Kinetics of glucose transport in rat skeletal muscle membrane vesicles: effects of insulin and contractions

To study the mechanism of acceleration of glucose transport in skeletal muscle after stimulation with insulin and contractions, we isolated a subcellular vesicular membrane fraction, highly enriched in the plasma membrane enzyme K(+)-stimulated p-nitrophenylphosphatase and also enriched in some intracellular membranes. Protein recovery, morphology, lipid content, marker enzyme activities, total intravesicular volume, Western blot quantitation of GLUT-1, and glucose-inhibitable cytochalasin B binding were identical in membrane fractions from control, insulin-stimulated, contraction-stimulated, and insulin- and contraction-stimulated muscle. Time course of D-[3H]glucose entry in membrane vesicles at equilibrium exchange conditions showed that initial rate of transport at 30 mM of glucose was increased 19-fold and that equilibrium distribution space was increased 4-fold in vesicles from maximum stimulated muscle. The effects of insulin and contractions on initial rate of transport as well as on equilibrium distribution space were additive, and stimulation increased the substrate saturability of glucose transport. Furthermore, cytochalasin B binding to membranes prepared by using less centrifugation time than usual showed that, after stimulation with insulin and contractions, at least 35% of the total number of glucose transporters were redistributed from one kind of vesicles to a more slowly sedimenting kind of vesicles, probably reflecting translocation within the membrane preparation from intracellular vesicles to the plasma membrane upon stimulation. In the present membrane preparation the effects of insulin and/or contractions on glucose transport resemble those seen in intact muscle, and the effects are thus not dependent on cellular integrity.(ABSTRACT TRUNCATED AT 250 WORDS)

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Glucose transport in skeletal muscle membrane vesicles from control and exercised rats

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.6.c1128 ◽

1989 ◽

Vol 257 (6) ◽

pp. C1128-C1134 ◽

Cited By ~ 52

Author(s):

P. A. King ◽

M. F. Hirshman ◽

E. D. Horton ◽

E. S. Horton

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Glucose Uptake ◽

Glucose Transport ◽

Active Form ◽

Membrane Vesicles ◽

Intrinsic Activity ◽

Muscle Membrane ◽

Affinity Constant ◽

Turnover Number

Skeletal muscle responds to exercise by increasing the rate of glucose uptake. Recent studies have indicated that these changes occur via mechanisms modulating the number of transporters in the plasma membrane and/or transporter intrinsic activity. In the present study, a protocol was developed for measuring the initial rate of glucose uptake by rat hindlimb skeletal muscle plasma membrane vesicles. Membranes were isolated from sedentary (control) and acutely exercised rats, and the initial rates of D- and L-glucose influx were assayed under equilibrium exchange conditions to obtain the kinetic constants for carrier-mediated transport. These values were compared with the values for transporter number measured by cytochalasin B binding, and the carrier turnover numbers were calculated. The maximum velocity (Vmax) for carrier-mediated glucose influx was increased 3.7-fold by exercise, from 3.5 nmol.mg protein-1.s-1 for the membranes from control rats to 13 nmol.mg protein-1.s-1 for the membranes from exercised animals. The mean affinity constant (K0.5; approximately 20 mM) was not different between the two groups. The number of transporters in the plasma membrane was increased to a lesser degree, 5.4 to 9.4 pmol/mg protein. As a result, the average carrier turnover number was increased almost twofold by exercise, 719 s-1 in the controls vs. 1,380 s-1 in the exercised rats. These data indicate that the response of glucose transport to exercise involves an increase in the average carrier intrinsic activity as well as a recruitment of transporters to the plasma membrane. Whether the increase in carrier turnover number is due to activation of the transporters or recruitment of a more “active” form of the carrier is unknown.

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Stimulation of glucose transport in skeletal muscle by hypoxia

Journal of Applied Physiology ◽

10.1152/jappl.1991.70.4.1593 ◽

1991 ◽

Vol 70 (4) ◽

pp. 1593-1600 ◽

Cited By ~ 203

Author(s):

G. D. Cartee ◽

A. G. Douen ◽

T. Ramlal ◽

A. Klip ◽

J. O. Holloszy

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Muscle Contraction ◽

Glucose Transport ◽

Glucose Transporter ◽

Cytochalasin B ◽

Sugar Transport ◽

Transport Activity ◽

Hindlimb Muscles ◽

Stimulation Of

Hypoxia caused a progressive cytochalasin B-inhibitable increase in the rate of 3-O-methylglucose transport in rat epitrochlearis muscles to a level approximately six-fold above basal. Muscle ATP concentration was well maintained during hypoxia, and increased glucose transport activity was still present after 15 min of reoxygenation despite repletion of phosphocreatine. However, the increase in glucose transport activity completely reversed during a 180-min-long recovery in oxygenated medium. In perfused rat hindlimb muscles, hypoxia caused an increase in glucose transporters in the plasma membrane, suggesting that glucose transporter translocation plays a role in the stimulation of glucose transport by hypoxia. The maximal effects of hypoxia and insulin on glucose transport activity were additive, whereas the effects of exercise and hypoxia were not, providing evidence suggesting that hypoxia and exercise stimulate glucose transport by the same mechanism. Caffeine, at a concentration too low to cause muscle contraction or an increase in glucose transport by itself, markedly potentiated the effect of a submaximal hypoxic stimulus on sugar transport. Dantrolene significantly inhibited the hypoxia-induced increase in 3-O-methylglucose transport. These effects of caffeine and dantrolene suggest that Ca2+ plays a role in the stimulation of glucose transport by hypoxia.

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Chronic growth hormone treatment in normal rats reduces post-prandial skeletal muscle plasma membrane GLUT1 content, but not glucose transport or GLUT4 expression and localization

Biochemical Journal ◽

10.1042/bj3150959 ◽

1996 ◽

Vol 315 (3) ◽

pp. 959-963 ◽

Cited By ~ 13

Author(s):

Raffaele NAPOLI ◽

Antonio CITTADINI ◽

Jesse C. CHOW ◽

Michael F. HIRSHMAN ◽

Robert J. SMITH ◽

...

Keyword(s):

Skeletal Muscle ◽

Growth Hormone ◽

Plasma Membrane ◽

Glucose Transport ◽

Glucose Transporter ◽

Growth Hormone Treatment ◽

Membrane Vesicles ◽

Hormone Treatment ◽

Plasma Membrane Vesicles ◽

Muscle Glucose Transport

Whether skeletal muscle glucose transport system is impaired in the basal, post-prandial state during chronic growth hormone treatment is unknown. The current study was designed to determine whether 4 weeks of human growth hormone (hGH) treatment (3.5 mg/kg per day) would impair glucose transport and/or the number of glucose transporters in plasma membrane vesicles isolated from hindlimb skeletal muscle of Sprague–Dawley rats under basal, post-prandial conditions. hGH treatment was shown to have no effect on glucose influx (Vmax or Km) determined under equilibrium exchange conditions in isolated plasma membrane vesicles. Plasma membrane glucose transporter number (Ro) measured by cytochalasin B binding was also unchanged by hGH treatment. Consequently, glucose transporter turnover number (Vmax/Ro), a measure of average glucose transporter intrinsic activity, was similar in hGH-treated and control rats. hGH did not change GLUT4 protein content in whole muscle or in the plasma membrane, and muscle content of GLUT4 mRNA also was unchanged. In contrast, GLUT1 protein content in the plasma membrane fraction was significantly reduced by hGH treatment. This was associated with a modest, although not significant, decrease in muscle content of GLUT1 mRNA. In conclusion, high-dose hGH treatment for 4 weeks did not alter post-prandial skeletal muscle glucose transport activity. Neither the muscle level nor the intracellular localization of GLUT4 was changed by the hormone treatment. On the contrary, the basal post-prandial level of GLUT1 in the plasma membrane was reduced by hGH. The mRNA data suggest that this reduction might result from a decrease in the synthesis of GLUT1.

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Glycaemia regulates the glucose transporter number in the plasma membrane of rat skeletal muscle

Biochemical Journal ◽

10.1042/bj2840341 ◽

1992 ◽

Vol 284 (2) ◽

pp. 341-348 ◽

Cited By ~ 45

Author(s):

D Dimitrakoudis ◽

T Ramlal ◽

S Rastogi ◽

M Vranic ◽

A Klip

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Glucose Transport ◽

Transport System ◽

Plasma Membranes ◽

Glucose Transporter ◽

Cytochalasin B ◽

Glucose Transporters ◽

Mrna Levels ◽

Rat Skeletal Muscle

The number of glucose transporters was measured in isolated membranes from diabetic-rat skeletal muscle to determine the role of circulating blood glucose levels in the control of glucose uptake into skeletal muscle. Three experimental groups of animals were investigated in the post-absorptive state: normoglycaemic/normoinsulinaemic, hyperglycaemic/normoinsulinaemic and hyperglycaemic/normoinsulinaemic made normoglycaemic/normoinsulinaemic by phlorizin treatment. Hyperglycaemia caused a reversible decrease in total transporter number, as measured by cytochalasin B binding, in both plasma membranes and internal membranes of skeletal muscle. Changes in GLUT4 glucose transporter protein mirrored changes in cytochalasin B binding in plasma membranes. However, there was no recovery of GLUT4 levels in intracellular membranes with correction of glycaemia. GLUT4 mRNA levels decreased with hyperglycaemia and recovered only partially with correction of glycaemia. Conversely, GLUT1 glucose transporters were only detectable in the plasma membranes; the levels of this protein varied directly with glycaemia, i.e. in the opposite direction to GLUT4 glucose transporters. This study demonstrates that hyperglycaemia, in the absence of hypoinsulinaemia, is capable of down-regulating the glucose transport system in skeletal muscle, the major site of peripheral resistance to insulin-stimulated glucose transport in diabetes. Furthermore, correction of hyperglycaemia causes a complete restoration of the transport system in the basal state (determined by the transporter number in the plasma membrane), but possibly only an incomplete recovery of the transport system's ability to respond to insulin (since there is no recovery of GLUT4 levels in the intracellular membrane insulin-responsive transporter pool). Finally, the effect of hyperglycaemia is specific for glucose transporter isoforms, with GLUT1 and GLUT4 proteins varying respectively in parallel and opposite directions to levels of glycaemia.

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Expression and cellular localization of glucose transporters (GLUT1, GLUT3, GLUT4) during differentiation of myogenic cells isolated from rat foetuses

Journal of Cell Science ◽

10.1242/jcs.107.3.487 ◽

1994 ◽

Vol 107 (3) ◽

pp. 487-496 ◽

Cited By ~ 3

Author(s):

I. Guillet-Deniau ◽

A. Leturque ◽

J. Girard

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Glucose Transport ◽

Cell Fusion ◽

Cellular Localization ◽

Glucose Transporter ◽

Glucose Transporters ◽

Short Exposure ◽

Myoblast Differentiation ◽

Igf I

Skeletal muscle regeneration is mediated by the proliferation of myoblasts from stem cells located beneath the basal lamina of myofibres, the muscle satellite cells. They are functionally indistinguishable from embryonic myoblasts. The myogenic process includes the fusion of myoblasts into multinucleated myotubes, the biosynthesis of proteins specific for skeletal muscle and proteins that regulates glucose metabolism, the glucose transporters. We find that three isoforms of glucose transporter are expressed during foetal myoblast differentiation: GLUT1, GLUT3 and GLUT4; their relative expression being dependent upon the stage of differentiation of the cells. GLUT1 mRNA and protein were abundant only in myoblasts from 19-day-old rat foetuses or from adult muscles. GLUT3 mRNA and protein, detectable in both cell types, increased markedly during cell fusion, but decreased in contracting myotubes. GLUT4 mRNA and protein were not expressed in myoblasts. They appeared only in spontaneously contracting myotubes cultured on an extracellular matrix. Insulin or IGF-I had no effect on the expression of the three glucose transporter isoforms, even in the absence of glucose. The rate of glucose transport, assessed using 2-[3H]deoxyglucose, was 2-fold higher in myotubes than in myoblasts. Glucose deprivation increased the basal rate of glucose transport by 2-fold in myoblasts, and 4-fold in myotubes. The cellular localization of the glucose transporters was directly examined by immunofluorescence staining. GLUT1 was located on the plasma membrane of myoblasts and myotubes. GLUT3 was located intracellularly in myoblasts and appeared also on the plasma membrane in myotubes. Insulin or IGF-I were unable to target GLUT3 to the plasma membrane. GLUT4, the insulin-regulatable glucose transporter isoform, appeared only in contracting myotubes in small intracellular vesicles. It was translocated to the plasma membrane after a short exposure to insulin, as it is in skeletal muscle in vivo. These results show that there is a switch in glucose transporter isoform expression during myogenic differentiation, dependent upon the energy required by the different stages of the process. GLUT3 seemed to play a role during cell fusion, and could be a marker for the muscle's ability to regenerate.

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Membrane domain localization of pH-regulating transporters in frog skeletal muscle membrane vesicles

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.4.c1367 ◽

1996 ◽

Vol 271 (4) ◽

pp. C1367-C1379 ◽

Cited By ~ 2

Author(s):

R. W. Putnam ◽

P. B. Douglas ◽

N. A. Ritucci

Keyword(s):

Skeletal Muscle ◽

Atpase Activity ◽

Surface Membrane ◽

Membrane Vesicles ◽

Muscle Membrane ◽

Frog Skeletal Muscle ◽

Membrane Domain ◽

Ph Response ◽

Kinase C ◽

Inside Out

The distribution of pH-regulating transporters in surface and transverse (T) tubular membrane (TTM) domains of frog skeletal muscle was studied. 2',7'-Bis(carboxyethyl)-5(6)- carboxyfluorescein-loaded giant sarcolemmal vesicles, containing surface membrane, exhibited reversible Na+/H+ exchange. A microsomal vesicle fraction was shown to be enriched in TTM on the basis of high Na(+)-K(+)-ATPase and Mg(2+)-ATPase activity, high ouabain and nitrendipine binding, and low Ca(2+)-ATPase activity. TTM vesicles were well sealed and oriented inside out. Vesicles were loaded with the pH-sensitive dye pyranine. In response to an inwardly directed Na+ gradient, vesicles displayed virtually no alkalinization unless monensin was present. No pH response to an imposed Na+ gradient was seen regardless of the direction of the pH gradient across the vesicles, after phosphorylation of the vesicles with protein kinase C, or when exposed to guanosine 5'-O-(3-thiotriphosphate). In the presence of CO2, addition of Na+ or Cl- had no effect on vesicle pH. These data indicate that the TTM lacks functional pH-regulating transporters [Na+/H+ and (Na+ + HCO3-)/Cl- exchangers], suggesting that pH-regulating transporters are localized only to the surface membrane domain in frog muscle.

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Effects of phenylarsine oxide on stimulation of glucose transport in rat skeletal muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1990.258.4.c648 ◽

1990 ◽

Vol 258 (4) ◽

pp. C648-C653 ◽

Cited By ~ 32

Author(s):

E. J. Henriksen ◽

J. O. Holloszy

Keyword(s):

Skeletal Muscle ◽

Glucose Transport ◽

Cytochalasin B ◽

Contractile Activity ◽

Transport Activity ◽

Rat Skeletal Muscle ◽

Phenylarsine Oxide ◽

Muscle Glucose Transport ◽

Glucose Analogue ◽

Stimulation Of

The trivalent arsenical phenylarsine oxide (PAO) inhibits insulin-stimulated glucose transport in adipocytes and skeletal muscle through direct interactions with vicinal sulfhydryls. In muscle, glucose transport is also activated by contractile activity and hypoxia. It was therefore the purpose of the present study to investigate whether vicinal sulfhydryls are involved in the stimulation of glucose transport activity in the isolated rat epitrochlearis muscle by hypoxia or contractions. PAO (greater than 5 microM) caused a twofold increase in rate of transport of the nonmetabolizable glucose analogue 3-O-methylglucose (3-MG) that was completely prevented by cytochalasin B, the vicinal dithiol dimercaptopropanol, dantrolene, or 9-aminoacridine, both inhibitors of sarcoplasmic reticulum Ca2+ release, or omission of extracellular Ca2+. Although PAO treatment (greater than or equal to 20 microM) prevented approximately 80% of the increase in 3-MG transport caused by insulin, it resulted in only a approximately 50% inhibition of the stimulation of 3-MG transport by either hypoxia or contractile activity. PAO treatment (40 microM) of muscles already maximally stimulated by insulin, contractile activity, or hypoxia did not reverse the enhanced rate of 3-MG transport. These data suggest that vicinal sulfhydryls play a greater role in the activation of glucose transport by insulin than by muscle contractions or hypoxia. The finding that PAO inhibits the stimulation of glucose transport, but does not affect glucose transport after it has been stimulated, provides evidence that vicinal sulfhydryls are involved in the pathways for glucose transport activation in muscle, but not in the glucose transport mechanism itself.

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Na+-independent D-glucose transport in rabbit renal basolateral membranes

AJP Renal Physiology ◽

10.1152/ajprenal.1988.254.5.f711 ◽

1988 ◽

Vol 254 (5) ◽

pp. F711-F718 ◽

Cited By ~ 3

Author(s):

P. T. Cheung ◽

M. R. Hammerman

Keyword(s):

Glucose Uptake ◽

Glucose Transport ◽

Brush Border ◽

Glucose Transporter ◽

Cytochalasin B ◽

Basolateral Membrane ◽

Membrane Vesicles ◽

Rabbit Kidney ◽

Basolateral Membrane Vesicles ◽

Proximal Tubular

To define the mechanism by which glucose is transported across the basolateral membrane of the renal proximal tubular cell, we measured D-[14C]glucose uptake in basolateral membrane vesicles from rabbit kidney. Na+-dependent D-glucose transport, demonstrable in brush-border vesicles, could not be demonstrated in basolateral membrane vesicles. In the absence of Na+, the uptake of D-[14C]glucose in basolateral vesicles was more rapid than that of L-[3H]glucose over a concentration range of 1-50 mM. Subtraction of the latter from the former uptakes revealed a saturable process with apparent Km of 9.9 mM and Vmax of 0.80 nmol.mg protein-1.s-1. To characterize the transport component of D-glucose uptake in basolateral vesicles, we measured trans stimulation of 2 mM D-[14C]glucose entry in the absence of Na+. Trans stimulation could be effected by preloading basolateral vesicles with D-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose, but not with L-glucose or alpha-methyl-D-glucoside. Trans-stimulated D-[14C]glucose uptake was inhibited by 0.1 mM phloretin or cytochalasin B but not phlorizin. In contrast, Na+-dependent D-[14C]glucose transport in brush-border vesicles was inhibited by phlorizin but not phloretin or cytochalasin B. Our findings are consistent with the presence of a Na+-independent D-glucose transporter in the proximal tubular basolateral membrane with characteristics similar to those of transporters present in nonepithelial cells.

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