scholarly journals Biochemical and functional characterization of the rat liver glucose-transport system Comparisons with the adipocyte glucose-transport system

1986 ◽  
Vol 240 (1) ◽  
pp. 115-123 ◽  
Author(s):  
T P Ciaraldi ◽  
R Horuk ◽  
S Matthaei
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Glucose Transport ◽  
Transport System ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Cell Types ◽  
Low Density ◽  
Lower Affinity ◽  
Glucose Transport System

The properties of the glucose-transport systems in rat adipocytes and hepatocytes were compared in cells prepared from the same animals. Hormones and other agents which cause a large stimulation of 3-O-methylglucose transport in adipocytes were without acute effect in hepatocytes. Hepatocytes displayed a lower affinity for 3-O-methylglucose (20 mM) and alternative substrates than adipocytes (6 mM), whereas inhibitor affinities were similar in both cell types. The concentration and distribution of glucose transporters were determined by Scatchard analysis of D-glucose-inhibitable [3H]cytochalasin B binding to subcellular fractions. In liver, most of the transporters were located in the plasma membrane (42 +/- 5 pmol/mg of protein) with a small amount (4 +/- 3 pmol/mg) in the low-density microsomal fraction (‘microsomes’), the reverse of the situation in adipocytes. Glucose transporters were covalently labelled with [3H]cytochalasin B by using the photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate and analysed by SDS/polyacrylamide-gel electrophoresis. A single D-glucose-inhibitable peak with a molecular mass of 40-50 kDa was seen in both plasma membrane and low-density microsomes. This peak was further characterized by isoelectric focusing and revealed a single peak of specific [3H]cytochalasin B binding at pI 6.05 in both low-density microsomes and plasma membrane, compared with peaks at pI 6.4 and 5.6 in adipocyte membranes. In summary: the glucose-transport system in hepatocytes has a lower affinity and higher capacity than that in adipocytes, and is also not accurately modulated by insulin; the subcellular distribution of glucose transporters in the liver suggests that few intracellular transporters would be available for translocation; the liver transporter has a molecular mass similar to that of the adipocyte transporter; the liver glucose transporter exists as a single charged form (pI 6.05), compared with the multiple forms in adipocytes. This difference in charge could reflect a functionally important difference in molecular structure between the two cell types.

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.

scholarly journals RCO-3 and COL-26 form an external-to-internal module that regulates the dual-affinity glucose transport system in Neurospora crassa

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Jinyang Li ◽  
Qian Liu ◽  
Jingen Li ◽  
Liangcai Lin ◽  
Xiaolin Li ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neurospora Crassa ◽  
Glucose Transport ◽  
Transport System ◽  
Rational Design ◽  
Glucose Sensor ◽  
Glucose Transporters ◽  
Glucose Fluctuation ◽  
High Affinity ◽  
Glucose Transport System

Abstract Background Low- and high-affinity glucose transport system is a conserved strategy of microorganism to cope with environmental glucose fluctuation for their growth and competitiveness. In Neurospora crassa, the dual-affinity glucose transport system consists of a low-affinity glucose transporter GLT-1 and two high-affinity glucose transporters HGT-1/HGT-2, which play diverse roles in glucose transport, carbon metabolism, and cellulase expression regulation. However, the regulation of this dual-transporter system in response to environmental glucose fluctuation is not yet clear. Results In this study, we report that a regulation module consisting of a downstream transcription factor COL-26 and an upstream non-transporting glucose sensor RCO-3 regulates the dual-affinity glucose transport system in N. crassa. COL-26 directly binds to the promoter regions of glt-1, hgt-1, and hgt-2, whereas RCO-3 is an upstream factor of the module whose deletion mutant resembles the Δcol-26 mutant phenotypically. Transcriptional profiling analysis revealed that Δcol-26 and Δrco-3 mutants had similar transcriptional profiles, and both mutants had impaired response to a glucose gradient. We also showed that the AMP-activated protein kinase (AMPK) complex is involved in regulation of the glucose transporters. AMPK is required for repression of glt-1 expression in starvation conditions by inhibiting the activity of RCO-3. Conclusions RCO-3 and COL-26 form an external-to-internal module that regulates the glucose dual-affinity transport system. Transcription factor COL-26 was identified as the key regulator. AMPK was also involved in the regulation of the dual-transporter system. Our findings provide novel insight into the molecular basis of glucose uptake and signaling in filamentous fungi, which may aid in the rational design of fungal strains for industrial purposes.

Potential mechanism of insulin resistance in ageing: impaired insulin-stimulated glucose transport due to a depletion of the intracellular pool of glucose transporters in Fischer rat adipocytes

1990 ◽  
Vol 126 (1) ◽  
pp. 99-107 ◽  
Author(s):  
S. Matthaei ◽  
H. Benecke ◽  
H. H. Klein ◽  
A. Hamann ◽  
G. Kreymann ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Insulin Binding ◽  
Low Density ◽  
Intracellular Pool ◽  
Subcellular Membrane ◽  
Impaired Insulin ◽  
In Cells

ABSTRACT To examine the cellular mechanism responsible for impaired insulin action in ageing, we determined various in-vitro parameters involved in the pathogenesis of insulin resistance, i.e. basal and insulin-stimulated [14C]3-O-methylglucose transport (30MG), 125I-labelled insulin binding, activation of insulin receptor kinase (IRKA) in intact cells, and number and subcellular distribution of glucose transporters in subcellular membrane fractions of adipocytes from 6- (FR-6) and 24- (FR-24) month-old Fischer rats. Ageing had no effect on basal 30MG (12±4 vs 13±3 fmol/5 × 104 cells, means ± s.e.m.); in contrast, in FR-24 rats insulin-stimulated 30MG was markedly decreased by 43% when compared with that in FR-6 rats (158±14 vs 90±8 fmol/5 × 104 cells; P < 0·01). Insulin binding to adipocytes from FR-6 rats was 2·40±0·38% compared with 2·28±0·47% in FR-24 (P not significant). Moreover, ageing had no significant effect on IRKA, as determined by insulin-stimulated (0, 1, 4 and 500 ng insulin/ml) 32P-incorporation into histone 2B. In subcellular membrane fractions, low density microsomes and plasma membranes, glucose transporter numbers were determined using [3H]cytochalasin B binding and immunodetection using an antiserum against the C-terminal peptide of the hepatoma-G2-glucose transporter. Cytochalasin B binding revealed that in the basal state the intracellular pool of glucose transporters was depleted in FR-24 by about 39% compared with low density microsomes from FR-6: (48·6±7·2 vs 29·8±5·5 pmol/mg membrane protein; P < 0·01). In consequence, in FR-24 there were fewer glucose transporters available for insulin-induced translocation to the plasma membrane (insulin-treated plasma membrane: 23·9±4·2 (FR-6) vs 14·4±3·1 (FR-24) pmol/mg membrane protein; P < 0·01). These results were confirmed by immunoblotting. In conclusion, (1) maximal insulin-stimulated 30MG was decreased by 43% in cells from FR-24 rats compared with those from FR-6 rats, while basal 30MG was similar in both groups, (2) neither insulin binding nor IRKA were significantly altered in cells from FR-24 rats, and (3) impaired insulin-stimulated 30MG was associated with reduced numbers of glucose transporters in the plasma membrane as a consequence of a depletion of the intracellular pool of glucose transporters in cells from FR-24 rats. Journal of Endocrinology (1990) 126, 99–107

scholarly journals RCO-3 and COL-26 form an External-to-Internal Module that Regulates the Dual-Affinity Glucose Transport System in Neurospora Crassa

2020 ◽  
Author(s):  
Jinyang Li ◽  
Qian Liu ◽  
Jingen Li ◽  
Liangcai Lin ◽  
Xiaolin Li ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neurospora Crassa ◽  
Glucose Transport ◽  
Transport System ◽  
Rational Design ◽  
Glucose Sensor ◽  
Glucose Transporters ◽  
Glucose Fluctuation ◽  
High Affinity ◽  
Glucose Transport System

Abstract Background Low- and high-affinity glucose transport system is a conserved strategy of microorganism to cope with environmental glucose fluctuation for their growth and competitiveness. In Neurospora crassa, the dual-affinity glucose transport system consists of a low-affinity glucose transporter GLT-1 and two high-affinity glucose transporters HGT-1/HGT-2, which play diverse roles in glucose transport, carbon metabolism, and cellulase expression regulation. However, the regulation of this dual-transporter system in response to environmental glucose fluctuation is not yet clear.Results In this study, we report that a regulation module consisting of a downstream transcription factor COL-26 and an upstream non-transporting glucose sensor RCO-3 regulates the dual-affinity glucose transport system in N. crassa. COL-26 directly binds to the promoter regions of glt-1, hgt-1, and hgt-2, whereas RCO-3 is an upstream factor of the module whose deletion mutant resembles the Δcol-26 mutant phenotypically. Transcriptional profiling analysis revealed that Δcol-26 and Δrco-3 mutants had similar transcriptional profiles, and both mutants had impaired response to a glucose gradient. We also showed that the AMP-activated protein kinase (AMPK) complex is involved in regulation of the glucose transporters. AMPK is required for repression of glt-1 expression in starvation conditions by inhibiting the activity of RCO-3.Conclusions RCO-3 and COL-26 form an external-to-internal module that regulates the glucose dual-affinity transport system. Transcription factor COL-26 was identified as the key regulator. AMPK was also involved in the regulation of the dual transporter system. Our findings provide novel insight into the molecular basis of glucose uptake and signaling in filamentous fungi, which may aid in the rational design of fungal strains for industrial purposes.

scholarly journals In vitro analysis of the glucose-transport system in GLUT4-null skeletal muscle

1999 ◽  
Vol 342 (2) ◽  
pp. 321-328 ◽  
Author(s):  
Jeffrey W. RYDER ◽  
Yuichi KAWANO ◽  
Alexander V. CHIBALIN ◽  
Jorge RINCÓN ◽  
Tsu-Shuen TSAO ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transport ◽  
Transport System ◽  
Soleus Muscle ◽  
Cytochalasin B ◽  
Transport Activity ◽  
Wild Type ◽  
Glucose Transport System ◽  
Vitro Analysis

We have characterized the glucose-transport system in soleus muscle from female GLUT4-null mice to determine whether GLUT1, 3 or 5 account for insulin-stimulated glucose-transport activity. Insulin increased 2-deoxyglucose uptake 2.8- and 2.1-fold in soleus muscle from wild-type and GLUT4-null mice, respectively. Cytochalasin B, an inhibitor of GLUT1- and GLUT4-mediated glucose transport, inhibited insulin-stimulated 2-deoxyglucose uptake by > 95% in wild-type and GLUT4-null soleus muscle. Addition of 35 mM fructose to the incubation media was without effect on insulin-stimulated 3-O-methylglucose transport activity in soleus muscle from either genotype, whereas 35 mM glucose inhibited insulin-stimulated (20 nM) 3-O-methylglucose transport by 65% in wild-type and 99% in GLUT4-null mice. We utilized the 2-N-4-1-(1-azi-2,2,2-t r i f l u o r o e t h y l ) b e n z o y l - 1, 3 - b i s (D - m a n n o s e - 4 - y l o x y ) - 2 - p ro p y lamine (ATB-BMPA) exofacial photolabel to determine if increased cell-surface GLUT1 or GLUT4 content accounted for insulin-stimulated glucose transport in GLUT4-null muscle. In wild-type soleus muscle, cell-surface GLUT4 content was increased by 2.8-fold under insulin-stimulated conditions and this increase corresponded to the increase in 2-deoxyglucose uptake. No detectable cell-surface GLUT4 was observed in soleus muscle from female GLUT4-null mice under either basal or insulin-stimulated conditions. Basal cell-surface GLUT1 content was similar between wild-type and GLUT4-null mice, with no further increase noted in either genotype with insulin exposure. Neither GLUT3 nor GLUT5 appeared to account for insulin-stimulated glucose-transport activity in wild-type or GLUT4-null muscle. In conclusion, insulin-stimulated glucose-transport activity in female GLUT4-null soleus muscle is mediated by a facilitative transport process that is glucose- and cytochalasin B-inhibitable, but which is not labelled strongly by ATB-BMPA.

Exercise training and the glucose transport system in obese SHHF/Mcc-fa cprats

1996 ◽  
Vol 81 (4) ◽  
pp. 1670-1676 ◽  
Author(s):  
Cynthia M. Ferrara ◽  
W. Michael Sherman ◽  
Nicole Leenders ◽  
Sylvia A. McCune ◽  
Karla Roehrig
Keyword(s):  
Plasma Membrane ◽  
Exercise Training ◽  
Glucose Transport ◽  
Transport System ◽  
Glucose Transporter ◽  
Citrate Synthase ◽  
Training Stimulus ◽  
Peak Oxygen Uptake ◽  
Glucose Transport System ◽  
Obese Male

Ferrara, Cynthia M., W. Michael Sherman, Nicole Leenders, Sylvia A. McCune, and Karla Roehrig. Exercise training and the glucose transport system in obese SHHF/ Mcc-fa cprats. J. Appl. Physiol. 81(4): 1670–1676, 1996.—The effects of a similar exercise training stimulus on maximal insulin-stimulated (MIS) plasma membrane glucose transporter number and glucose transport were determined in lean and obese SHHF/ Mcc-fa cprats. Six-week-old lean and obese male rats were randomly divided into four groups: lean sedentary (LSed), obese sedentary (OSed), lean exercise (LEx), and obese exercise (OEx). An 8- to 12-wk treadmill running program equalized daily muscular work for LEx and OEx. Plasma membranes were isolated from control and MIS muscles of mixed fiber types. MIS significantly increased glucose transport (3.4- and 2.8-fold) in LSed and OSed, respectively. MIS significantly increased glucose transporter number (2.5-fold) in LSed, but there was no increase in glucose transporter number in OSed. Peak oxygen uptake and citrate synthase activity were increased a similar amount for LEx and OEx groups, demonstrating a similar training stimulus. MIS significantly and similarly increased glucose transport in LEx and OEx (4.4- and 5.1-fold, respectively). The effects of MIS on plasma membrane glucose transporter number in the exercise-trained rats were similar to the responses observed in the sedentary lean and obese groups. MIS significantly increased glucose transporter number (2.6-fold) in LEx, whereas there was no increase in glucose transporter number in OEx. The reduction in MIS glucose transport in OSed appears to be related to a defect in the processes associated with the translocation of glucose transporters to the plasma membrane. Exercise training of the obese rats apparently did not alter this defect. Similar increases in peak oxygen uptake, citrate synthase, and MIS glucose transport in LEx and OEx groups suggest that insulin resistance does not limit the ability of the glucose transport system to adapt to exercise training in the obese male SHHF/ Mcc-fa cprats.

scholarly journals Glucose transporters in isolated chromaffin cells. Effects of insulin and secretagogues

1987 ◽  
Vol 243 (2) ◽  
pp. 541-547 ◽  
Author(s):  
E G Delicado ◽  
M T Miras Portugal
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Adrenal Medulla ◽  
Chromaffin Cells ◽  
Cytochalasin B ◽  
Insulin Receptors ◽  
Neural Tissue ◽  
Glucose Transporters ◽  
High Affinity ◽  
Bovine Adrenal Medulla

1. Isolated chromaffin cells from bovine adrenal medulla were used to study glucose transport in a homogeneous neural tissue. 2. The affinity of glucose transporters was 1.20 +/- 0.52 mM by the infinite-cis technique and 1.02 +/- 0.09 mM by the direct transport experiments. 3. The affinity for 2-deoxyglucose of these transporters was 2.3 mM. 4. The glucose transporters, quantified by [3H]cytochalasin B binding, were 419,532 +/- 120,740 receptors/cell, which corresponds to about 7.2 +/- 2 pmol/mg of protein, with KD = 0.1 microM. 5. High-affinity insulin receptors with KD = 3.95 nM were present at a density of 68,400 +/- 7500 per cell. 6. Insulin and secretagogues increased glucose transport, raising the transporter number at the plasma membrane without changes in the affinity.

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