A method for uni-directional reconstitution of human erythrocyte glucose transporter

STUDY OBJECTIVE: To determine whether patient factors other than body weight would better predict patients' initial antifactor Xa heparin activity (HA) after start of unfractionated heparin (UFH) therapy. DESIGN: Case series. SETTING: A 625-bed, adults-only, private, tertiary care teaching hospital. PATIENTS: Ninety-two patients requiring UFH therapy. INTERVENTIONS: Patients received initial UFH bolus doses of 72-80 U/kg ideal weight and initial UFH infusions of 19.1-21.2 U/kg ideal weight. MEASUREMENTS AND MAIN RESULTS: Fifty-five percent of the first 6-hour HA measurements were supratherapeutic (> 0.7 U/ml antifactor Xa activity). Patient weight was inferior to a combination of age and estimated plasma volume in predicting initial HA. A predictive model including these two factors accounted for 38.5% of variation in first HA levels compared with 17.7% with actual weight alone. CONCLUSION: Weight-based UFH dosing may frequently result in nontherapeutic initial HA levels. Initial UFH dosing might be improved if protocols based on patient age and estimated plasma volume were developed.     

Overexpression of the human erythrocyte glucose transporter occurs as a late event in human colorectal carcinogenesis and is associated with an increased incidence of lymph node metastases

Energy metabolism of human colon cancer in vivo relies predominantly on glucose. Although studies have revealed increased expression of Glut1 mRNA in colon cancer, Glut1 protein (Glut1) expression in the large intestine and its significance are still unknown. The objective of this work was to determine whether Glut1 is present in human colorectal neoplasms and whether that presence is of biological significance. Formalin-fixed, paraffin-embedded tissue sections of 53 colonic adenocarcinomas, 82 adenomas, 46 hyperplastic polyps, and 38 normal colon samples were immunostained with the anti-Glut1 antibody MYM. The localization was carried out using the avidin-biotin immunoperoxidase technique. No Glut1 immunoreactivity was present in normal colonic mucosa or in hyperplastic polyps, whereas 8 (10%) of 82 adenomas showed such immunoreactivity. The frequency of Glut1 expression in adenomas increased with villous morphology and with the size of the adenoma. Forty-four (83%) of 53 colorectal adenocarcinomas expressed Glut1, and, of these, tumors in which >50% of the cancer cells expressed Glut1 had a significantly higher incidence of metastasis to the lymph nodes (P = 0.0001). It is concluded that (a) Glut1 is expressed as a late event in the carcinogenesis process in human colorectal cancer, and (b) expression of Glut1 in a high proportion of cancer cells is associated with a high incidence of lymph node metastases.     

The glucose transporter of human erythrocytes--working hypothesis for its functional mechanism

Cloning of rat cadherin-8 cDNA demonstrated two types of cDNAs. The overall structure of the protein defined by one type of the cDNA is essentially the same as that of classic cadherins, whereas the protein defined by the other type of cDNA ends near the N-terminus of the fifth repeat of the extracellular domain (EC5) and contains a short unique sequence at the C-terminus. The same truncated type of cDNA was also obtained from a human cDNA library. In Northern blot analysis of rat brain mRNA, a probe for EC5 detected multiple bands of about 3.5-4.3 knt, whereas a probe for the alternative form hybridized with a band of about 3.5 knt. Western blot experiments showed that an antibody against the extracellular domain of rat cadherin-8 stained a band of about 95 kDa and a faint band of about 130 kDa in rat brain extract. These results suggest that cadherin-8 is expressed in two forms, a complete form and a truncated form without a transmembrane domain or cytoplasmic domain, in brain. The complete form of cadherin-8 expressed in L cells was about 130 kDa in molecular mass and was located at the cell periphery, mainly at the cell-cell contact sites. However, we failed to express the truncated form in L cells. The transfectants of the complete form showed weak cell adhesion activity. The complete form of cadherin-8 was sensitive to trypsin digestion, and Ca2+ did not protect cadherin-8 from digestion, in contrast to the classic cadherins. The complete form of cadherin-8 coprecipitated with beta-catenin, but did not immunoprecipitate well with alpha-catenin or gamma-catenin. Cadherin-8, as well as cadherin-11, was mapped to a specific region of chromosome 8 that also includes cadherins-1, -3, and -5.     

Glycaemic regulation of glucose transporter expression and activity in the human placenta

To determine whether the expression and activity of glucose transporters in human trophoblast are regulated by glucose, syncytiotrophoblast cells, choriocarcinoma cells, and villous fragments were incubated with a range of glucose concentrations (0-20 mM, 24 h). Expression of GLUT1 and GLUT3 glucose transporters was measured by immunoblotting, while glucose transporter activity was determined by [3H]2-deoxyglucose uptake in the cultured cells. GLUT1 expression in syncytial cells was enhanced following incubation in absence of glucose, reduced by incubation in 20 mM glucose but was not altered by incubation at 1 or 12 mM glucose. Transporter activity was inversely related to extracellular glucose over the entire range of concentrations tested (0-20 mM). Incubation of villous fragments in 20 mM glucose produced a limited suppression of GLUT1 expression, but no effects were noted following incubation at 0 or 1 mM glucose. Neither GLUT1 expression in JAr and JEG-3 choriocarcinoma cells nor transport activity in JEG-3 cells was affected by extracellular glucose concentration. Unlike syncytial cells, JAr, JEG-3 and BeWo all expressed GLUT3 protein in addition to GLUT1. These results show that while syncytiotrophoblast GLUT1 expression is altered at the extremes of extracellular glucose concentration, it is refractory to glucose alone at lower concentrations. By contrast, an inverse relationship exists between glucose transporter activity and extracellular glucose. This suggests that there are post-translational regulatory mechanisms which may respond to changes in extracellular glucose concentration.     

Characterization of glucose transporter isoforms in the adult and developing human eye

The expression of glucose transporter isoforms (Glut 1, Glut 3, Glut 4, and Glut 5) in the human eye was investigated at various ages ranging between 8 weeks gestation (first trimester) and adult using Western blot and immunohistochemical analyses. Glut 1 and Glut 3 expression and cellular localization patterns were similar to those of human brain. Glut 1 (50-kilodalton protein) was expressed by epithelial cells (retinal pigmented epithelium, choroidal, iridial, and pars planus), which form the blood-eye barrier, retinal Mueller cells, the lens fiber cells, iridial microvascular endothelial cells, and to a lesser extent by the outer segments of the photoreceptor cells in the adult eye. This pattern was conserved throughout development and was evident as early as 8 weeks gestation. In addition, the endothelial cells of vitreous hyaloid vessels expressed Glut 1 at 8 weeks gestation. Glut 3 (50 to 55-kilodalton protein) immunoreactivity was observed only in the adult inner synaptic layer of the retina. Neither Glut 4 nor Glut 5 was expressed in any occular tissue at any age examined. These results suggest that Glut 1 is the main glucose transporter of the human eye and that it is ontogenically conserved. In contrast, Glut 3 is associated with selective neuronal processes, and its expression is developmentally altered.     

Crystal structure of the unliganded alkaline protease from Pseudomonas aeruginosa IFO3080 and its conformational changes on ligand binding

Since volatile anesthetics, barbiturates, and local anesthetics have been reported to inhibit endothelium-dependent relaxation, we hypothesized that any drug with anesthetic action would suppress this relaxation. In the present study, using rat thoracic aortae, we attempted to determine whether nonbarbiturate intravenous anesthetics, including midazolam, propofol, and ketamine, suppress endothelium-dependent relaxation, and to clarify the mechanism(s) involved. Acetylcholine-induced, endothelium-dependent relaxation was significantly attenuated by propofol and ketamine, but was unaffected by midazolam. Sodium nitroprusside (SNP)-induced relaxation was attenuated by propofol, but not by midazolam or ketamine. The acetylcholine-stimulated 3',5'-cyclic guanosine monophosphate (cGMP) level was reduced by pretreatment with propofol and ketamine but not by midazolam, and that stimulated by SNP was reduced by propofol but not by ketamine or midazolam. We conclude that propofol and ketamine suppress endothelium-dependent relaxation, whereas midazolam has no influence. Moreover, the suppressive effect of ketamine on endothelium-dependent relaxation is mediated by suppression of nitrous oxide (NO) formation, whereas that of propofol may be mediated at least partly by suppression of NO function.     

Unexpected expression of glucose transporter 4 in villous stromal cells of human placenta

Glucose transporter 4 (GLUT4) protein expression was characterized in human and rodent term placentas. A 50-kDa protein was detected, by immunoblotting, in term human placenta at levels averaging 25% of those found in white adipose tissue. It was also present, albeit at lower levels, in mouse and rat placentas. The specificity of the 50-kDa signal was established by using skeletal muscle and placental tissues obtained from GLUT4-null mice as controls. Indirect immunohistochemistry, performed in human placentas, showed that intravillous stromal cells were conspicuously labeled by GLUT4 and revealed colocalization of GLUT4 transporters with insulin receptors. This study provides the first evidence that the insulin-responsive GLUT4 glucose transporter is present in human and rodent hemochorial placentas. Placental GLUT4 gene and protein levels were not modified in human pregnancy complicated by insulin-dependent diabetes mellitus. The significance of the high level of GLUT4 protein in human placenta remains to be elucidated, because, so far, this organ was not considered to be insulin-sensitive, with regard to glucose transport.     

FDG uptake, GLUT-1 glucose transporter and cellularity in human pancreatic tumors

We previously reported that grading of GLUT-1 glucose transporter expression was related closely to FDG accumulation in FDG PET in human cancers. But in this strong GLUT-1 expression group, there was an enormous range of standardized uptake values (SUVs) within them. METHODS: To evaluate other factors determining the FDG PET uptake, FDG PET was performed in 36 preoperative patients (mean age 62.0 yr) suspected of having pancreatic tumors, including 33 malignant and 3 benign neoplastic tumors. FDG uptake at 50 min after injection of 185 MBq 18F-FDG with > 5 hr fasting condition was semiquantitatively analyzed as SUVs. The GLUT-1 expression was studied by immunohistochemistry of paraffin sections from these tumors after the operation using the antiGLUT-1 antibody. The number of tumor cells within a 5- x 5-mm square was counted manually using x200 magnification photographs and was graded immunohistochemically as strong, weak or negative. RESULTS: In all 36 cases there were 3 cases of GLUT-1 negative, 8 of GLUT-1 weak positive and 25 of GLUT-1 strong positive. In all cases, the total number of tumor cells had no significant value for SUVs. Among 33 GLUT-1 positive cases, the number of GLUT-1 positive tumor cells correlated significantly with SUVs (p < 0.01). Only in 25 strong grade cases, the number of GLUT-1 strong positive tumor cells had a more significant value for SUVs (p < 0.005). Computational multivariate analysis using multiple regression for SUVs was performed evaluating the five variables as follows: tumor size, GLUT-1 immunohistochemical grading, number of total tumor cells, number of total GLUT-1 positive tumor cells and number of GLUT-1 strong positive cells. This analysis revealed that only the variable, the number of GLUT-1 strong positive cells, had a significant regression coefficient for SUVs (standard regression coefficient = 0.855, p < 0.0001). CONCLUSION: These data indicate that GLUT-1 expression plays an essential role in higher FDG accumulation in pancreatic tumor FDG PET, and the cellularity has a significant influence on SUVs only in the condition of GLUT-1 strong positive expression.     

Excitability changes and glucose metabolism in experimentally induced focal cortical dysplasias

Malformations of cortical development are increasingly recognized in association with severe epileptic syndromes, neuropsychological disorders and mental retardation. Several clinical and experimental studies suggest that functional consequences of cortical dysplasias are not restricted to the area of the dysplastic lesion but also involve remote brain regions. In the present study cortical malformations were induced in newborn rats at day of birth by intracerebral injection of the glutamatergic agonist ibotenate. The resulting cytoarchitectonic lesion associates neuronal depopulation of deep cortical layers, ectopic neurons in superficial layers and sulcus formation, mimicking human polymicrogyria and migration disorders. Electrophysiological recordings of evoked field potentials in slice preparations of adult animals reveal hyperexcitability in widespread cortical regions surrounding the dysplasia. Low-intensity stimulation induced epileptiform activity consisting of long-lasting, multiphasic and N-methyl-D-aspartate-dependent field responses. They appeared with high variability as all-or-none events. These widespread changes in excitability were not observed in sham-operated animals with small superficial ectopias but intact deep cortical layers, indicating that focal loss of these layers induces extended alterations in cortical connectivity and imbalance of excitation and inhibition. Restricted zones of increased excitability were also found in the forelimb and hindlimb representation cortex in sham-operated and control animals, demonstrating that this activity has to be considered as an intrinsic property of specific cortical areas. Deoxyglucose autoradiography showed that the widespread hyperexcitability in ibotenate-injected animals was not accompanied by alterations in glucose metabolism, although in the area of structural abnormality a typical metabolic pattern was found, revealing an increased glucose uptake in layer I. Hypometabolism as described for many types of human dysplastic lesions was not observed. This difference between the experimental and clinical data may be due to the absence of behavioral seizures in this model. However, it can be hypothesized that in patients with developmental malformations, additional pathogenic factors contribute to the manifestation of seizure disorders.     

Two modes of ligand binding in maltose-binding protein of Escherichia coli. Electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling

Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closure of its two lobes. We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels. Using EPR spectroscopy, we examined the changes, caused by the ligand binding, in distance between the two spin labels, hence between the two lobes. The binding of both maltose and maltotetraose induced a considerable closure of the N- and C-terminal lobes of MBP. Little closure occurred upon the binding of maltotetraitol or beta-cyclodextrin. Previous study by fluorescence and UV differential absorbance spectroscopy (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609) showed that maltose and a large portion of maltotetraose bound to MBP via one mode (R mode or "end-on" mode), which is physiologically active and leads to the subsequent transport of the ligands across the cytoplasmic membrane. In contrast, maltotetraitol and beta-cyclodextrin bound to MBP via a different mode (B mode or "middle" mode), which is physiologically inactive. The present work suggests that the B mode is nonproductive because ligands binding in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interacting properly with the inner membrane-associated transporter complex.     

Insulin action on whole body glucose utilization and on muscle glucose transporter translocation in mice

Murine models of insulin resistance and diabetes are versatile and have been used to investigate genetic and metabolic disorders. However, the principal assays to assess insulin action, i.e., the euglycemic-hyperinsulinemic clamp and subcellular distribution of glucose transporters, have not been implemented in this species. Here we describe procedures which allow these methods to be adapted to mice. When normal C57bl/6j mice were infused with graded doses of insulin (1, 3, 10 or 30 mU/kg/min) during a euglycemic-hyerinsulinemic clamp, the glucose infusion rate necessary to maintain euglycemia increased in a dose-dependent manner (7.4 +/- 1.7, 13.1 +/- 3.6, 24.1 +/- 2.3 or 34.8 +/- 7.5 mg/kg/min), respectively. Hindlimb muscles were isolated and samples of 2-3 g were subjected to subcellular fractionation finalizing on 25%, 30% and 35% sucrose gradients. Fraction F25 (plasma membranes) was enriched in alpha 2 Na+/K(+)-ATPase and GLUT1 glucose transporters, whereas fraction F35 (intracellular membranes) was enriched in Ca(2+)-ATPase and GLUT4 glucose transporters. Following insulin treatment, GLUT4 increased in F25 and decreased in F35. Insulin treatment had no effect on GLUT1 in F25. However, unlike in rat skeletal muscle, GLUT1 was detectable in F35 and its content decreased in this fraction following insulin treatment. The results demonstrate that whole-body glucose utilization can be assessed in mice using euglycemic-hyperinsulinemic clamps and demonstrate how subcellular fractionation procedures can be applied to murine muscle. Murine muscle GLUT4 translocates from an intracellular storage site to the plasma membrane in response to insulin.     

Manganese transport through human erythrocyte membranes. An EPR study

Manganese uptake by human erythrocytes was investigated in the concentration range 0.5-20 mM in the suspending solution, by using the EPR technique. S shaped dependencies of manganese influx on manganese doping solution concentration for both fresh and vanadate treated erythrocytes were found, with maximum influx values of 4.1 +/- 1.9 x 10(-10) mol/m2 x s and 2.1 +/- 0.3 x 10(-9) mol/m2 x s, respectively. At low manganese concentrations (< 2 mM) the manganese permeability coefficient increases with increasing the doping concentration, the ions cooperate for achieving a transport event. For high manganese concentration (> 5 mM) the permeability coefficient decreases with increasing the doping concentration, the ions competing for the limited amount of transport system. A similar increase in manganese uptake as in vanadate treated erythrocytes was measured for 'in vitro' aged erythrocytes. These results might suggest that human erythrocytes possess an active transport mechanism by which, they oppose to manganese influx. This hypothesis is also supported by the 10-15 min time lag between the moment of doping and the start of the manganese influx into the fresh erythrocytes. The manganese uptake inhibition by nifedipine, a calcium channel blocker, for the case of vanadate treated erythrocytes, suggests that, at least partially, manganese uptake by the cells occurs via the 'calcium channels'.     

Effects of ethanol on glucose transporter expression in cultured hippocampal neurons

Glucose transport was studied in primary hippocampal neuron cultures exposed to ethanol. Immunofluorescent staining with antibodies against neuron-specific enolase and glial fibrillary acidic protein identified approximately 95% of the cultured cells as neurons. Western blot analysis was conducted with polyclonal antisera to glucose transporter isoforms GLUT1 and GLUT3. As previously seen in astrocytes, GLUT1 protein was regulated by the culture medium glucose content. Exposure to 50 and 100 mM of ethanol for 5 hr induced dose-dependent reductions in GLUT1 and GLUT3 protein. In contrast, GLUT1 mRNA abundance was increased relative to controls under the same conditions. Glucose uptake, measured with the nonmetabolized analog, 2-deoxy-D-glucose, was reduced by 50 and 100 mM of ethanol in four experiments. These results indicate a direct effect of ethanol on neuronal glucose transporter expression, which may play a role in the neurotoxic effects of alcohol.     

Monoclonal antibodies inducing conformational changes on the antigen molecule

Monoclonal antibodies (MoAbs) are extensively used as biological tools because of their invariable specificity. However, the interpretation of results can be misled by the behaviour of MoAb displaying allosteric effects, i.e. long-range conformational changes on the antigen (Ag). It has been shown that some MoAbs are able to modify the spatial structure of the corresponding protein Ag, affecting in this way its biological activity as well as its binding to a second MoAb. Thus, a researcher using a MoAb as a tool to investigate some features of an antigenic molecule must be aware of the possible positive or negative allosteric properties of the antibody.     

Dissection of drug-binding-induced conformational changes in P-glycoprotein

OBJECTIVES: We tested the prognostic value of a new electrocardiographic (ECG) method (Perugia score) for diagnosis of left ventricular hypertrophy (LVH) in essential hypertension and compared it with five standard methods (Cornell voltage, Framingham criterion, Romhilt-Estes point score, left ventricular strain, Sokolow-Lyon voltage). BACKGROUND: Several standard ECG methods for assessment of LVH are used in the clinical setting, but a comparative prognostic assessment is lacking. METHODS: A total of 1,717 white hypertensive subjects (mean age 52 years; 51% men) were prospectively followed up for up to 10 years (mean 3.3). RESULTS: At entry, the prevalence of LVH was 17.8% (Perugia score), 9.1% (Cornell), 3.9% (Framingham), 5.2% (Romhilt-Estes), 6.4% (strain) and 13.1% (Sokolow-Lyon). During follow-up there were 159 major cardiovascular morbid events (33 fatal). The event rate was higher in the subjects with than in those without LVH (all p < 0.001) according to all methods except the Sokolow-Lyon method. By multivariate analysis, an independent association between LVH and cardiovascular disease risk was maintained by the Perugia score (hazard ratio [HR] 2.04, 95% confidence interval [CI] 1.5 to 2.8) and the Framingham (HR 1.91, 95% CI 1.1 to 3.2), Romhilt-Estes (HR 2.63, 95% CI 1.7 to 4.1) and strain methods (HR 2.11, 95% CI 1.4 to 3.2). The Perugia score showed the highest population-attributable risk for cardiovascular events, accounting for 15.6% of all cases, whereas the Framingham, Romhilt-Estes and strain methods accounted for 3.0%, 7.4% and 6.8% of all events, respectively. LVH diagnosed by the Perugia score was also associated with an increased risk of cardiovascular mortality (HR 4.21, 95% CI 2.1 to 8.7), with a population-attributable risk of 37.0%. CONCLUSIONS: The Perugia score carried the highest population-attributable risk for cardiovascular morbidity and mortality compared with classic methods for detection of LVH. Traditional interpretation of standard electrocardiography maintains an important role for cardiovascular risk stratification in essential hypertension.     

Age-related changes on glucose transport and utilization of human erythrocytes: effect of oxidative stress

BACKGROUND: Normal aging is associated with an impairment in glucose homeostasis. METHODS: In order to investigate the effect of aging on glucose transport and utilization in erythrocytes, the transport and utilization of glucose were measured in erythrocytes from 10 young (mean age 26 +/- 3 years) and 10 elderly (mean age 70 +/- 7 years) healthy individuals. In addition, the glucose transport and utilization were also measured in the presence of cumene hydroperoxide (CumOOH), a toxic organic hydroperoxide that is known to induce oxidative stress. RESULTS: The levels of glucose transport and utilization were significantly lower in the elderly group than the young group (p < 0.05). The glucose transport and utilization were not altered by CumOOH treatment in either young or elderly individuals. CONCLUSION: These results indicate an age-related decrease in the both glucose transport and utilization in erythrocytes.     

ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.