Alterations in hexose monophosphate shunt during lymphoblastic transformation

This study characterized the alteration in hexose monophosphate shunt (HMPS) activity occurring during lymphoblastic transformation to nonspecific mitogens. Average HMPS activity was threefold higher in phytohemagglutinin (PHA)-stimulated cultures incubated for 68 hr when compared to unstimulated cultures. Serial measurement of HMPS activity indicated that this increased activity was delayed for several hours after the addition of the mitogen and reached a peak of fivefold higher activity during the second day of culture. Glycolysis increased before HMPS activity and was increased during the entire culture period although it was also maximal during the second day of culture. Pokeweed mitogen also resulted in increased glucose metabolism. The increase was of lesser magnitude than PHA and probably reflected the lower degree of lymphoblastic transformation of pokeweed cultures.These studies demonstrate that lymphocytes undergoing blastic transformation have increased HMPS activity which follows a predictable temporal pattern as does glucose consumption. Also, this study demonstrates that the ionization chamber electrometer technique for the direct measurement of ¹⁴CO₂ can be applied to the study of long-term tissue cultures.     

Leucocyte energy metabolism. V. Glycolytic and hexose monophosphate shunt enzymes in leucocytes from adults and newborn infants.

The activities of ATP-consuming and ATP-producing steps of the Embden-Meyerhof pathway, as well as other glycolytic enzymes (phosphoglucomutase and enolase) and glucose-6-phosphate dehydrogenase are lower in leucocytes from cord blood than in white cells from adults. These results are related to previous observations (reduced anaerobic glycolysis and nitroblue tetrazolium-test in leucocytes from newborn infants) and discussed in connection with the fact that newborn infants are more susceptible to infections than normal adults.     

Regulation and rate of the hexose monophosphate shunt in Rana ridibunda erythrocytes

1. Resting rates of Rana ridibunda erythrocyte glucose consumption and 14CO2 production from 1-14C-glucose were found to be significantly lower than the respective values in human erythrocytes. 2. In the presence of 1-14C-glucose Methylene Blue stimulated 14CO2 production 7-fold, while in the presence of 6-14C-glucose Methylene Blue stimulated 14CO2 production 1.2-fold. 3. The Km of G-6-PD for G-6-P and NADP were 29 and 12 microM, respectively while the Km of 6-PGD for 6-PG and NADP were 83 and 32 microM, respectively. The Ki of G-6-PD and 6-PGD for NADPH were 80 and 12 microM, respectively. 4. Excess amounts of NADP resulted in a significant decrease of 14CO2 production from 1-14C-glucose in total haemolysates. 5. ATP, ADP and fructose diphosphate inhibited both G-6-PD and 6-PGD, the latter being more sensitive than G-6-PD to their inhibitory effect, 2,3-DPG and reduced and oxidized glutathione showed a marked inhibitory effect on 6-PGD, while the phosphorylated trioses inhibited only G-6-PD. 6. Physiological concentrations of oxidized glutathione decreased the inhibition exercised by NADPH on G-6-PD. 7. The possible role of the two dehydrogenases in the regulation of the HMS is discussed.     

Erythrocyte hexose monophosphate shunt is intact in healthy aged humans

The integrity of the erythrocyte (RBC) hexose monophosphate shunt was investigated in a group of 33 healthy elderly individuals by determining their RBC glutathione content, glucose-6-phosphate dehydrogenase activity and glutathione regeneration. When these parameters were compared with those of the controls, 44 young healthy adults, no significant differences were found. This study indicates that the RBC hexose monophosphate shunt in healthy elderly individuals is intact. Factors other than senescence per se should be sought in elderly individuals who exhibit dysfunction of this shunt.     

Mixed function oxidation of hexobarbital and generation of NADPH by the hexose monophosphate shunt in isolated rat liver cells.

Mixed function oxidation of hexobarbital and the generation of NADPH by the hexose monophosphate shunt were studied in isolated rat liver parenchymal cells from phenobarbital-pretreated and untreated animals. In cells isolated from untreated rats, a maximal rate of hexobarbital oxidation of 17 μmol·g^?1 liver wet weight·(60 min)^?1 was observed, while in cells isolated from phenobarbital-pretreated rats a maximal rate of 29 μmol·g^?1 liver wet weight·(60 min)^?1 has been obtained. On the basis of the specific radioactivity at carbon atom 1 of glucose 6-phosphate, fructose 6-phosphate and 6-phosphogluconate, determined by enzymatic decarboxylation, a ratio between NADPH formation via the hexose monophosphate shunt and NADH utilization for hexobarbital oxidation of 6:1 in untreated and 9.5:1 in pretreated cells has been obtained. With phenazine methosulfate the stimulation of NADPH generation via the hexose monophosphate shunt exceeded that observed in the presence of hexobarbital by 329 and 160%, respectively, indicating that the capacity of this pathway is sufficient to provide more reducing equivalents than are required for maximal rates of mixed function oxidation.     

The effect of hexose monophosphate shunt inhibitors on adenohypophyseal and ceruloplasmin reactions to oestrogen.

Oestradiol benzoate, as an aqueous microcrystal suspension, was administered i.m. to rats in doses of 1 mg twice a week; it induced adenohypophyseal hyperplasia and an increase of the thyroxine-binding capacity of the adenohypophyseal proteins in vitro and raised the blood ceruloplasmin level. The simultaneous administration of a hexose monophosphate shunt inhibitor--6-aminonicotinamide (200 microgram/rat/day in food) or oxythiamine (8 mg/rat/day in food)--did not modify the reaction of the adenohypophysis; the hexose monophosphate shunt thus probably does not play a significant role in the adenohypophyseal reaction to oestrogens. By themselves, both inhibitors raised the blood ceruloplasmin level and their effect summated with that of oestradiol. The mechanism of action of the inhibitors is not known, but a nonspecific stress effect leading to an increase in the ceruloplasmin level as an "acute phase protein" is considered to be the most likely.     

Roles of cAMP and free fatty acids on the activity of the hexose monophosphate shunt

Summary Basal glucose utilization by isolated rat adipocytes have been found to be increased ten times in the presence of certain preparations of albumin. In these conditions the effects of several adrenergic agonists and related compounds on glucose oxidation, lipolysis and triacylglycerol synthesis in isolated fat cells have been studied. Oxidation of D(1-¹⁴C) glucose in rat adipocytes was almost completely inhibited by norepinephrine and isoproterenol when added to incubated fat cells. Agents able to modify intracellular AMP cyclic levels by different mechanisms display a similar ability to imitate the effect of lipolytic agents. The inhibition of glucose oxidation due to norepinephrine and isoproterenol is partially reverted by propanolol. Under the same conditions in which norepinephrine and isoproterenol markedly reduced glucose conversion to ¹⁴CO₂, they stimulated lipolysis and triacylglycerol synthesis and in this case propanolol also reverted those actions. However, in these experimental conditions, norepinephrine and isoproterenol did not raise CAMP levels 10 min after hormone addition.It is concluded from these data that glucose oxidation through hexose monophosphate shunt, activation of lipolysis and triacylglycerol synthesis in isolated rat fat cells by lipolytic agents occurs by a mechanism(s) that depend(s) on intracellular free fatty acids levels.     

Stimulation of rat-kidney hexose monophosphate shunt dehydrogenase activity by chronic metabolic acidosis

The effect of chronic metabolic acidosis on the kinetic behaviour of renal glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been investigated. Acidosis induced a significant increase in both enzyme activities at all substrate concentrations used. Saturation curves of both dehydrogenases were hyperbolic with no evidence of sigmoidicity. Maximum activities were found after 7 days of acidosis with no significant change in the Km values. The results suggest that stimulated renal hexose monophosphate dehydrogenases activities are probably due to an increased intracellular concentration of these enzymes. The relationship between these changes and those generated in the metabolic acidosis are also discussed.     

Quinone induced stimulation of hexose monophosphate shunt activity in the guinea pig lens: role of zeta-crystallin.

The response of the hexose monophosphate shunt (HMS) in organ-cultured guinea pig lens to 1,2-naphthoquinone and 5-hydroxy-1,4-naphthoquinone (juglone) has been investigated. Both these compounds, which are substrates of guinea pig lens zeta-crystallin (NADPH:quinone oxidoreductase), were found to cause increases in the rate of 14CO2 production from 1-14C-labelled glucose. Exposure of lenses to 15 microM 1,2-naphthoquinone or 20 microM juglone yielded 5.9- and 7-fold stimulation of HMS activity, respectively. Unlike hydrogen peroxide-induced stimulation of HMS activity, these effects were not abolished by preincubation with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1 nitrosourea (BCNU). While hydrogen peroxide produced substantial decrements in lens glutathione (GSH) levels, incubation with quinones was not associated with a similar reduction in GSH concentration. Protein-bound NADPH content in quinone-exposed guinea pig lenses was decreased, with a concomitant increase in the amounts of free NADP+. This finding supported the involvement of zeta-crystallin bound NADPH in the in vivo enzymic reduction of quinones. Hydrogen peroxide, on the other hand, caused decreases in the level of free NADPH alone, serving to confirm our earlier inference that quinone stimulated increases in the guinea pig lens HMS could be mediated through zeta-crystallin NADPH:quinone oxidoreductase activity.     

Dynamic assessment of hexose monophosphate shunt activity in the intact rabbit lens by proton NMR spectroscopy

A proton nuclear magnetic resonance technique is demonstrated for ascertaining the real-time contribution of the hexose monophosphate shunt to glucose metabolism in the intact incubated rabbit lens. This measurement requires incubation of the tissue in medium supplemented with [1-13C]glucose, and depends on the presence of the 13C label in the methyl position of lactate which creates satellite resonances by way of 13C - 1H spin-spin scalar coupling. The assumptions required to make the measurement are presented. For lenses maintained under control conditions, a basal level corresponding to 5% hexose monophosphate shunt activity was determined. An eight-fold increase in activity was observed under conditions known to stimulate the shunt.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Effects of the hexose monophosphate shunt as mediated by glutathione and ascorbate.

Lipid peroxidation and haemoglobin degradation were the two extremes of a spectrum of oxidative damage in red cells exposed to t-butyl hydroperoxide. The exact position in this spectrum depended on the availability of glucose and the ligand state of haemoglobin. In red cells containing oxy- or carbonmono-oxy-haemoglobin, hexose monophosphate-shunt activity was mainly responsible for metabolism of t-butyl hydroperoxide; haem groups were the main scavengers in red cells containing methaemoglobin. Glutathione, via glutathione peroxidase, accounted for nearly all of the hydroperoxide metabolizing activity of the hexose monophosphate shunt. Glucose protection against lipid peroxidation was almost entirely mediated by glutathione, whereas glucose protection of haemoglobin was only partly mediated by glutathione. Physiological concentrations of intracellular or extracellular ascorbate had no effect on consumption of t-butyl hydroperoxide or oxidation of haemoglobin. Ascorbate was mainly involved in scavenging chain-propagating species involved in lipid peroxidation. The protective effect of intracellular ascorbate against lipid peroxidation was about 100% glucose-dependent and about 50% glutathione-dependent. Extracellular ascorbate functioned largely without a requirement for glucose metabolism, although some synergistic effects between extracellular ascorbate and glutathione were observed. Lipid peroxidation was not dependent on the rate or completion of t-butyl hydroperoxide consumption but rather on the route of consumption. Lipid peroxidation appears to depend on the balance between the presence of initiators of lipid peroxidation (oxyhaemoglobin and low concentrations of methaemoglobin) and terminators of lipid peroxidation (glutathione, ascorbate, high concentrations of methaemoglobin).