scholarly journals Effects of insulin on glucose transport and glucose transporters in rat heart

1988 ◽  
Vol 250 (1) ◽  
pp. 277-283 ◽  
Author(s):  
D Zaninetti ◽  
R Greco-Perotto ◽  
F Assimacopoulos-Jeannet ◽  
B Jeanrenaud
Keyword(s):  
Glucose Transport ◽  
Rat Heart ◽  
Plasma Membranes ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Hill Coefficient ◽  
Maximal Velocity ◽  
Microsomal Membranes ◽  
The Hill ◽  
Intracellular Glucose

The effect of insulin on glucose transport and glucose transporters was studied in perfused rat heart. Glucose transport was measured by the efflux of labelled 3-O-methylglucose from hearts preloaded with this hexose. Insulin stimulated 3-O-methylglucose transport by: (a) doubling the maximal velocity (Vmax); (b) decreasing the Kd from 6.9 to 2.7 mM; (c) increasing the Hill coefficient toward 3-O-methylglucose from 1.9 to 3.1; (d) increasing the efficiency of the transport process (k constant). Glucose transporters in enriched plasma and microsomal membranes from heart were quantified by the [3H]cytochalasin-B-binding assay. When added to normal hearts, insulin produced the following changes in the glucose transporters: (a) it increased the translocation of transporters from an intracellular pool to the plasma membranes; (b) it increased (from 1.6 to 2.7) the Hill coefficient of the transporters translocated into the plasma membranes toward cytochalasin B, suggesting the existence of a positive co-operativity among the transporters appearing in these membranes; (c) it increased the affinity of the transporters (and hence, possibly, of glucose) for cytochalasin B. The data provide evidence that the stimulatory effect of insulin on glucose transport may be due not to the sole translocation of intracellular glucose transporters to the plasma membrane, but to changes in the functional properties thereof.

scholarly journals Insulin modifies the properties of glucose transporters in rat brown adipose tissue

1987 ◽  
Vol 247 (1) ◽  
pp. 63-68 ◽  
Author(s):  
R Greco-Perotto ◽  
F Assimacopoulos-Jeannet ◽  
B Jeanrenaud
Keyword(s):  
Adipose Tissue ◽  
Plasma Membrane ◽  
Brown Adipose Tissue ◽  
Cytochalasin B ◽  
Fold Increase ◽  
Glucose Transporters ◽  
Hill Coefficient ◽  
Brown Adipose ◽  
Microsomal Membranes ◽  
The Hill

The properties of glucose transporters associated with plasma and microsomal membranes have been studied in brown adipose tissue of rats after treatment by saline infusion or hyperinsulinaemic/euglycaemic clamp. In this tissue, insulin produces a 40-fold increase in glucose utilization as measured by the 2-deoxy-D-glucose technique, and therefore a 40-fold increase in the rate-limiting glucose transport. This increase, promoted by insulin, is associated with: (a) translocation of the transporters from a pool associated with the microsomal fraction to the plasma membrane without modification of the total number of transporters; (b) an increase in the Hill coefficient of the plasma-membrane glucose transporters for cytochalasin B from 1.1 to 2.5, indicating the presence of positive co-operativity; (c) a decrease in the Kd (apparent dissociation constant) of the transporters towards cytochalasin B from 148 to 82 nM; (d) no change in the Hill coefficient or Kd for the transporters associated with the microsomal membranes. These data indicate that, in addition to causing translocation of the glucose transporters, insulin modifies their properties and behaviour towards cytochalasin B. This may reflect modifications in their properties and behaviour towards glucose, and by this contribute to bringing about the marked effect of this hormone on glucose transport in brown adipose tissue.

scholarly journals Cycloheximide decreases glucose transporters in rat adipocyte plasma membranes without affecting insulin-stimulated glucose transport

1988 ◽  
Vol 251 (2) ◽  
pp. 491-497 ◽  
Author(s):  
S Matthaei ◽  
J M Olefsky ◽  
E Karnieli
Keyword(s):  
Protein Synthesis ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Electrophoretic Analysis ◽  
Adipocyte Plasma Membranes ◽  
Inhibitor Of Protein Synthesis ◽  
Adipose Cells

This study examines the relationship between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters in isolated rat adipocytes. Adipose cells were incubated with or without cycloheximide, a potent inhibitor of protein synthesis, for 60 min and then for an additional 30 min with or without insulin. After the incubation we measured 3-O-methylglucose transport in the adipose cells, and subcellular membrane fractions were prepared. The numbers of glucose transporters in the various membrane fractions were determined by the cytochalasin B binding assay. Basal and insulin-stimulated 3-O-methylglucose uptakes were not affected by cycloheximide. Furthermore, cycloheximide affected neither Vmax. nor Km of insulin-stimulated 3-O-methylglucose transport. In contrast, the number of glucose transporters in plasma membranes derived from cells preincubated with cycloheximide and insulin was markedly decreased compared with those from cells incubated with insulin alone (10.5 +/- 0.8 and 22.2 +/- 1.8 pmol/mg of protein respectively; P less than 0.005). The number of glucose transporters in cells incubated with cycloheximide alone was not significantly different compared with control cells. SDS/polyacrylamide-gel-electrophoretic analysis of [3H]cytochalasin-B-photolabelled plasma-membrane fractions revealed that cycloheximide decreases the amount of labelled glucose transporters in insulin-stimulated membranes. However, the apparent molecular mass of the protein was not changed by cycloheximide treatment. The effect of cycloheximide on the two-dimensional electrophoretic profile of the glucose transporter in insulin-stimulated low-density microsomal membranes revealed a decrease in the pI-6.4 glucose-transporter isoform, whereas the insulin-translocatable isoform (pI 5.6) was decreased. Thus the observed discrepancy between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters strongly suggests that a still unknown protein-synthesis-dependent mechanism is involved in insulin activation of glucose transport.

scholarly journals Role of facilitative glucose transporters in diffusional water permeability through J774 cells.

1993 ◽  
Vol 102 (5) ◽  
pp. 897-906 ◽  
Author(s):  
J D Loike ◽  
L Cao ◽  
K Kuang ◽  
J C Vera ◽  
S C Silverstein ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Water Permeability ◽  
Plasma Membranes ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Osmotic Gradient ◽  
Water Permeation ◽  
J774 Cells ◽  
First Time ◽  
Water Passage

We have reported previously that in the presence of an osmotic gradient, facilitative glucose transporters (GLUTs) act as a transmembrane pathway for water flow. Here, we find evidence that they also allow water passage in the absence of an osmotic gradient. We applied the linear diffusion technique to measure the diffusional permeability (Pd) of tritiated water (3H-H2O) through plasma membranes of J774 murine macrophage-like cells. Untreated cells had a Pd of 30.9 +/- 1.8 microns/s; the inhibitors of facilitative glucose transport cytochalasin B (10 microM) and phloretin (20 microM) reduced that value to 15.3 +/- 1.8 (50%) and 11.0 +/- 0.7 (62%) microns/s, respectively. In contrast, no significant effect on Pd was observed in cells treated with dihydrocytochalasin B (Pd = 28.4 +/- 1.5 microns/s). PCMBS (3 mM) inhibited glucose uptake by greater than 95%, and 3H-H2O diffusion by approximately 30% (Pd = 22.9 +/- 1.5 microns/s). The combination of cytochalasin B plus pCMBS reduced Pd by about 87% (Pd = 3.9 +/- 0.3 microns/s). Moreover, 1 mM pCMBS did not affect the osmotic water permeability in Xenopus laevis oocytes expressing the brain/erythroid form of facilitative glucose transporters (GLUT1). These results indicate for the first time that about half of the total Pd of J774 cells may be accounted for by water passage across GLUTs. Hence, they highlight the multifunctional properties of these transporters serving as conduits for both water and glucose. Our results also suggest for the first time that pCMBS blocks glucose transport without affecting water permeation through GLUTs. Lastly, because pCMBS decreases the Pd of J774 cells, this suggests the presence in their plasma membranes of another protein(s) exhibiting water channel properties.

Seasonal changes in plasma membrane glucose transporters enhance cryoprotectant distribution in the freeze-tolerant wood frog

1995 ◽  
Vol 73 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Patricia A. King ◽  
Mary N. Rosholt ◽  
Kenneth B. Storey
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Glucose Transport ◽  
Seasonal Changes ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Wood Frog ◽  
Maximal Velocity ◽  
Liver Membranes

One of the critical adaptations for freeze tolerance by the wood frog, Rana sylvatica, is the production of large quantities of glucose as an organ cryoprotectant during freezing exposures. Glucose export from the liver, where it is synthesized, and its uptake by other organs is dependent upon carrier-mediated transport across plasma membranes by glucose-transporter proteins. Seasonal changes in the capacity to transport glucose across plasma membranes were assessed in liver and skeletal muscle of wood frogs; summer-collected (June) frogs were compared with autumn-collected (September) cold-acclimated (5 °C for 3–4 weeks) frogs. Plasma membrane vesicles prepared from liver of autumn-collected frogs showed 6-fold higher rates of carrier-mediated glucose transport than vesicles from summer-collected frogs, maximal velocity (Vmax) values for transport being 72 ± 14 and 12.0 + 2.9 nmol∙mg protein−1∙s−1, respectively (at 10 °C). However, substrate affinity constants for carrier-mediated glucose transport (K1/2) did not change seasonally. The difference in transport rates was due to greater numbers of glucose transporters in liver plasma membranes from autumn-collected frogs. The total number of transporter sites, as determined by cytochalasin B binding, was 8.5-fold higher in autumn than in summer. Glucose transporters in wood frog liver membranes cross-reacted with antibodies to the rat GluT-2 glucose transporter (the mammalian liver isoform), and Western blots further confirmed a large increase in transporter numbers in liver membranes from autumn- versus summer-collected frogs. By contrast with the liver, however, there were no seasonal changes in glucose-transporter activity or numbers in plasma membranes isolated from skeletal muscle. We conclude that an enhanced capacity for glucose transport across liver, but not muscle, plasma membranes during autumn cold-hardening is an important adaptation that anticipates the need for rapid export of cryoprotectant from liver during natural freezing episodes.

Effects of ventromedial hypothalamic stimulation on glucose transport system in rat tissues

1992 ◽  
Vol 263 (6) ◽  
pp. R1228-R1234
Author(s):  
A. Takahashi ◽  
M. Sudo ◽  
Y. Minokoshi ◽  
T. Shimazu
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Insulin Treatment ◽  
Sympathetic Innervation ◽  
Glucose Transporters ◽  
Rat Tissues ◽  
Brown Adipose ◽  
Microsomal Membranes

Electrical stimulation of the ventromedial hypothalamus (VMH) increased the rate constant of glucose uptake in rat heart and brown adipose tissue (BAT), as measured in vivo by the 2-deoxy-D-[3H]glucose method. The increase in glucose uptake in BAT was abolished by local sympathetic denervation. To analyze the mechanism of this hypothalamic modulation, the effects of VMH stimulation and insulin treatment on the number and dissociation constant (Kd) of glucose transporters in the plasma and microsomal membranes were examined by means of [3H]cytochalasin B binding. VMH stimulation did not alter either the number or Kd value of glucose transporters in plasma and microsomal membranes prepared from heart and BAT, whereas insulin treatment increased the number of glucose transporters in the plasma membranes and decreased those in the microsomal membranes. D-Glucose transport activity was also measured with the same plasma membrane vesicles. An apparent functional activity of transporters was detected to be increased in the heart and BAT plasma membranes after VMH stimulation but not after insulin treatment. These results suggest that VMH stimulation enhances glucose utilization in heart and BAT via sympathetic innervation and that the mechanism by which VMH stimulation increases tissue glucose uptake is different from that of insulin, possibly causing an activation of glucose transporters present in the plasma membrane.

Effect of insulin and glucocorticoids on glucose transporters in rat adipocytes

1987 ◽  
Vol 252 (4) ◽  
pp. E441-E453 ◽  
Author(s):  
C. Carter-Su ◽  
K. Okamoto
Keyword(s):  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Insulin Binding ◽  
Maximal Response ◽  
Transport Activity ◽  
Rat Adipocytes ◽  
Dose Dependent Fashion

The ability of glucocorticoids to modify the effect of insulin on glucose transport was investigated in both intact isolated rat adipocytes and in membranes isolated from hormone-treated adipocytes. In intact adipocytes, dexamethasone, a potent synthetic glucocorticoid, inhibited insulin-stimulated 3-O-methylglucose transport at all concentrations of insulin tested (0-2,000 microU/ml). Insulin sensitivity, as well as the maximal response to insulin, was decreased by dexamethasone in the absence of a change in insulin binding. The inhibition was observed regardless of which hormone acted first, was blocked by actinomycin D, and resulted from a decrease in Vmax rather than an increase in Kt of transport. In plasma membranes isolated from insulin-treated adipocytes, glucose transport activity and the amount of glucose transporter covalently labeled with [3H]cytochalasin B were increased in parallel in a dose-dependent fashion. The amount of labeled transporter in a low-density microsomal fraction (LDMF) was decreased in a reciprocal fashion. In contrast, addition of dexamethasone to insulin-stimulated cells caused decreases in both transport activity and amount of labeled transporter in the plasma membranes. This was accompanied by a small increase in the amount of [3H]cytochalasin B incorporated into the glucose transporter in the LDMF. These results are consistent with both insulin and glucocorticoids altering the distribution of glucose transporters between the plasma membrane and LDMF, in opposite directions.

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

1992 ◽  
Vol 284 (2) ◽  
pp. 341-348 ◽  
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.

Glucose transport by isolated plasma membranes of the bovine blood-brain barrier

1997 ◽  
Vol 272 (5) ◽  
pp. C1552-C1557 ◽  
Author(s):  
W. J. Lee ◽  
D. R. Peterson ◽  
E. J. Sukowski ◽  
R. A. Hawkins
Keyword(s):  
Glucose Transport ◽  
Blood Brain Barrier ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Extracellular Fluid ◽  
Brain Barrier ◽  
Maximal Velocity ◽  
Plasma Membrane Vesicles ◽  
Cerebral Microvessels ◽  
Blood Brain

Luminal and abluminal endothelial plasma membrane vesicles were isolated from bovine cerebral microvessels, the site of the blood-brain barrier. Glucose transport across each membrane was measured using a rapid-filtration technique. Glucose transport into luminal vesicles occurred by a stereospecific energy-independent transporter [Michaelis-Menten constant (K(m)) = 10.3 +/- 2.8 (SE) mM and maximal velocity (Vmax) = 8.6 +/- 2.0 nmol.mg protein(-1).min-1]. Kinetic analysis of abluminal vesicles also showed a transport system with characteristics similar to the luminal transporter (K(m) = 12.5 +/- 2.3 mM and Vmax = 10.0 +/- 1.0 nmol.mg protein-1.min-1). These functional, facilitative glucose transporters were symmetrically distributed between the luminal and abluminal membrane domains, providing a mechanism for glucose movement between blood and brain. The studies also revealed a Na-dependent transporter on the abluminal membrane with a higher affinity and lower capacity than the facilitative transporters (K(m) = 130 +/- 20 microM and Vmax = 1.59 +/- 0.44 nmol.mg protein-1.min-1. The abluminal Na-dependent glucose transporter is in a position to transport glucose from the brain extracellular fluid into the endothelial cells of the blood-brain barrier. The functional significance of its presence there remains to be determined.

SERCA1a can functionally substitute for SERCA2a in the heart

1999 ◽  
Vol 276 (1) ◽  
pp. H89-H97 ◽  
Author(s):  
Yong Ji ◽  
Evgeny Loukianov ◽  
Tanya Loukianova ◽  
Larry R. Jones ◽  
Muthu Periasamy
Keyword(s):  
Turnover Rate ◽  
Ph Dependence ◽  
Fold Increase ◽  
Enzymatic Properties ◽  
Hill Coefficient ◽  
Maximal Velocity ◽  
Apparent Affinity ◽  
Serca Pump ◽  
Fast Twitch ◽  
The Hill

We recently generated a transgenic (TG) mouse model in which the fast-twitch skeletal muscle sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1a) is overexpressed in the heart. Ectopic overexpression of SERCA1a results in remodeling of the cardiac SR containing 80% SERCA1a and 20% endogenous SERCA2a with an ∼2.5-fold increase in the total amount of SERCA protein (E. Loukianov et al. Circ. Res. 83: 889–897, 1998). We have analyzed the Ca2+ transport properties of membranes from SERCA1a TG hearts in comparison to control hearts. Our data show that the maximal velocity of SR Ca2+ transport was significantly increased (∼1.9-fold) in TG hearts, whereas the apparent affinity of the SERCA pump for Ca2+ was not changed. Addition of phospholamban antibody in the Ca2+ uptake assays increased the apparent affinity for Ca2+ to the same extent in TG and non-TG (NTG) hearts, suggesting that phospholamban regulates the SERCA1a pump in TG hearts. Analysis of SERCA enzymatic properties in TG hearts revealed that the SERCA pump affinity for ATP, the Hill coefficient, the pH dependence of Ca2+ uptake, and the effect of acidic pH on Ca2+ transport were similar to those of NTG hearts. Interestingly, the rate constant of phosphoenzyme decay (turnover rate of SERCA enzyme) was also very similar between TG and NTG hearts. Together these findings suggest that 1) the SERCA1a pump can functionally substitute for SERCA2a and is regulated by endogenous phospholamban in the heart and 2) SERCA1a exhibits several enzymatic properties similar to those of SERCA2a when expressed in a cardiac setting.

Discrepancy between glucose transport and transporters in human femoral adipocytes

1989 ◽  
Vol 256 (1) ◽  
pp. E179-E185 ◽  
Author(s):  
E. Karnieli ◽  
R. Moscona ◽  
R. Rafaeloff ◽  
Y. G. Illouz ◽  
M. Armoni
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Intact Cell ◽  
Transport Activity ◽  
Intact Cells ◽  
Insulin Resistant ◽  
Large Size ◽  
Microsomal Membranes ◽  
Subcutaneous Adipose

Obesity is known to be associated with insulin resistance in human and rat adipocytes. However, it is not known what are the perturbations in insulin action that contribute to disproportional femoral obesity. Thus femoral subcutaneous adipose tissue was obtained from lean women with various degrees of disproportional obesity, by liposuction. 3-O-methylglucose (3-O-methyl-D-glucopyranose) transport was measured in intact cells, and glucose transporter levels in plasma and low-density microsomal membranes were assessed using the cytochalasin B binding assay. A sixfold cellular enlargement was associated with increase in both basal and insulin-stimulated glucose transport activity in the intact cell, and a 300-600% increase in insulin stimulating effect per se. However, when glucose transporter levels were assessed, this cellular enlargement was accompanied by a 40-70% transporter depletion (in largest cells compared with smallest ones) in both subcellular fractions examined, from either basal or insulin-stimulated cells. This discrepancy, between increasing cellular glucose transport rates and relative depletion of transporter levels, suggests that these cells are not insulin resistant, as could be expected from their large size. A role for other factor(s), additional to glucose transporter levels, in the regulation of cellular glucose uptake rate is thus suggested.

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