Acute and chronic stress-induced oxidative gastrointestinal mucosal injury in rats and protection by bismuth subsalicylate

To determine the role of translocation vs. activation of Glut1 in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation, we measured the abundance of myc-tagged Glut1 in plasma membrane of stably transfected Clone 9 cells, a rat liver cell line expressing only the Glut1 isoform. The myc epitope-tag is located between Ile56 and Pro57 in the putative first extracellular loop of Glut1. Under basal conditions, transfected cells expressed ~3 fold higher levels of Glut1 and exhibited a ~3 fold higher rate of glucose transport than non-transfected cells. To delineate the mechanism mediating the stimulation of glucose transport by a azide we employed two strategies: (1) mild cell surface biotinylation followed by isolation of plasma membranes and quantitation of Glut1 sites in Western blots employing anti-Glut1 and anti-myc antibodies, and (2) quantitative immunofluorescence of myc epitopes in plasma membrane sheets. The rate of glucose transport increased 2.9 ± 0.5 fold in transfected cells exposed to 5 mM azide for 1 h. Exposure to azide, however, resulted in no significant increase in Glut1 content of plasma membranes using anti-Glut1 or anti-myc antibodies in Western blots (1.0 ± 0.1 and 0.9 ± 0.2 fold, respectively; azide/control), and was associated with no detectable increase in immunofluorescence using either anti-Glut1 or anti-myc antibodies (p > 0.1 for both measurements). Treatment of cells with cobalt chloride (employed as a positive control) resulted in marked increases in glucose transport, cell and plasma membrane Glut1 content, and immunofluorescence of plasma membrane sheets (8-10 fold increase in each parameter). We conclude that the stimulation of glucose transport by azide results mainly from activation of Glut1 transporters pre-existing in the plasma membrane.     

Differential accumulation of Glut1 in the non-DRM domain of the plasma membrane in response to the inhibition of oxidative phosphorylation

Approximately 50% of Glut1 in the plasma membrane of Clone 9 cells is localized to the detergent-resistant membrane (DRM) fraction. Acute exposure (90 min) to 5mM azide stimulated glucose transport by approximately 4.7-fold and increased the abundance of Glut1 in the non-DRM fraction of the plasma membrane by approximately 2.9-fold while the abundance of Glut1 in the DRMs was not changed. In parallel experiments, approximately 17 h exposure to azide further increased the rate of glucose transport over that observed at 90 min by approximately 33% and increased plasma membrane Glut1 content by approximately 3.5-fold over control. The increase in total plasma membrane Glut1 reflected a approximately 4.7-fold increase of Glut1 content in the non-DRM fraction and a approximately 2.6-fold increase in the DRMs. We conclude that acute exposure to azide increases Glut1 content in the non-DRM fractions, while prolonged exposure to azide increases the Glut1 content in both non-DRM and DRM fractions. These changes may play an important role in the stimulation of glucose transport in response to the inhibition of oxidative phosphorylation.     

Distribution of Glut1 in detergent-resistant membranes (DRMs) and non-DRM domains: effect of treatment with azide

We have previously shown that the acute stimulation of glucose transport in Clone 9 cells in response to azide is mediated by activation of Glut1 and that stomatin, a Glut1-binding protein, appears to inhibit Glut1 function. In Clone 9 cells under basal conditions, 38% of Glut1, 70% of stomatin, and the bulk of caveolin-1 was localized in the detergent-resistant membrane (DRM) fraction; a significant fraction of Glut1 is also present in DRMs of 3T3-L1 fibroblasts and human red blood cells (RBCs). Acute exposure to azide resulted in 40 and 50% decreases in the content of Glut1 in DRMs of Clone 9 cells and 3T3-L1 fibroblasts, respectively, whereas the distribution of stomatin and caveolin-1 in Clone 9 cells remained unchanged. In addition, treatment of Clone 9 cells with azide resulted in a 50% decrease in the content of Glut1 in the DRM fraction of plasma membranes. We conclude that 1) a significant fraction of Glut1 is localized in DRMs, and 2) treatment of cells with azide results in a partial redistribution of Glut1 out of the DRM fraction. stomatin; caveolin-1; transferrin receptor; sucrose density fractionation; lipid raft     

Stimulation of AMP-activated protein kinase (AMPK) is associated with enhancement of Glut1-mediated glucose transport

In cells expressing only the Glut1 isoform of glucose transporters, we have shown that glucose transport is markedly stimulated in response to hypoxia or inhibition of oxidative phosphorylation, conditions that would be expected to cause a stimulation of AMP-activated protein kinase (AMPK) activity. In the present study we tested the hypothesis that the stimulation of AMPK activity might be accompanied by an enhancement of Glut1-mediated glucose transport. Exposure of Clone 9 cells, 3T3-L1 preadipocytes, and C(2)C(12) myoblasts (cells that express only the Glut1 isoform) to 5-aminoimidazole-4-carboxamideribonucleoside (AICAR), an adenosine analog that stimulates AMPK activity, resulted in a marked increase in the rate of glucose transport (ranging from four- to sixfold) that was accompanied by activation of AMPK. This stimulation of AMPK activity was associated with an increase in the phosphorylation of threonine 172 on the activation loop of its alpha subunit, with the predominant change being in the alpha-2 isoform. Exposure of Clone 9 cells to 5-iodotubercidin, an inhibitor of adenosine kinase, abolished the accumulation of AICAR-5'-monophosphate (ZMP), stimulation of AMPK, and the enhancement of glucose transport in response to AICAR. There was no significant increase in the content of Glut1 in plasma membranes of Clone 9 cells exposed to AICAR. We conclude that stimulation of AMPK activity is associated with enhancement of Glut1-mediated glucose transport, and that the glucose transport response is mediated by activation of Glut1 transporters preexisting in the plasma membrane.     

Stimulation of glucose transport in response to activation of distinct AMPK signaling pathways

AMP-activated protein kinase (AMPK) plays a critical role in the stimulation of glucose transport in response to hypoxia and inhibition of oxidative phosphorylation. In the present study, we examined the signaling pathway(s) mediating the glucose transport response following activation of AMPK. Using mouse fibroblasts of AMPK wild type and AMPK knockout, we documented that the expression of AMPK is essential for the glucose transport response to both azide and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). In Clone 9 cells, the stimulation of glucose transport by a combination of azide and AICAR was not additive, whereas there was an additive increase in the abundance of phosphorylated AMPK (p-AMPK). In Clone 9 cells, AMPK wild-type fibroblasts, and H9c2 heart cells, azide or hypoxia selectively increased p-ERK1/2, whereas, in contrast, AICAR selectively stimulated p-p38; phosphorylation of JNK was unaffected. Azide's effect on p-ERK1/2 abundance and glucose transport in Clone 9 cells was partially abolished by the MEK1/2 inhibitor U0126. SB 203580, an inhibitor of p38, prevented the phosphorylation of p38 and the glucose transport response to AICAR and, unexpectedly, to azide. Hypoxia, azide, and AICAR all led to increased phosphorylation of Akt substrate of 160 kDa (AS160) in Clone 9 cells. Employing small interference RNA directed against AS160 did not inhibit the glucose transport response to azide or AICAR, whereas the content of P-AS160 was reduced by approximately 80%. Finally, we found no evidence for coimmunoprecipitation of Glut1 and p-AS160. We conclude that although azide, hypoxia, and AICAR all activate AMPK, the downstream signaling pathways are distinct, with azide and hypoxia stimulating ERK1/2 and AICAR stimulating the p38 pathway.     

Enhancement of Glucose transport in clone 9 cells by exposure to alkaline pH: Studies on potential mechanisms

Summary Incubation of a nontransformed rat liver cell line. Clone 9, at pH 8.5 resulted in an 16-fold stimulation of cytochalasin B-inhibitable 3-O-methylglucose (3-OMG) transport, an effect that was independent of the presence of serum. Exposure to 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated 3-OMG uptake, and the enhancement was not additive to that produced by incubation at pH 8.5. In cells depleted of protein kinase C activity by a 20-hr exposure to TPA, however, the stimulation of 3-OMG transport in response to incubation at alkaline pH was still fully demonstrable. In control and alkaline pH-exposed cells, the inhibition of 3-OMG uptake by cytochalasin B was consistent with a single-site ligand binding model (K ₁10^–7 m). Northern blot analysis demonstrated the presence of only the human erythrocyte/rat brain/HepG2 cell glucose transporter-mRNA isoform (EGT), and the abundance of this mRNA was unchanged following exposure to alkaline pH. Immunoblot analysis, using polyclonal antibodies directed against the carboxy-terminal dodecapeptide of EGT, demonstrated and 2.0-fold increase in the abundance of transporters in partially purified plasma membrane fractions following incubation at pH 8.5, while EGT abundance was unchanged in whole-cell extracts. It is concluded that the stimulation of glucose transport in response to incubation of Clone 9 cells at alkaline pH does not require the presence of serum or activation of protein kinase C, and that the response is at least in part mediated by an increase in the number of glucose transporters in the plasma membrane.     

Monosaccharide uptake in common carp (Cyprinus carpio) EPC cells is mediated by a facilitative glucose carrier

In most animal cells, transport of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUT). Mammals express at least five distinct transporters (GLUTs 1--5), which are well characterised both functionally and genetically. In contrast, the glucose transport system of fish remains poorly studied. Here we report studies of hexose uptake in carp EPC cells and cloning of a glucose transporter cDNA from these cells. Transport of radio-labelled methylglucose (3-OMG) followed Michaelis--Menten kinetics with a K(m) value (8.5 mM) similar to that of mammalian cells. The inhibition of transport by cytochalasin B and phloretin, but not by phloridzin or cyanide, strongly suggested the existence of a facilitative carrier. D-Glucose, 2-deoxyglucose, 3-OMG, D-mannose and D-xylose were competitive inhibitors of 3-OMG uptake, while L-glucose, mannitol, D-fructose, D-ribose and sucrose did not compete with 3-OMG. We cloned a carp glucose transporter (CyiGLUT1), using RT-PCR and RACE strategies. CyiGLUT1 was different from known carp and zebrafish EST sequences. The complete cDNA (3060 bp) contained one open reading frame encoding a predicted protein of 478 amino acids. The deduced amino acid sequence shared 78% identity with mammalian and avian GLUT1 proteins. Key amino acids involved in substrate selection and catalysis of mammalian GLUTs were conserved in the carp transporter.     

Glucose transport system in a facultative iron-oxidizing bacterium, Thiobacillus ferrooxidans

Properties of a heat-labile glucose transport system in Thiobacillus ferrooxidans strain AP-44 were investigated with iron-grown cells. [14C]glucose was incorporated into cell fractions, and the cells metabolized [14C]glucose to 14CO2. Amytal, rotenone, cyanide, azide, 2,4-dinitrophenol, and dicyclohexylcarbodiimide strongly inhibited [14C]glucose uptake activity, suggesting the presence of an energy-dependent glucose transport system in T. ferrooxidans. Heavy metals, such as mercury, silver, uranium, and molybdate, markedly inhibited the transport activity at 1 mM. When grown on mixotrophic medium, the bacteria preferentially utilized ferrous iron as an energy source. When iron was exhausted, the cells used glucose if the concentration of ferrous sulfate in the medium was higher than 3% (wt/vol). However, when ferrous sulfate was lower than 1%, both of the energy sources were consumed simultaneously.     

Non-insulin dependent diabetic patients have increased glucose uptake in red blood cells

The kinetic characteristics of 3-O-methyl glucose (3-OMG) uptake were examined in red blood cells (RBC) from seven normal individuals (controls) and nine patients with non-insulin-dependent diabetes mellitus (NIDDM) treated with diet and oral hypoglycemic medication. Comparison of rates of 3-OMG uptake at 5 different substrate concentrations revealed significantly higher overall 3-OMG uptake in the diabetic group (P less than 0.0001). Kinetic parameters obtained for individual subjects showed there was not a significant difference in the Km between the diabetic (3.17 +/- 0.45 mM; mean +/- SE) and the control (2.46 +/- 0.25 mM) groups. However, Vmax was significantly increased (61%; P less than 0.025) in the diabetics (217.8 +/- 28.9 pmol/2 sec per 10(6) cells) compared to controls (135.2 +/- 15.6 pmol/2 sec per 10(6) cells). There was no correlation between HbA1C levels in the diabetic patients and Vmax values for 3-OMG uptake, suggesting that the increased hexose uptake was not accounted for simply by increased glycosylation in these cells. Glucose transport in RBC in hyperglycemic states may be a useful model for delineating the regulation of the non-insulin-mediated disposal of glucose in diabetes.     

Functional studies of threonine 310 mutations in Glut1: T310I is pathogenic, causing Glut1 deficiency

We have previously reported on a patient with the Glut1 deficiency syndrome (Online Mendelian Inheritance in Man number 606777) carrying a heterozygous T310I missense mutation in the GLUT1 gene (Klepper, J., Wang, D., Fischbarg, J., Vera, J. C., Jarjour, I. T., O'Driscoll, K. R., and De Vivo, D. C. (1999) Neurochem. Res. 24, 587-594). To investigate the molecular basis for the associated functional deficit, we constructed T310A, T310S, and T310I human GLUT1 mutants for expression in Xenopus laevis oocytes via cRNA injection. For all mutants, glucose transport was decreased, and osmotic water permeability (Pf) was increased. Km values for 3-O-methylglucose (3-OMG) uptake under zero-trans influx and equilibrium exchange influx conditions were, respectively, 13 +/- 1 and 68 +/- 5 mm for wild-type Glut1, 5 +/- 1 and 25 +/- 6 mm for T310A, 6 +/- 3 and 30 +/- 6 mm for T310I, and 5 +/- 1 and 48 +/- 5 mm for T310S. Compared with wild-type Glut1, we determined the following. (a). Zero-trans and equilibrium exchange influx values of 3-OMG were significantly decreased, respectively, 15 and 5% in T310A, 8 and 3% in T310I, and 40 and 34% in T310S mutants. (b). Zero-trans efflux of 3-OMG and dehydroascorbic acid uptake were significantly decreased in mutants. (c). The relative Pf values for T310A, T310I, and T310S were increased 3-, 4.8-, and 3.5-fold compared with wild-type values. We found a very high negative correlation between the rate of glucose uptake and Pf (-0.93), and between hydropathy and uptake (-0.92), a moderate correlation between hydropathy and Pf (0.73), and a minimal correlation between uptake, Pf, and molecular weight. These findings are consistent with a central role for hydropathy rather than size at position 310 of this mutation.     

Association of stomatin (band 7.2b) with Glut1 glucose transporter

Employing a monoclonal antibody directed against the C-terminal peptide of glucose transporter molecule 1 (Glut1), we identified a approximately 30-kDa polypeptide which coimmunoprecipitated with Glut1 from sample of human red blood cells (RBC) membranes. The approximately 30-kDa polypeptide reacted with an antibody directed against stomatin, an integral plasma membrane protein which is also present at a high abundance in the human RBC plasma membrane. Likewise, employing anti-stomatin antibody, we found that Glut1 coimmunoprecipitated with stomatin from samples of RBC membranes. However, neither band 3, which is the most abundant integral membrane protein in the RBC, nor actin coimmunoprecipitated with Glut1, indicating a specific interaction between Glut1 and stomatin. Similar to the results obtained in the RBC, Glut1 and stomatin immunoprecipitated with each other in lysates of Clone 9 cells, a rat liver cell line in which Glut1 is expressed at approximately 1/200 the level present in RBC. Employing conditions that resulted in immunoprecipitation of approximately 10% of Glut1 in RBC membranes led to a approximately 3% coimmunoprecipitation of stomatin. A mixed population of Clone 9 cells stably transfected with a plasmid overexpressing the mouse stomatin exhibited 30 +/- 3% reduction in the basal rate of glucose transport compared to control cells or cells stably transfected with the empty vector. The above results suggest that stomatin is closely associated with Glut1 in the plasma membrane and that overexpression of stomatin results in a depression in the basal rate of glucose transport.     

紫云英根瘤菌109菌株的氢氧化电子传递链

H_2还原减去O_2氧化的差示光谱显示424,522,552,560,603nm峰,鱼腾酮(反竞争性抑制),DBMIB,HQNO,抗霉素A,氰化钠和叠氮化钠(非竞争性抑制)明显抑制吸氢活性,表明细胞色素c,b和a分别参与氢氧化的电子传递。以Dixon作图来确定抑制剂在电子传递链中结合位点数目,鱼腾酮和DBMIB为单位点结合,HQNO和氰化物为双位点结合,HQNO所引起的部份抑制,可使对氰化钠敏感的结合位点消逝。鱼腾酮与HQNO同时存在时,其叠加或累积抑制效果表明,两种类型的细胞色素b参与氢氧化的电子传递,由H_2到O_2的电子传递于细胞色素b处分叉,对氰化物抑制敏感性也有所不同。     

Critical role of 5'-AMP-activated protein kinase in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation

5'-AMP-activated protein kinase (AMPK) functions as an energy sensor to provide metabolic adaptation under conditions of ATP depletion, such as hypoxia and inhibition of oxidative phosphorylation. Whether activation of AMPK is critical for stimulation of glucose transport in response to inhibition of oxidative phosphorylation is unknown. Here we found that treatment of Glut1-expressing Clone 9 cells with sodium azide (5 mM for 2 h) or the AMPK activator 5'-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR, 2 mM for 2 h) stimulated the rate of glucose transport by two- to fourfold. Use of small interference RNA (siRNA) directed against AMPK₁ or AMPK₁ + AMPK₂ (total AMPK) resulted in a significant inhibition of the glucose transport response and the content of phosphorylated AMPK₁ + phosphorylated AMPK₂ (total p-AMPK) and phosphorylated acetyl-CoA carboxylase (p-ACC) in response to azide. Transfection with siRNA directed against AMPK₂ did not affect the glucose transport response. The efficacy of transfection with siRNAs in reducing AMPK content was confirmed by Western blotting. Incubation of cells with compound C, an inhibitor of AMPK, abrogated the glucose transport response and abolished the increase in total p-AMPK in azide-treated or hypoxia-exposed cells. Simultaneous exposure to azide and AICAR did not augment the rate of transport in response to AICAR alone. There was no evidence of coimmunoprecipitation of total p-AMPK with Glut1. However, LKB1 was associated with total p-AMPK. We conclude that activation of AMPK plays both a sufficient and a necessary role in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation. small interference RNA; compound C; hypoxia     

Electron transport components of the MnO2 reductase system and the location of the terminal reductase in a marine Bacillus.

The response of MnO2 reduction by uninduced and induced whole cells and cell extracts of Bacillus 29 to several electron transport inhibitors was compared. MnO2 reduction with glucose by uninduced whole cells and cell extracts was strongly inhibited at 0.1 mM dicumarol, 100 mM azide, and 8 mM cyanide but not by atebrine or carbon monoxide, suggesting the involvement of a vitamin K--type quinone and a metalloenzyme in the electron transport chain. MnO2 reduction with ferrocyanide by uninduced cell extracts was inhibited by 5 mM cyanide and 100 mM azide but not by atebrine, dicumarol, or carbon monoxide, suggesting that the metalloenzyme was associated with the terminal oxidase activity. MnO2 reduction with glucose by induced whole cells and cell extracts, was inhibited by 1 mM atebrine, 0.1 mM dicumarol, and 10 mM cyanide but not by antimycin A, 2n-nonyl-4-hydroxyguinoline-N-oxide) (NOQNO), 4,4,4-trifluoro-1-(2-thienyl),1,3-butanedione, or carbon monoxide. Induced cell extract was also inhibited by 100 mM azide, but stimulated by 1 mM and 10 mM azide. Induced whole cells were stimulated by 10 mM and 100 mM azide. These results suggested that electron transport from glucose to MnO2 in induced cells involved such components as flavoprotein, a vitamin K-type quinone, and metalloenzyme. The stimulatory effect of azide on induced cells was explained on the basis of a branching in the terminal part of the electron transport chain, one branch involving a metalloenzyme for the reduction of MnO2 and the other involving a metalloenzyme for the reduction of oxygen. The latter was assumed to be the more azide sensitive. Spectral studies showed the presence of a-, b-, and c-type cytochromes in membrane but not in soluble fractions. Of these cytochromes, only the c type may be involved in electron transport of MnO2, owing to the lack of inhibition by antimycin A or 2n-nonyl-4-hydroxyquinoline-N-oxide. The terminal MnO2 reductase appears to be loosely attached to the cell membrane of Bacillus 29 because of cell fractionation it is found associated with both particulate and soluble fractions. Electron photomicrographs of bacilli attached to synthetic Fe-Mn oxide revealed an intimate contact of the cell walls with the oxide particles.     

Electron transport components of the MnO2 reductase system and the location of the terminal reductase in a marine Bacillus.

The response of MnO2 reduction by uninduced and induced whole cells and cell extracts of Bacillus 29 to several electron transport inhibitors was compared. MnO2 reduction with glucose by uninduced whole cells and cell extracts was strongly inhibited at 0.1 mM dicumarol, 100 mM azide, and 8 mM cyanide but not by atebrine or carbon monoxide, suggesting the involvement of a vitamin K--type quinone and a metalloenzyme in the electron transport chain. MnO2 reduction with ferrocyanide by uninduced cell extracts was inhibited by 5 mM cyanide and 100 mM azide but not by atebrine, dicumarol, or carbon monoxide, suggesting that the metalloenzyme was associated with the terminal oxidase activity. MnO2 reduction with glucose by induced whole cells and cell extracts, was inhibited by 1 mM atebrine, 0.1 mM dicumarol, and 10 mM cyanide but not by antimycin A, 2n-nonyl-4-hydroxyguinoline-N-oxide) (NOQNO), 4,4,4-trifluoro-1-(2-thienyl),1,3-butanedione, or carbon monoxide. Induced cell extract was also inhibited by 100 mM azide, but stimulated by 1 mM and 10 mM azide. Induced whole cells were stimulated by 10 mM and 100 mM azide. These results suggested that electron transport from glucose to MnO2 in induced cells involved such components as flavoprotein, a vitamin K-type quinone, and metalloenzyme. The stimulatory effect of azide on induced cells was explained on the basis of a branching in the terminal part of the electron transport chain, one branch involving a metalloenzyme for the reduction of MnO2 and the other involving a metalloenzyme for the reduction of oxygen. The latter was assumed to be the more azide sensitive. Spectral studies showed the presence of a-, b-, and c-type cytochromes in membrane but not in soluble fractions. Of these cytochromes, only the c type may be involved in electron transport of MnO2, owing to the lack of inhibition by antimycin A or 2n-nonyl-4-hydroxyquinoline-N-oxide. The terminal MnO2 reductase appears to be loosely attached to the cell membrane of Bacillus 29 because of cell fractionation it is found associated with both particulate and soluble fractions. Electron photomicrographs of bacilli attached to synthetic Fe-Mn oxide revealed an intimate contact of the cell walls with the oxide particles.     

The mechanism of pantothenate transport by rat liver parenchymal cells in primary culture

The mechanism of pantothenate transport across the plasma membrane was investigated with initial velocity studies of [14C]pantothenate uptake and efflux in rat liver parenchymal cells maintained in primary culture. At 116 mM sodium, double-reciprocal plots of the initial velocity of uptake versus [pantothenate] were linear from 0.3 to 36.5 microM pantothenate and gave an apparent Km,pant of 11 +/- 2 microM. The rate of pantothenate uptake at 0 [sodium] was about 14% of the rate at 116 mM sodium, and the reciprocal of the apparent Km,pant was a linear function of [sodium]. Vmax obtained by extrapolation to infinite [pantothenate] was independent of [sodium]. Ouabain, gramicidin D, cyanide, azide, and 2,4-dinitrophenol inhibited uptake, but preloading cells with pantothenate did not. Pantothenate derivatives or carboxylic acids were only weak inhibitors of uptake. Efflux was measured in cells preloaded with [14C]pantothenate. The apparent Km for efflux was 85 +/- 29 microM, and the rate of efflux was unaffected by addition of pantothenate, sodium, ouabain, gramicidin D, or 2,4-dinitrophenol to the external medium. These features are consistent with a mechanism for pantothenate transport in which sodium and pantothenate are cotransported in a 1:1 ratio on a carrier highly specific for pantothenate; sodium decreases the apparent Km for pantothenate, and a sodium-carrier complex forms only on the intracellular side of the membrane.     

Role of 5'-AMP-activated protein kinase in stimulation of glucose transport in response to inhibition of oxidative phosphorylation

Glucose transport is stimulated in a variety of cells and tissues in response to inhibition of oxidative phosphorylation. However, the underlying mechanisms and mediating steps remain largely unknown. In the present study we first tested whether a decrease in the redox state of the cell per se and the resultant increase in generation of reactive oxygen species (ROS) lead to stimulation of glucose transport. Clone 9 cells (expressing the Glut1 isoform of facilitative glucose transporters) were exposed to azide, lactate, and ethanol for 1 h. Although all three agents stimulated glucose transport and increased cell NADH-to-NAD⁺ ratio and phospho-ERK1/2, signifying increased ROS generation, the response to the stimuli was not blocked by N-acetyl-L-cysteine (an agent that counteracts ROS); moreover, the response to azide was not blocked by diamide (an intracellular sulfhydryl oxidizing agent). We then found that cell AMP-to-ATP and ADP-to-ATP ratios were increased and 5'-AMP-activated protein kinase (AMPK) was stimulated by all three agents, as evidenced by increased phosphorylation of AMPK and acetyl-CoA carboxylase. We conclude that although azide, lactate, and ethanol increase NADH-to-NAD⁺ ratios and ROS production, their stimulatory effect on glucose transport is not mediated by increased ROS generation. However, all three agents increased cell AMP-to-ATP ratio and stimulated AMPK, making it likely that the latter pathway plays an important role in the glucose transport response. 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside; extracellular signal related-kinase 1/2; phospho-extracellular signal related-kinase 1/2; N-acetyl-L-cysteine; diamide; acetyl-CoA carboxylase; phospho-acetyl-CoA carboxylase     

Description of glucose transport in isolated bovine mammary epithelial cells by a three-compartment model

Initial rates of glucose entry into isolated bovine mammary epithelial cells display moderate degrees of asymmetry and cooperative interactions between export and import sites. The present study examined the hypothesis that these kinetic features are due to compartmentalization of intracellular glucose. Net uptake of 3-O-methyl-d-[1-(3)H]glucose (3-OMG) by isolated bovine mammary epithelial cells was measured at 37 degrees C. The time course of 3-OMG net uptake was better fitted by a double-exponential equation than by a single- or triple-exponential equation. Compartmental analysis of the time course curve suggested that translocated 3-OMG is distributed into two compartments with fractional volumes of 32.6 +/- 5.7% and 67.4 +/- 5.7%, respectively. The results support the view that glucose transport in bovine mammary epithelial cells is a multistep process consisting of two serial steps: fast, carrier-mediated, symmetric translocation of sugar across the cell plasma membrane into a small compartment and subsequent slow exchange of posttranslocated sugar between two intracellular compartments. A three-compartment model of this system successfully simulated the observed time course of 3-OMG net uptake and the observed dependence of unidirectional entry rates on intra- and extracellular 3-OMG concentrations. Simulations indicated that backflux of radiolabeled sugar from the small compartment to extracellular space during 15 s of incubation gives rise to the apparent asymmetry, trans-stimulation, and cooperativity of mammary glucose transport kinetics. The fixed-site carrier model overestimated the rate of glucose accumulation in cells, and its features can be accounted for by the compartmentalization of intracellular sugar.     

Relationship between insulin stimulation and endogenous regulation of 2-deoxyglucose uptake in 3T3-L1 adipocytes

The occurrence of the endogenous regulatory response to high rates of 2-deoxyglucose (2-DG) uptake, as previously described for C6 glioma cells during incubation with 2 mM 2-DG (Lange et al.: J. Cell. Physiol., 1989), was studied in 3T3-L1 preadipocytes and adipocytes, and the influence of insulin on this endogenous uptake regulation was examined. In contrast to 3T3-L1 preadipocytes, insulin-sensitive differentiated 3T3-L1 adipocytes displayed the time-dependent cyclic pattern of 2-DG uptake rates characteristic of the membrane-limited and endogenously regulated cellular state of hexose utilization. Although insulin induced a threefold stimulation of 2-DG tracer uptake in adipocytes, the hormone did not additionally stimulate the uptake rates or affect the periodic response: maximum and minimum levels of uptake remained unchanged. Scanning electron microscopy (SEM) revealed that the acquirement of the differentiated state is accompanied by a conspicuous transformation of the smooth surface of undifferentiated 3T3-L1 cells into a surface covered by numerous microvilli of uniform size and appearance. Treatment with insulin (10 mU/ml; 10 minutes) converted these microvilli into voluminous saccular membrane protrusions of the same type as had been formed during incubation of 3T3-L1 adipocytes with 2 mM 2-DG, and which have previously been shown to be involved in the endogenous uptake regulation of C6 glioma cells (Lange et al.: J. Cell. Physiol., 1989). These insulin-induced saccated membrane areas appeared to become integrated into the cell surface. Accordingly, insulin treatment caused a twofold increase of the intracellular distribution space of 3-O-methylglucose (3-OMG) in 3T3-L1 adipocytes. This insulin-induced increase of the 3-OMG distribution space exhibited the same time (t1/2 = 2-2.5 minutes) and dose dependence (EC50 = 20 nM) as the insulin-induced stimulation of 3-OMG transport. Glucose deprivation during the differentiation period inhibited the outgrowth of microvilli from the cell surface. Glucose starvation (18 hours at less than 0.5 mM) induced a conspicuous reduction of the length of microvilli on differentiated 3T3-L1 cells. In this state, the stalks of the microvilli are almost invisible and the enlarged spherical tips of the microvilli (with an average diameter of 370 nm compared to 230 nm of fed cells) appeared to protrude directly out of the cell surface. Starvation-induced shortening of microvilli was accompanied by a threefold increase of the basal 3-OMG transport rate and a greater than twofold increase of the intracellular 3-OMG distribution space as compared to fed cells (10 mM; 18 hours).(ABSTRACT TRUNCATED AT 400 WORDS)