Pentose Phosphate Cycle Activity Research Articles

Features of the Hemostasis and Platelets Enzyme Activity in Patients with Different Sensitivity to Acetylsalicylic Acid by the Acute Coronary Syndrome

Aim. To study the features of the state of hemostasis and platelet enzymes activity in acetylsalicylic acid (ASA) sensitive and resistant patients with acute coronary syndrome (ACS). Material and methods. The study included 53 patients (25 men and 28 women) with ACS during the first 24 hours and after 10 days. The control group included 50 healthy volunteers. Before treatment the patients were tested on the sensitivity and resistance to ASA. The indicators of vascularplatelet and plasma hemostasis were evaluated as well as the NAD(P)- dependent dehydrogenases activity in platelets was assessed by the bioluminescent method in the first day of ACS before antiplatelet therapy and on day 10. Results. Increase in spontaneous [1.72 U (1.28-2.72 U) and 1.60 U (1.49-2.78 U), respectively] and ADP-induced [24.4% (21.1-29.8%) and 19.2% (16.1-22.9%), respectively] platelet aggregation, von Willebrand factor activity [159.0% (108.0-190.0%) and 155.0% (149.0-185.1%), respectively] was found in ASA-resistant patients with ACS in 1 and 10 day. Besides the ASA-resistant patients with ACS had very low pentose phosphate cycle and lactate dehydrogenase aerobic activity. They also demonstrated, compared with ASA-sensitive patients, higher intensity of aerobic respiration and the level of NADP-dependent substrate exchange between the tricarboxylic acid cycle and reactions of amino acid metabolism. Conclusion. Despite the dual antiplatelet therapy with ASA and clopidogrel, risk of thrombotic events is saved in ASA resistant patients with ACS. The metabolic changes in platelet influence their aggregation activity and cause an inadequate response to the antiplatelet therapy in ACS patients.

Characterization of the metabolic phenotype of chronically activated lymphocytes

Activated lymphocytes proliferate, secrete cytokines, and can make antibodies. Normally activated B and T cells meet the bioenergetic demand for these processes by up-regulating aerobic glycolysis. In contrast, several lines of evidence suggest that pathogenic lymphocytes in autoimmune diseases like lupus meet ATP demands through oxidative phosphorylation. Using (13)C-glucose as a stable tracer, we found that splenocytes from mice with lupus derive the same fraction of lactate from glucose as control animals, suggesting comparable levels of glycolysis and non-oxidative ATP production. However, lupus splenocytes increase glucose oxidation by 40% over healthy control animals. The ratio between pentose phosphate cycle (PPC) activity and glycolysis is the same for each group, indicating that increased glucose oxidation is due to increased activity of the TCA cycle in lupus splenocytes. Repetitive stimulation of cultured human T cells was used to model chronic lymphocyte activation, a phenotype associated with lupus. Chronically activated T cells rely primarily on oxidative metabolism for ATP synthesis suggesting that chronic antigen stimulation may be the basis for the metabolic findings observed in lupus mice. Identification of disease-related bioenergetic phenotypes should contribute to new diagnostic and therapeutic strategies for immune diseases.

Non‐oxidative cerebral carbohydrate metabolism

Kety and Schmidt's application of the Fick Principle in the 1940s has served as the technical basis for the human cerebral metabolic studies still in use today. In this technique, arterial and jugular venous blood samples are collected, analysed for oxygen, glucose and lactate, and an arterial-jugular venous difference (AVD) is calculated. The cerebral metabolic rate is then calculated as the product of the AVD and the simultaneous measurement of cerebral blood flow. To further understand the relationship between the cerebral metabolic components, an oxygen–carbohydrate index (OCI), calculated by , or a metabolic ratio (OGI), calculated by , is used to determine how much fuel substrate is oxidized, where a value of 6 represents the complete oxidation of glucose. Glucose is considered to be the preferred cerebral oxidative fuel source under normal resting conditions, with 90% of brain glucose used oxidatively. The remaining 10% of glucose goes to non-oxidative uses, such as glycogen formation and other metabolic processes (Siesjo, 1978). On the other hand, in normal resting volunteers, the arterial jugular venous difference for lactate (AVDlac) is negative, indicating that the brain is producing and releasing lactate primarily through glycolysis. However, strenuous exercise or traumatic brain injury studies reveal that the brain has a positive AVDlac, and is therefore taking up lactate (Ide et al. 1999; Glenn et al. 2003; Soustiel & Sviri, 2007; Dalsgaard, 2006). Furthermore, the OCI/OGI can be influenced by brain activation or brain insult, with values dropping significantly due to a greater uptake of glucose and lactate relative to oxygen. In this issue of The Journal of Physiology, Larsen et al. (2009) have examined the role that the catecholamines adrenaline and noradrenaline may play in cerebral carbohydrate uptake and metabolism in normal healthy subjects. This work is a continuation of earlier studies, which found that the non-selective β-adrenergic antagonist propranolol blocked the reduction of the OCI in exercising humans (Larsen et al. 2008). In Larsen's current study, subjects were infused with either adrenaline or noradrenaline, and arterial and jugular venous blood was sampled at fixed times, allowing for the calculation of variables such as AVD, OGI and OCI. The overall findings of the current study revealed that adrenaline caused an increase in glucose uptake and a small uptake of lactate. On the other hand, noradrenaline caused an increase in glucose uptake alone, and only at the lower doses. Furthermore, adrenaline, but not noradrenaline, caused a decrease in both OCI and OGI. Therefore, their results indicate that a β2-adrenergic mechanism is responsible for the increased carbohydrate uptake. In conclusion, several important questions are raised by this research. Does adrenaline cross the blood brain barrier? As the authors suggest, are the changes in cerebral metabolism from earlier brain activation studies of Fox et al. (1988) caused by a catecholamine stress response? And ultimately, what is the fate of the excess carbohydrate uptake, i.e. where is the lactate going and through which pathways is glucose being metabolized? As the authors state, studies using stable isotopes, such as 13C-labelled glucose and lactate, are needed to further define and help answer these questions. In a recent paper by Dusick et al. (2007), [1,2-13C2]glucose was infused into traumatically brain injured patients and normal subjects. In this study, the pentose phosphate cycle activity was significantly increased in the trauma patient group as compared to normal controls, accounting in part for the increased glucose uptake seen during decreased OGI. Thus, Larsen's current study has important implications for understanding catecholamine induced cerebral responses, extending beyond exercise physiology. In disease states, such as trauma, where catecholamine levels are elevated, these findings may shed new light on the complexity of cerebral carbohydrate metabolism.

Character of Viscero- and Somatomotor Interactions in Rat Pups at Changes of the Level of Activity of Adrenergic Structures

In the 5–10-day old rat pups, in the course of simultaneous recording of ECG, respiration rate (RR), and spontaneous periodic motor activity (SPMA) there were determined parameters of the studied processes and their correlations at changes of levels of activity of adrenergic structures. As adrenoactive agents, the sympathomimetic amphetamine, α- and β-adrenolytics phentolamine and propranolol, as well as the dopamine receptor blocker haloperidol were used. It has been established that injection of amphetamine significantly increases intensity of the decasecond rhythm in the activity pattern of excitable structures. However, the action both on the α- and β-adrenoreceptors and on dopamine receptors produces no significant changes of this rhythm parameters. Based on this, it can be suggested that an increase of the decasecond rhythm is due to the ability of amphetamine to inhibit the pentose phosphate cycle activity. Administration of adrenolytics decreases RR and heart rate (HR). Amphetamine restores them by a simultaneous increase of pathological disturbances of the respiration rhythm, which appear at injections of sympatholytics. Blockade of dopamine receptors leads to replacement of motor activity fires by jerks following in the rhythm close to the previous one. As a rule, at the blockade of α-adrenoreceptors the SPMA pattern is deprived of obvious rhythmic components, whereas at the blockade of β-adrenoreceptors, they are preserved. Analysis of the obtained data indicates that the respiration frequency modulation that increases at action on adrenoreactive structures correlates with the motor activity changes. However, there are several peculiarities due to action on various types of adrenoreceptors. Of the leading significance in the normal intersystem interaction is preservation of balance between individual adrenergic structures.

Reduction and uptake of methylene blue by human erythrocytes.

A thiazine dye reductase has been described in endothelial cells that reduces methylene blue (MB), allowing its uptake into cells. Because a different mechanism of MB uptake in human erythrocytes has been proposed, we measured MB uptake and reduction in this cell type. Oxidized MB (MB(+)) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced MB was then taken up by the cells and partially oxidized to MB(+). Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB(+) <5 microM selectively oxidized NAD(P)H, higher concentrations also oxidized both glutathione (GSH) and ascorbate, especially in the absence of d-glucose. MB(+)-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB(+)-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and it did not affect cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB(+), which was abolished by pretreating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB(+), that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB(+) reduction may correspond to MB(+)-dependent NAD(P)H reductase activity in erythrocyte ghosts.

Heat shock inactivates cellular antioxidant defenses against hydrogen peroxide: protection by glucose

Hyperthermia is used in cancer treatment and potentiates the cytotoxicity of radiation and certain chemotherapy drugs. The mechanism(s) of heat killing and those involved in heat potentiation of cytotoxic modalities are not understood. This study examines whether heat shock causes a redox imbalance, leading to oxidative changes in Chinese hamster ovary cells. Decreases in the GSH/GSSG ratio reflected an oxidative imbalance in heated (42°C) and in H 2O 2-challenged cells. Glucose provided protection against these changes. Glucose also protected cells against cytotoxicity of H 2O 2 and/or hyperthermia (42 to 43°C). Glucose appears to protect cells against H 2O 2 and heat shock by providing NADPH through its metabolism via the pentose phosphate cycle (PC). When cells were deprived of glucose, there was a marked decrease in the GSH/GSSG ratio and in NADPH levels, indicating a severe redox imbalance. Glucose deprivation caused cell death, which was consistent with increased accumulation of H 2O 2, since three distinct H 2O 2-detoxifying systems (N-acetyl-L-cysteine, sodium pyruvate, and catalase) rescued cells against cytotoxicity. Nontoxic levels of H 2O 2 stimulated a corresponding increase in both PC activity and NADPH levels. NADPH levels and basal activity of the PC increased at 42°C. However, the oxidant-stimulated increases in PC activity and NADPH levels were lost in heated cells. Therefore, heat shock inactivates an important cellular defense mechanism against oxidants. These findings suggest that heat shock may enhance the cytotoxicity of oxidants by inhibiting increases in PC activity following oxidative stress. These data are potentially relevant to understanding the potentiation of cytotoxicity of radiation and oxidant-generating drugs by heat shock, used in combined modality cancer treatment.

Glucose-6-phosphate dehydrogenase and the oxidative pentose phosphate cycle protect cells against apoptosis induced by low doses of ionizing radiation.

The initial and rate-limiting enzyme of the oxidative pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), is inhibited by NADPH and stimulated by NADP(+). Hence, under normal growth conditions, where NADPH levels exceed NADP(+) levels by as much as 100-fold, the activity of the pentose phosphate cycle is extremely low. However, during oxidant stress, pentose phosphate cycle activity can increase by as much as 200-fold over basal levels, to maintain the cytosolic reducing environment. G6PD-deficient (G6PD(-)) cell lines are sensitive to toxicity induced by chemical oxidants and ionizing radiation. Compared to wild-type CHO cells, enhanced sensitivity to ionizing radiation was observed for G6PD(-) cells exposed to single-dose or fractionated radiation. Fitting the single-dose radiation response data to the linear-quadratic model of radiation-induced cytotoxicity, we found that the G6PD(-) cells exhibited a significant enhancement in the alpha component of radiation-induced cell killing, while the values obtained for the beta component were similar in both the G6PD(-) and wild-type CHO cell lines. Here we report that the enhanced alpha component of radiation-induced cell killing is associated with a significant increase in the incidence of ionizing radiation-induced apoptosis in the G6PD(-) cells. These data suggest that G6PD and the oxidative pentose phosphate shunt protect cells from ionizing radiation-induced cell killing by limiting the incidence of radiation-induced apoptosis. The sensitivity to radiation-induced apoptosis was lost when the cDNA for wild-type G6PD was transfected into the G6PD(-) cell lines. Depleting GSH with l-BSO enhanced apoptosis of K1 cells while having no effect in the G6PD(-) cell line

Correlation between Glutathione and Stimulation of the Pentose Phosphate Cyclein Situin Chinese Hamster Ovary Cells Exposed to Hydrogen Peroxide

The effect of glutathione on stimulation of pentose phosphate cycle activity during oxidative challenge was evaluated in intact Chinese hamster ovary cellsin situ.Glutathione was depleted to varying levels withL-buthionine-[S,R] sulfoximine. The level of stimulation of pentose phosphate cycle activity by exogenous H2O2(4 μmol/107cells) was dependent on the time of pretreatment withL-buthionine-[S,R] sulfoximine and was proportional to the total glutathione concentration. This was not related to the amount of GSSG, since its level was exceedingly low under conditions where H2O2stimulated pentose phosphate cycle activity. The amount of GSSG in cells increased after exposure to 10-fold higher concentrations of H2O2under conditions where there was no stimulation of pentose phosphate cycle activity above the basal level. Paraquat caused stimulation of pentose phosphate cycle activity which was independent ofL-buthionine-[S,R] sulfoximine pretreatment and of the glutathione content of cells. The stimulatory effects of both oxidants on pentose phosphate cycle activity appeared to be independent of glutathione reductase activity since they were unaffected in cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea. The inhibitory effect ofL-buthionine-[S,R] sulfoximine on stimulation of pentose phosphate cycle activity by H2O2did not appear to be due to the inhibitor itself, but rather to the overall level of glutathione. Glutathione could have a role in maintaining activity of the pentose phosphate cycle at a level which is appropriate for the severity of the oxidative challenge as well as for the capacity of the cellular antioxidant defenses.

The Role of Glucose in Cellular Defenses Against Cytotoxicity of Hydrogen Peroxide in Chinese Hamster Ovary Cells

The presence of glucose in the cellular environment influences the response of cells to hydrogen peroxide. This study examines the effect of glucose on clonogenic cell survival of Chinese hamster ovary cells exposed to micromolar concentrations of exogenous hydrogen peroxide. Exposure to hydrogen peroxide (20 μmol/10 7 cells) resulted in considerable cytotoxicity that was unaffected by the presence or absence of glucose. However, glucose protected the cells from killing induced by milder exposure (1 μmol/10 7 cells) to the oxidant causing a shift in the dose-response curve. This effect was considered in terms of glucose metabolism via the pentose phosphate cycle. A low nontoxic concentration of hydrogen peroxide (0.1 μmol/10 7 cells) markedly increased pentose phosphate cycle activity in normal cells. Clonogenic survival and activity of the cycle for cells depleted of total glutathione to about 8.6% of its initial value and/or with catalase activity reduced to about 10% of control levels were also determined. Neither of these modifications alone completely abolished the protective effect of glucose. Efficacy of glucose protection against cytotoxicity of hydrogen peroxide diminished in cells depleted of glutathione, and this was not accompanied by any detectable increase in pentose phosphate cycle activity above the control level. Cells depleted of catalase alone had a profile of survival and pentose phosphate cycle activity similar to that of control cells when exposed to hydrogen peroxide. Cells depleted of both glutathione and catalase were almost as sensitive to hydrogen peroxide as the cells incubated without glucose. They also did not express any detectable increase in pentose phosphate cycle activity. Survival of those cells, when exposed to hydrogen peroxide, was almost the same regardless if glucose was present or not. These results demonstrate an important role for the glutathione redox cycle, catalase, and the pentose phosphate cycle in protection against hydrogen peroxide in Chinese hamster ovary cells. They confirm the essential role of glucose and pentose phosphate cycle activity for the detoxification of hydrogen peroxide via the glutathione redox cycle. The data suggest that the ability of catalase to metabolise peroxide may also depend on metabolism of glucose via the pentose phosphate cycle. A clear understanding of the protective mechanisms in cells against hydrogen peroxide has many applications since this common reactive oxygen species is implicated in several pathophysiologies and in the action of certain chemotherapeutic drugs.

Glucose-6-phosphate: A key compound in glycogenosis I and favism leading to hyper- or hypolipidaemia

The glycogen storage disorders (GSD)-I, -III, -VI and -VIII are associated with hypertriglyceridaemia or mixed hyperlipidaemia which poses the question whether these patients have an increased risk for atherosclerosis. The atherogenicity of triglycerides has remained controversial, while increased plasma cholesterol levels are generally accepted as a significant risk factor for coronary heart disease. However, clinical data show that one has to differentiate between metabolic conditions where triglycerides are atherogenic and those which are not significantly related to early onset of atherosclerosis but may cause other disorders such as pancreatitis. Among the disorders of carbohydrate metabolism patients with diabetes mellitus frequently have enhanced plasma triglycerides associated with a higher risk for coronary heart disease, while patients with certain types of glycogen storage disease have high triglyceride levels but do not seem to have an enhanced risk for atherosclerosis. Here we have compared the biochemical abnormalities and the atherogenic risk of three different disorders of glucose metabolism including GSD-I (glucose-6-phosphatase deficiency), favism (glucose-6-phosphate dehydrogenase deficiency), and diabetes mellitus which are related to either hyper- or hypolipidaemia. The available data indicate that glucose-6-phosphate (Glc-6-P) is a central molecule in cellular glucose metabolism which critically influences pentose phosphate cycle activity and, via NADPH2-generation, regulates glutathione peroxidase activity for radical detoxification and also cholesterol and triglyceride synthesis. Radical detoxification is a major protective factor for cell membrane integrity and together with an appropriate renewal of membrane lipids may protect against the development of atherosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The pentose phosphate cycle is regulated by [formula omitted] ratio in rat liver

The changes in the activity of the pentose phosphate cycle produced by the activation or inhibition of different NADPH-consuming pathways have been studied. The inhibition of fatty acid synthesis by kynurenate produced to the same extent, inhibition of the pentose phosphate cycle activity and an increase (about twofold) in the NADPH NADP ratio. The addition of ter-butyl-hydroperoxide or paraquat, which is metabolized via NADPH-consuming pathways, produced the activation of the pentose phosphate cycle and a decrease in the NADPH/NADP ratio (about threefold). The plot of the NADPH NADP ratio versus the pentose phosphate cycle activity gave a straight line with a regression index of 0.999. The regulation of the pentose phosphate cycle mainly by the intracellular NADPH NADP ratio is discussed.

Glutathione oxidation and activation of pentose phosphate cycle during hydroperoxide metabolism. A comparison of livers from fed and fasted rats.

Perfusion of livers from fed and fasted rats with 0.07--0.1 mM t-butyl hydroperoxide for 15 min decreased the levels of reduced glutathione (GSH) by 1.5 mumol/g liver in both nutritional states. Glutathione disulfide (GSSG) was increased by 70 and 140 nmol/g liver and glutathione mixed disulfides enhanced by 45 and 150 nmol/g liver in livers from fed and fasted animals, respectively. The ratio of GSH/GSSG was decreased from 243 to 58 in fed animals, and from 122 to 8 in fasted animals. The increase of GSSG and the mixed disulfides was nearly parallel until an apparently critical low GSH content of 1.5 mumol/g was reached. Only in livers from fasted rats 14CO2-production from [1-14C]glucose was stimulated upon t-butyl hydroperoxide infusion at the employed rates. Flux of glucose through pentose phosphate cycle rose from 8 to 12% of glucose utilization via glycolysis, whereas in livers from fed animals this portion remained unchanged at 8% Dithio-erythritol reversed pentose phosphate cycle activity as well as GSSG and protein-bound glutathione contents to the original levels. In livers from fasted rats the activity of glucose-6-phosphate dehydrogenase was increased by 34% by t-butyl hydroperoxide infusion.

The effect of insulin-stimulated pentose phosphate cycle activity on cellular glutathione content in rat adipocytes.

Insulin is known to stimulate intracellular H2O2 production in rat adipocytes. This H2O2 could in turn stimulate the pentose phosphate cycle by oxidizing GSH and shifting the redox state of the cells. However, insulin had no effects on cell GSH content or GSSG in buffer other than as related to changes in medium glucose. On the contrary, in the presence of an active pentose phosphate cycle, insulin tended to reverse the fall in glutathione content induced with the catalase inhibitor 3-amino-1,2,4-triazole, the oxidant t-butyl hydroperoxide and the sulfhydryl blocker N-ethylmaleimide. It was also found that insulin-stimulated H2O2 production could be blocked under conditions in which the effect of the hormone on the pentose phosphate cycle persisted. These results suggest that stimulation of the pentose phosphate cycle by insulin is not related to increased H2O2 generation, rather that activation of the pentose phosphate cycle by the hormone may provide NADPH for regeneration of depleted GSH.

The mechanism of the insulin-like effects of ionic zinc.

The insulin-like effects of ionic zinc (Zn2+) were studied in isolated rat adipocytes. Concentrations of Zn2+ between 250 and 1000 microM stimulated 3-O-methylglucose transport and glucose metabolism to CO2, glyceride-fatty acid, and glyceride-glycerol. Selective stimulation of the pentose phosphate cycle was observed since a Zn2+-induced increase in glucose carbon 1 oxidation persisted even when glucose transport was blocked with 50 microM cytochalasin B or when transport was no longer rate-limiting for metabolism at high concentrations of glucose. Enhanced pentose phosphate cycle activity may have been due to a selective inhibition of glutathione reductase by the ion, which was also accompanied by a fall in cellular glutathione content. Zn2+ also inhibited lipolysis stimulated by the beta-adrenergic agent ritodrine in the absence of glucose. The effects of Zn2+ on glucose oxidation and stimulated rates of lipolysis were inhibited by extracellular catalase, indicating that they were largely a result of H2O2 generation. The H2O2 production appeared for the most part to be caused by zinc-catalyzed autoxidation of sulfhydryl groups present on external cell membranes, although involvement of sulfhydryl groups on bovine serum albumin in the buffer could also have contributed. The insulin-like effects of Zn2+ in adipocytes are therefore caused not only by direct effects of the ion on intracellular metabolism but also by indirect effects related to H2O2 generation.

Reductive Pentose Phosphate Cycle and Oxidative Carbohydrate Metabolic Activities in Pea Chloroplast Stroma Extracts

Oxidative and reductive carbohydrate metabolism was studied in reaction mixtures based on chlorophyll-free stromal extracts from chloroplasts of Pisum sativum. A new assay system for the reductive pentose phosphate cycle was characterized.When provided with ATP, an enzymic ATP-regenerating system and reduced pyridine nucleotide, substantial rates of CO(2) fixation and pyridine nucleotide oxidation were observed following the addition of millimolar concentrations of reductive pentose phosphate cycle intermediates. The reduced pyridine nucleotide requirement could be met either by NADPH, or by NADH plus the added enzymes NAD(+)-glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase. When the assay system was primed with small amounts of reductive pentose phosphate cycle intermediates, lower rates of pyridine nucleotide oxidation were observed, but turnover of the complete cycle was demonstrated. Autocatalytic effects were not evident. The optimum pH and Mg concentrations for cycle turnover were similar to those believed to exist in the stroma of intact chloroplasts in the light.Oxidative carbohydrate metabolism was studied by supplying oxidized pyridine nucleotide and measuring its rate of reduction in the presence of sugar phosphates. Glycolytic activity, estimated as the rate of fructose-6-phosphate entry to the phosphofructokinase reaction was 2.7 micromoles per milligram chlorophyll per hour when fructose-6-phosphate was provided as substrate. Evidence based on glucose-6-phosphate and ribose-5-phosphate-dependent NADP(+) reduction showed that the oxidative pentose phosphate cycle was also active. Apparent oxidative pentose phosphate cycle turnover in the presence of ribose-5-phosphate, estimated as the rate of glucose-6-phosphate entry to the glucose-6-phosphate dehydrogenase reaction, was 1.7 micromoles per milligram chlorophyll per hour.It was concluded that under the defined conditions, reductive pentose phosphate cycle activity could be measured without interference from oxidative carbohydrate metabolism in this experimental system.

The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 m m) had higher GSH contents (3.2 μg/10 6 cells) than in the absence of glucose (2.3 μg/10 6 cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H 2O 2 to a concentration of 60 μ m to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 m m glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 m m GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 m m NADP + was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP +] ratios may play a significant role in short-term control of the pentose phosphate cycle.

Relationship of Malic Enzyme Activity to Fatty Acid Synthesis and the Pathways of Glucose Catabolism in Developing Rat Liver

The rates of fatty acid synthesis and the activity of several enzymes involved in lipid and carbohydrate metabolism were determined in livers from fetal, suckling, weanling and maternal rats. An estimate of the proportion of glucose catabolized via the pentose phosphate cycle and the Embden-Meyerhoff pathway was made using 14C- and 3H-labeled glucose. The contribution of pentose phosphate cycle-generated reducing equivalents to fatty acid synthesis was assessed using glucose-3-3H. Developmental changes in the activity of hepatic malic enzyme were not related to developmental changes in the rate of fatty acid synthesis as might be expected if this enzyme functioned to provide NADPH for fatty acid synthesis. Malic enzyme activity did not correlate with pentose phosphate cycle activity or with utilization for fatty acid synthesis of NADPH generated via this pathway.

Reversible inhibition of the calvin cycle and activation of oxidative pentose phosphate cycle in isolated intact chloroplasts by hydrogen peroxide

Hydrogen peroxide (6x10(-4) M) causes a 90% inhibition of CO2-fixation in isolated intact chloroplasts. The inhibition is reversed by adding catalase (2500 U/ml) or DTT (10 mM). If hydrogen peroxide is added to a suspension of intact chloroplasts in the light, the incorporation of carbon into hexose- and heptulose bisphosphates and into pentose monophosphates is significantly increased, whereas; carbon incorporation into hexose monophosphates and ribulose 1,5-bisphosphate is decreased. At the same time formation of 6-phosphogluconate is dramatically stimulated, and the level of ATP is increased. All these changes induced by hydrogen peroxide are reversed by addition of catalase or DTT. Additionally, the conversion of [(14)C]glucose-6-phosphate into different metabolites by lysed chloroplasts in the dark has been studied. In presence of hydrogen peroxide, formation of ribulose-1,5-bisphosphate is inhibited, whereas formation of other bisphosphates,of triose phosphates, and pentose monophosphates is stimulated. Again, DTT has the opposite effect. The release of (14)CO2 from added [(14)C]glucose-6-phosphate by the soluble fraction of lysed chloroplasts via the reactions of oxidative pentose phosphate cycle is completely inhibited by DTT (0.5 mM) and re-activated by comparable concentrations of hydrogen peroxide. These results indicate that hydrogen peroxide interacts with reduced sulfhydryl groups which are involved in the light activation of enzymes of the Calvin cycle at the site of fructose- and sedoheptulose bisphophatase, of phosphoribulokinase, as well as in light-inactivation of oxidative pentose phosphate cycle at the site of glucose-6-phosphate dehydrogenase.

6-Phosphogluconate/glucose-6-phosphate ratio in rat pancreatic islets during inhibition of insulin release by exogenous insulin.

Inhibition of glucose-stimulated insulin release by exogenous insulin has been demonstrated in pancreatic islets to be associated with a decrease of the NADPH/NADP ratio and the pentose-phosphate cycle activity. Batches of five islets were incubated for 15 and 90 minutes in 1 ml. of KRB buffer with 2 per cent albumin containing 3 mg./ml. glucose and 0, 200, 400, or 800 microU./ml. of rat insulin, and the glucose-6-phosphate (G6P) and 6-phosphogluconate (6PG) contents were determined by enzymatic cycling. In response to a rise in the concentration of insulin, the 6PG/G6P ratio decreased. A close relationship was observed between this decrease of 6PG/G6P ratio and the net insulin release, the absolute rate of glucose oxidation via the pentose phosphate cycle, and the NADPH/NADP ratios measured under similar conditions. The results suggest that exogenous insulin, directly or indirectly, regulates the pentose cycle activity in the pancreatic islets at the G6P dehydrogenase step.

Thiamin Deficiency and the Pentose Phosphate Cycle in Rats: Intracerebral Mechanisms

Previous studies have shown that transketolase activity is decreased in the brains of thiamin deficient rats. This study assesses the effect of decreased transketolase levels on the activity of the pentose phosphate cycle in murine thiamin deficient cortex and brainstem. Thiamin deficiency was produced in newborn and adult rats by either pyrithiamin administration or by feeding a low thiamin diet. Newborn rats were killed at 22 days of age, and adults were killed at the onset of moderate to severe nurological signs. Cortices and brainstems from thiamin deficient and control rats were analyzed for activity of the two regulatory enzymes of the pentose phosphate cycle, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Flux through the pathway was measured by the differentially labeled glucose technique in the brainstems of deficient and control adult rats. In both the brainstem and cortex of thiamin dificient rats, areas in which transketolase activity was decreased up to 65%, the activities of the two regulatory enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were unaltered. Further, flux through the pentose phosphate cycle was not decreased as compared to pair-fed control rats. These data do not support the hypothesis that in thiamin dificient rats, a decrease in cerebral transketolase activity leads to a diminished pentose phosphate cycle activity.