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Gluc ose Metab olism in Trisomy 1 Op a nd Normal Controls
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Red blood cell glucose metabolism was investigated in a male patient with de novo trisomy 10p. According to previous evidence, when assigning hexokinase gene locus in the 10p11 leads to pter region, a triplex dosage effect of hexokinase activity (HK) was found, while all the other erythrocyte glycolytic enzymes were in the normal values range. Red...
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The association between high selenium (Se) intake and metabolic disorders such as type 2 diabetes has raised great concern, but the underlying mechanism remains unclear.
Objective:
Through targeted metabolomics analysis, we examined the liver sugar and acylcarnitine metabolism responses to supranutritional selenomethionine (SeMet) su...
Citations
... In eukaryotic as well as in some prokaryotic cells, a considerable part of the intracellular glucose is catabolized via the PPP. In mature human erythrocytes nearly 5-10 percent of the total glucose consumption of 1-2 µmol/hr/ml erythrocyte at 37°C (Brin and Yonemoto 1958, Magnani et al. 1983, Murphy 1960, in adipose tissue nearly 15 percent of the total glucose in the absence and 23 percent in the presence of insulin (Landau and Katz 1964), and in growing E. coli approximately 12 percent of the total glucose (Fuhrer et al. 2005). The PPP is divided into two branches, namely the oxidative (irreversible) and non-oxidative (reversible) PPP. ...
... The HMP-shunt assay used in this study is simple and requires only 50 pl of red cells per assay. The basal HMP-shunt activities of the total population measured with I4C-1-labelled glucose are higher than reported by Magnani et al (22) and Davidson & Tanaka (18), but comparable with the results of Trotta et al (23). The MB-stimulated HMP-shunt activity of the total population is more than 2-fold higher than reported by Magnani (22) or Davidson & Tanaka (1 8), but about equal to the values found by Rogias et a1 (24) and Albrecht et al (25). ...
... The basal HMP-shunt activities of the total population measured with I4C-1-labelled glucose are higher than reported by Magnani et al (22) and Davidson & Tanaka (18), but comparable with the results of Trotta et al (23). The MB-stimulated HMP-shunt activity of the total population is more than 2-fold higher than reported by Magnani (22) or Davidson & Tanaka (1 8), but about equal to the values found by Rogias et a1 (24) and Albrecht et al (25). ...
Erythrocytes were separated by age using a combination of density centrifugation and counterflow centrifugation and tested for basal activity of the hexose monophosphate shunt (HMP-shunt) as well as the methylene blue-stimulated maximal capacity by measuring CO2 production. No significant differences were found in basal HMP-shunt activity, but the maximal methylene blue-stimulated activity of old erythrocytes reached only half of the activity of the total cell population. The maximal HMP-shunt activity showed a significant correlation with hexokinase activity, but not with glucose-6-phosphate dehydrogenase activity in all but the youngest cells. The sensitivity to oxidative stress was tested by measuring the kinetics of pyruvate kinase isolated from erythrocytes incubated in presence and absence of methylene blue. Pyruvate kinase kinetics were affected more in the old cell population than in the total cell population: the K0.5 for phosphoenol-pyruvate increased four times in the unseparated cells and eight times in old cells.
The aim of this chapter is to survey phenomena relating to the regulation of metabolism in intact cells and how NMR spectroscopy can contribute to a greater understanding of these phenomena. The emphasis of the chapter is on isolated erythrocytes and hemolysates, so it is not a comprehensive review of in vivo NMR spectroscopy, and many of the examples which have been chosen come from our own particular work on human erythrocytes. The aspect of erythrocyte metabolism which has been subjected to most scrutiny by NMR spectroscopy is the pentose phosphate pathway, so work relating to this group of reactions is presented in greater detail than that from other areas; the work will serve as an example of the type of applications to which NMR can be put in studies of cells. The general physical characteristics of the intracellular milieu of the human erythrocyte, which can be measured using NMR spectroscopy, such as pH, viscosity, rotational and translational diffusion coefficients, and metabolite concentrations, are derived from generic experiments that can in principle be applied in studies of any cell type.
The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37 degrees C (pH 7.2) was 10.23 +/- 1.90 mumol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg X ATP2-. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 microM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP4- and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94 +/- 0.02 mumol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92 +/- 0.3 mumol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.
The synthesis and turnover of hexokinase has been measured in Zajdela hepatoma ascites cells labeled for short periods with [35S]methionine. Digitonin fractionation of the labeled cells into a soluble and a membrane fraction showed that only a small part of the newly labeled hexokinase is transferred to mitochondrial binding sites. The soluble enzyme disappears, however, with a half-life of less than 2 h. Glucose had no effect on the stability of the soluble enzyme in intact cells. Our experiments suggest that Zajdela cell hexokinase is synthesized in excess of binding sites and that the excess enzyme is not stable.
In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.
Human erythrocytes were loaded with homogeneous hexokinase purified from human placenta (an enzyme species apparently identical to the erythrocyte enzyme), using a procedure of encapsulation based on hypotonic hemolysis, isotonic resealing and reannealing. The hexokinase-overloaded erythrocytes contained 4.77 +/- 0.75 IU of hexokinase activity per ml of packed erythrocytes, a value 15-times higher than that of corresponding unloaded or native red cells. The hexokinase-loaded erythrocytes were found to metabolize twice the amount of glucose consumed by the unloaded cells through a nearly doubled glycolytic activity, while the activity of the hexose monophosphate shunt pathway was unmodified. Estimates of glycolytic intermediates showed increased levels of most metabolites with respect to the unloaded erythrocytes, while the intracellular concentrations of adenine nucleotides and 2,3-bisphosphoglycerate were unaffected by entrapment of hexokinase. The new steady-state condition characterized by improved glycolytic function was demonstrated to be directly related to enhanced levels of hexokinase activity and not to the use of a rejuvenation solution during the procedure of entrapment. These results are consistent with suggestions by several investigators that glucose metabolism in human erythrocytes is regulated by hexokinase, and they open new perspectives for manipulating erythrocytes with the ultimate aim of improving their survival under different storage conditions.
In addition to the well known effect of phenylhydrazine on red blood cells (methaemoglobin and Heinz body formation, autologous IgG binding, lipid peroxidation, etc.) an increased glucose utilization was observed. Measurement of 14CO2 formation from [1-14C]-glucose showed a maximum value at 2mM phenylhydrazine followed by a progressive inhibition on increasing the drug concentration to 16 mM. Concomitantly we found a reduction in the reduced glutathione concentration but not a corresponding increase in the level of oxidized glutathione. Phenylhydrazine also causes ATP depletion. The ATP is in part dephosphorylated to ADP and AMP and in part converted to inosine monophosphate and hypoxanthine. Measurement of the cell content of reduced and oxidized pyridine nucleotides was also performed and showed a progressive increase in the reduced forms of these coenzymes. Thus phenylhydrazine promotes cellular ATP depletion followed by adenine nucleotide catabolism that is not efficiently counteracted by an increase in glucose utilization. The relevance of these data to the mechanism of phenylhydrazine-induced anemia is discussed.
Human erythrocytes overloaded with homogeneous human hexokinase (up to 15-times the activity of normal RBC) show almost unmodified rates of glucose metabolized in the HMP, however hexokinase-loaded RBC are able to metabolize 1.5 fold more glucose than controls through the HMP when an oxidizing agent like methylene blue (5 to 100 microM) is present. Similarly, RBC loaded with inactivating anti-hexokinase IgG (12 +/- 3% residual hexokinase activity) show HMP rates unchanged under resting conditions, but only 12% of the HMP rate found in normal controls under oxidative stress. These data provide clear evidence that the HMP rate under conditions of oxidative stress is controlled by hexokinase activity and suggest that RBC from patients with hexokinase deficiency are not able to increase the HMP rate under oxidative stress like erythrocytes from individuals with G6PD deficiency.
Human red blood cell hexokinase exists in multiple molecular forms with different isoelectric points but similar kinetic and regulatory properties. All three major isoenzymes (HK Ia, Ib, and Ic) are inhibited competitively with respect to Mg.ATP by glucose 6-phosphate (Ki = 15 microM), glucose 1,6-diphosphate (Ki - 22 microM), 2,3-diphosphoglycerate (Ki = 4 mM), ATP (Ki = 1.5 mM), and reduced glutathione (Ki = 3 mM). All these compounds are present in the human erythrocyte at concentrations able to modify the hexokinase reaction velocity. However, the oxygenation state of hemoglobin significantly modifies their free concentrations and the formation of the Mg complexes. The calculated rate of glucose phosphorylation, in the presence of the mentioned compounds, is practically identical to the measured rate of glucose utilization by intact erythrocytes (1.43 +/- 0.15 mumol h-1 ml red blood cells-1). Hexokinase in young red blood cells is fivefold higher when compared with the old ones, but the concentration of many inhibitors of the enzyme is also cell age-dependent. Glucose 6-phosphate, glucose 1,6-diphosphate, 2,3-diphosphoglycerate, ATP, and Mg all decay during cell ageing but at different rates. The free concentrations and the hemoglobin and Mg complexes of both ATP and 2,3-diphosphoglycerate with hemoglobin in the oxy and deoxy forms have been calculated. This information was utilized in the calculation of glucose phosphorylation rate during cell ageing. The results obtained agree with the measured glycolytic rates and suggest that the decay of hexokinase during cell ageing could play a critical role in the process of cell senescence and destruction.
