp. E1019-E1026 ◽  
Author(s):  
Min Zhou ◽  
Gino Vallega ◽  
Konstantin V. Kandror ◽  
Paul F. Pilch
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Sedimentation Coefficient ◽  
Compensatory Mechanism ◽  
Muscle Denervation ◽  
Denervated Muscle ◽  
Glut 1 ◽  
Glut 4 ◽  
Cargo Proteins ◽  
Insulin Dependent

Skeletal muscle denervation decreases insulin-sensitive glucose uptake into this tissue as a result of marked GLUT-4 protein downregulation (∼20% of controls). The process of insulin-stimulated glucose transport in muscle requires the movement or translocation of intracellular GLUT-4-rich vesicles to the cell surface, and it is accompanied by the translocation of several additional vesicular cargo proteins. Thus examining GLUT-4 translocation in muscles from denervated animals allows us to determine whether the loss of a major cargo protein, GLUT-4, affects the insulin-dependent behavior of the remaining cargo proteins. We find no difference, control vs. denervated, in the insulin-dependent translocation of the insulin-responsive aminopeptidase (IRAP) and the receptors for transferrin and insulin-like growth factor II/mannose 6-phosphate, proteins that completely (IRAP) or partially co-localize with GLUT-4. We conclude that 1) denervation of skeletal muscle does not block the specific branch of insulin signaling pathway that connects receptor proximal events to intracellular GLUT-4-vesicles, and 2) normal levels of GLUT-4 protein are not necessary for the structural organization and insulin-sensitive translocation of its cognate intracellular compartment. Muscle denervation also causes a twofold increase in GLUT-1. In normal muscle, all GLUT-1 is present at the cell surface, but in denervated muscle a significant fraction (25.1 ± 6.1%) of this transporter is found in intracellular vesicles that have the same sedimentation coefficient as GLUT-4-containing vesicles but can be separated from the latter by immunoadsorption. These GLUT-1-containing vesicles respond to insulin and translocate to the cell surface. Thus the formation of insulin-sensitive GLUT-1-containing vesicles in denervated muscle may be a compensatory mechanism for the decreased level of GLUT-4.

Calorie restriction increases cell surface GLUT-4 in insulin-stimulated skeletal muscle

1998 ◽  
Vol 275 (6) ◽  
pp. E957-E964 ◽  
Author(s):  
David J. Dean ◽  
Joseph T. Brozinick ◽  
Samuel W. Cushman ◽  
Gregory D. Cartee
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Glucose Transport ◽  
Calorie Restriction ◽  
Glucose Transporter ◽  
Calorie Intake ◽  
Phosphatidylinositol 3 Kinase ◽  
Glut 4 ◽  
Pi3k Activity ◽  
Stimulation Of

Reduced calorie intake [calorie restriction (CR); 60% of ad libitum (AL)] leads to enhanced glucose transport without altering total GLUT-4 glucose transporter abundance in skeletal muscle. Therefore, we tested the hypothesis that CR (20 days) alters the subcellular distribution of GLUT-4. Cell surface GLUT-4 content was higher in insulin-stimulated epitrochlearis muscles from CR vs. AL rats. The magnitude of this increase was similar to the CR-induced increase in glucose transport, and GLUT-4 activity (glucose transport rate divided by cell surface GLUT-4) was unaffected by diet. The CR effect was specific to the insulin-mediated pathway, as evidenced by the observations that basal glucose transport and cell surface GLUT-4 content, as well as hypoxia-stimulated glucose transport, were unchanged by diet. CR did not alter insulin’s stimulation of insulin receptor substrate (IRS)-1-associated phosphatidylinositol 3-kinase (PI3K) activity. Muscle abundance of IRS-2 and p85 subunit of PI3K were unaltered by diet, but IRS-1 content was lower in CR vs. AL. These data demonstrate that, despite IRS-1-PI3K activity similar to AL, CR specifically increases insulin’s activation of glucose transport by enhancing the steady-state proportion of GLUT-4 residing on the cell surface.

Exercise training prevents maturation-induced decrease in insulin sensitivity

1996 ◽  
Vol 80 (6) ◽  
pp. 1963-1967 ◽  
Author(s):  
N. Nakai ◽  
Y. Shimomura ◽  
N. Ohsaki ◽  
J. Sato ◽  
Y. Oshida ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Insulin Sensitivity ◽  
Exercise Training ◽  
Infusion Rate ◽  
Glucose Transporter ◽  
Euglycemic Clamp ◽  
Glut 4 ◽  
Hindlimb Muscle ◽  
Female Wistar Rats ◽  
Sensitivity Improvement

We examined the effects of exercise training initiated before maturation or after maturation on insulin sensitivity and glucose transporter GLUT-4 content in membrane fractions of skeletal muscle. Female Wistar rats (4 wk of age) were divided into sedentary and exercise-trained groups. At 12 wk of age, a subset of the trained animals (Tr) was killed along with a subset of sedentary controls (Sed). One-half of the remaining sedentary animals remained sedentary (Sed-Sed) while the other half began exercise training (Sed-Tr). The remaining rats in the original trained group continued to train (Tr-Tr). Euglycemic clamp (insulin infusion rate at 6 mU.kg body wt-1. min-1) was performed at 4, 12, and 27 wk. After euglycemic clamp in all animals except the 4-wk-old, hindlimb (gastrocnemius and part of quadriceps) muscles were removed for preparation of membrane fractions. In sedentary rats, glucose infusion rate (GIR) during euglycemic clamp was decreased from 15.9 mg.kg-1.min-1 at 4 wk of age to 9.8 mg.kg-1.min-1 at 12 wk of age and 9.1 mg.kg-1.min-1 at 27 wk of age. In exercise-trained rats, the GIR was not significantly decreased by maturation (at 12 wk) and further aging (at 27 wk). Initiation of exercise after maturation restored the GIR at 27 wk of age to the same levels as these for the corresponding exercise-trained rats. GLUT-4 content in plasma and intracellular membrane fractions of hindlimb muscle obtained just after euglycemic clamp showed the same trend as the results of GIR. These results suggest that exercise training prevented the maturation-induced decrease in insulin sensitivity. Improvement of insulin sensitivity caused by exercise training was attributed, at least in part, to the increase in insulin-sensitive GLUT-4 on the plasma membrane in skeletal muscle.

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