The effects of arachidonic and other fatty acids on canine neutrophil metabolism

The effect of arachidonic acid on the metabolic activity and chemiluminesence of canine neutrophils was investigated to gain further insight into its role in the neutrophil metabolic burst. Arachidonic acid was found to stimulate metabolic activity and luminol-augmented chemiluminescence. The increased metabolic activity was detected by both oxygen uptake measurements and assays of hexose monophosphate shunt activity. An inhibitor of lipoxygenase and cyclooxygenase,5, 8, 11, 14-eicosatetraynoic acid prevented the hexose monophosphate shunt response to arachidonic acid. Aspirin or indomethacin, blockers of cyclooxygenase, inhibited chemiluminescence but failed to block the metabolic response to arachidonic acid. Since superoxide dismutase and 2-deoxyglucose, a blocker of glucose metabolism, inhibited the chemiluminescent response of neutrophils to arachidonic acid, it is likely that oxygen radicals produced via the hexose monophosphate shunt are required for the chemiluminescent reaction. In addition it was found that inhibition of cyclooxygenase activity blocked chemiluminescence but not the metabolic stimulation induced by sodium fluoride, suggesting that the chemiluminescence stimulated by sodium fluoride is associated with endogenous fatty acid stores. From these studies it can be concluded that arachidonic acid products of the cyclooxygenase pathway do not play a significant role in the metabolic response of neutrophils when arachidonic acid or sodium fluoride is the stimulant while the lipoxygenase pathway appears to be involved. The metabolic response is not linked to the chemical reaction that causes neutrophil, chemiluminesence, although the chemiluminescent response depends on hexose monophosphate shunt activity and presumably the oxygen radicals that ultimately result from that process.     

Increased heat production proportional to oxygen consumption in human neutrophils activated with phorbol-12-myristate-13-acetate

The heat produced by neutrophils was measured with a flow microcalorimeter. 02 consumption, ATP concentration, lactic acid production and 14CO2 production from oxidation of [1-(14)C]-glucose [6-(14)C]-glucose and [U-14C]-glucose were evaluated. Experiments were also carried out in the presence of the metabolic inhibitors, N-ethylmaleimide and NaF. Heat effects were correlated to the enthalpy change of aerobic and anaerobic glucose catabolism. Two different heat contributions related to two different nonmitochondrial 02 reduction pathways are present during the metabolic burst. Theoretical and experimental data indicate that the reducing power is derived from the catabolism of glucose both through the hexose monophosphate shunt and glycolysis.     

Carbohydrate metabolism in the rat peritoneal macrophages

Rat peritoneal macrophages derive energy differently from other tissues. Resting rat peritoneal macrophages have been taken for the present investigation. Lactate produced by extracellular glycolysis in the peritoneal lavage fluid, is readily converted into pyruvate by resting peritoneal macrophages and is oxidised in mitochondria. Glycolytic enzymes other than phosphoglucoisomerase and lactate dehydrogenase could not be substantially demonstrated. Glucose-6-phosphate dehydrogenase was detected. The presence of glucose-6-phosphate dehydrogenase along with phosphoglucoisomerase indicates the operation of the hexose monophosphate shunt as a pathway supplementary to glycolysis. Resting rat peritoneal macrophages thus appear to utilize extracellular lactate as their main energy source instead of glucose, bypass glycolysis and have active hexose monophosphate shunt.     

Mitochondrial proteome: cancer-altered metabolism associated with cytochrome c oxidase subunit level variation

Shifts in metabolism associated with tumorigenesis were first noted by Otto Warburg in the 1920s. In the ensuing decades many examples of the phenomenon have been elucidated while the underlying molecular mechanism has remained elusive. As the enzyme complex at the crux of oxidative phosphorylation, cytochrome c oxidase is uniquely positioned to have a very high impact on cellular metabolism. In this study, we test the hypothesis that there is a specific association between altered cytochrome c oxidase subunit levels and altered metabolism by combining the technique of reverse-phase protein microarray with radiolabeled glucose metabolic studies. Such a relationship is observed with five different cell lines, two of which (1542N and 1542T) are a matched set of normal and tumor-based lineages derived from the same prostate gland. By measuring the [(14)C]carbon dioxide production of a cell line metabolizing [1-(14)C]glucose and comparing those measurements to values obtained for the same cell line metabolizing [6-(14)C]glucose, we determined the relative utilization of the hexose monophosphate shunt and glycolysis progressing through the Krebs cycle metabolic pathway in each cell line. In all cases there is an increased utilization of hexose monophosphate shunt relative to glycolysis progressing through the Krebs cycle in tumor derived relative to normal derived cell lines. Additionally, there is an associated increase in the ratio of nuclear encoded cytochrome c oxidase subunits to mitochondrially encoded cytochrome c oxidase subunits in the tumor-derived cell lines. These results demonstrate an alteration in subunit levels of a single enzyme complex (cytochrome c oxidase) commensurate with tumor-altered metabolism.     

Hexose monophosphate shunt,the role of its metabolites and associated disorders: A review

The hexose monophosphate (HMP) shunt acts as an essential component of cellular metabolism in maintaining carbon homeostasis. The HMP shunt comprises two phases viz. oxidative and nonoxidative, which provide different intermediates for the synthesis of biomolecules like nucleotides, DNA, RNA, amino acids, and so forth; reducing molecules for anabolism and detoxifying the reactive oxygen species during oxidative stress. The HMP shunt is significantly important in the liver, adipose tissue, erythrocytes, adrenal glands, lactating mammary glands and testes. We have researched the articles related to the HMP pathway, its metabolites and disorders related to its metabolic abnormalities. The literature for this paper was taken typically from a personal database, the Cochrane database of systemic reviews, PubMed publications, biochemistry textbooks, and electronic journals uptil date on the hexose monophosphate shunt. The HMP shunt is a tightly controlled metabolic pathway, which is also interconnected with other metabolic pathways in the body like glycolysis, gluconeogenesis, and glucuronic acid depending upon the metabolic needs of the body and depending upon the biochemical demand. The HMP shunt plays a significant role in NADPH₂ formation and in pentose sugars that are biosynthetic precursors of nucleic acids and amino acids. Cells can be protected from highly reactive oxygen species by NADPH ₂. Deficiency in the hexose monophosphate pathway is linked to numerous disorders. Furthermore, it was also reported that this metabolic pathway could act as a therapeutic target to treat different types of cancers, so treatments at the molecular level could be planned by limiting the synthesis of biomolecules required for proliferating cells provided by the HMP shunt, hence, more experiments still could be carried out to find additional discoveries.     

Glycolysis in the eye tissues of the rabbit in ontogeny. I. The enzymes of glycolysis and hexosemonophosphate shunt]

The activity of the enzymes of glycolysis (phosphofructokinase, aldolase, pyruvate kinase, lactate dehydrogenase) and hexose monophosphate shunt (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) was determined in the eye tissues of the rabbit at different stages of ontogenesis. The activity of these enzymes in the retina was shown to be higher than in other eye tissues. In the uveal tract (iris, ciliary bodies, uvea) the activity of glycolytic enzymes changes with the age. The greatest changes in the activity of enzymes were found during the period of the opening of eyelids. The activity of the enzymes of hexose monophosphate shunt in the eye tissues increases with the age. The relative activity of dehydrogenases of the hexose monophosphate shunt after the establishment of visual function is, however, not high and does not exceed that of phosphofructokinase and pyruvate kinase in the eye tissues of the rabbit.     

Metabolomic and proteomic analysis of a clonal insulin-producing beta-cell line (INS-1 832/13)

Metabolites generated from fuel metabolism in pancreatic beta-cells control exocytosis of insulin, a process which fails in type 2 diabetes. To identify and quantify these metabolites, global and unbiased analysis of cellular metabolism is required. To this end, polar metabolites, extracted from the clonal 832/13 beta-cell line cultured at 2.8 and 16.7 mM glucose for 48 h, were derivatized followed by identification and quantification, using gas chromatography (GC) and mass spectrometry (MS). After culture at 16.7 mM glucose for 48 h, 832/13 beta-cells exhibited a phenotype reminiscent of glucotoxicity with decreased content and secretion of insulin. The metabolomic analysis revealed alterations in the levels of 7 metabolites derived from glycolysis, the TCA cycle and pentose phosphate shunt, and 4 amino acids. Principal component analysis of the metabolite data showed two clusters, corresponding to the cells cultured at 2.8 and 16.7 mM glucose, respectively. Concurrent changes in protein expression were analyzed by 2-D gel electrophoresis followed by LC-MS/MS. The identities of 86 spots corresponding to 75 unique proteins that were significantly different in 832/13 beta-cells cultured at 16.7 mM glucose were established. Only 5 of these were found to be metabolic enzymes that could be involved in the metabolomic alterations observed. Anticipated changes in metabolite levels in cells exposed to increased glucose were observed, while changes in enzyme levels were much less profound. This suggests that substrate availability, allosteric regulation, and/or post-translational modifications are more important determinants of metabolite levels than enzyme expression at the protein level.     

Trophoblast contact deactivates human neutrophils

Trophoblasts are fetal epithelial cells that form an interface between mother and offspring. To evaluate their anti-inflammatory capacity, we tested the hypothesis that trophoblasts deactivate neutrophils using single-cell assays. Several biophysical (Ca2+ and NAD(P)H oscillation frequency) and physiological (oxidant production) markers of activated neutrophils revert to a nonactivated phenotype as activated cells make contact with trophoblasts. Indistinguishable results were obtained using syncytiotrophoblasts and in experiments using trophoblasts and neutrophils from the same mother to recapitulate the semiallogeneic system. These changes suggest reduced hexose monophosphate shunt (HMS) activity. We discovered that two metabolic regulatory points, glucose transport and HMS enzyme trafficking, are affected by trophoblasts. This restriction in HMS activity deactivates neutrophils, thereby limiting oxidative DNA damage within trophoblasts.     

Oxidative Enzymes and Pathways of Hexose and Triose Metabolism in Chlorella

Cell-free preparations of Chlorella pyrenoidosa Chick, van Niel's strain, were assayed for oxidative enzymes, utilizing isotopic and spectrophotometric techniques. The enzyme activity of heterotrophic and autotrophic cells was compared. The study was divided into categories, one concerned with the spectrophotometric detection of enzymes involved in the initial reactions of glycolysis and the hexose monophosphate shunt, and the other with the direct oxidation of glucose as compared with that oxidized via glycolysis. The reduction of pyridine nucleotides in crude extracts was studied with glucose, glucose-6-phosphate, 6-phosphogluconate, and fructose-1-6-diphosphate as substrates. Enzymes detected in both heterotrophic and autotrophic cells were hexokinase, fructose-diphosphate-aldolase, NAD-linked 3-phosphoglyceraldchyde dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and a NADP-linked 3-phosphoglyceraldchyde dehydrogenase. In addition to isotopic studies designed to make an appraisal of the hexose monophosphate shunt, a comparison of the rate of reduction of NADP by glucose-6-phosphate and 6-phosphogluconate in relation to the reduction of NAD by 3-phosphoglyceraldehyde was made in light- and dark-grown cells. The rate of reduction of NADP appeared to be lowered in the light-grown cells, suggesting, as did also the isotopic studies, that the hexose monophosphate shunt is less active in autotrophic metabolism than in heterotrophic metabolism.     

Glucose metabolism of thraustochytrium roseum,a nonfilamentous marine phycomycete

Summary In uniformly labeled logarithmic-phase cells of Thraustochytrium roseum grown in isotopic glucose, 85% of the respiratory CO₂ was derived from endogenous reserves and only 15% was contributed by exogenous glucose. Experiments with asymetrically labeled glucose showed that the main portion of metabolic CO₂ came from carbon 1 of the glucose molecule, suggesting that the hexose monophosphate shunt is a major pathway for glucose dissimilation in the fungus. The presence of several enzymes of the hexose monophosphate shunt, the Embden-Meyerhof and glyoxylate pathways, and the tricarboxylic acid cycle were demonstrated.     

IFN-gamma primes RAW264 macrophages and human monocytes for enhanced oxidant production in response to CpG DNA via metabolic signaling: roles of TLR9 and myeloperoxidase trafficking

Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-gamma. We hypothesize that synergistic cellular responses to IFN-gamma and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.7 macrophages and human monocytes exhibited NAD(P)H autofluorescence oscillation periods of approximately 20 s. IFN-gamma increased the oscillatory amplitude, which was required for CpG DNA-mediated metabolic changes. These alterations in metabolic dynamics required the appropriate combinations of murine/human TLR9 and murine/human-specific CpG DNA. Other factors that also promoted an increase in metabolic oscillatory amplitude could substitute for IFN-gamma. Because recent studies have shown that the metabolic frequency is coupled to the hexose monophosphate shunt, and the amplitude is coupled to the peroxidase cycle, we tested the hypothesis that myeloperoxidase (MPO) participates in IFN-gamma priming for oxidant production. MPO inhibitors blocked cell responses to IFN-gamma and CpG DNA. In the absence of IFN-gamma exposure, the effects of CpG DNA could be duplicated by MPO addition to cell samples. Moreover, monocytes from MPO knockout mice were metabolically unresponsive to IFN-gamma and CpG DNA. NAD(P)H frequency doubling responses due to CpG DNA were blocked by an inhibitor of the hexose monophosphate shunt. Because NAD(P)H participates in electron trafficking to NO and superoxide anions, we tested oxidant production. Although CpG DNA alone had no effect, IFN-gamma plus CpG enhanced NO and reactive oxygen metabolite release compared with IFN-gamma treatment alone. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy.     

Hexose-specificity of hexokinase and ADP-dependence of pyruvate kinase play important roles in the control of monosaccharide utilization in freshly diluted boar spermatozoa

Incubation of boar sperm from fresh ejaculates in a minimal medium with 10 mM glucose induced a fast and intense activation of glycolysis, as indicated by the observed increases in the intracellular levels of glucose 6-phosphate (G 6-P) and ATP and the rate of formation of extracellular L-lactate. The effect of glucose was much more intense than that induced by fructose, sorbitol, and mannose. The greater utilization of glucose was related to a much greater sensitivity to hexokinase when compared with the other monosaccharides. Thus, the presence of 0.5 mM glucose induced total hexokinase activity in supernatants from sperm extracts of 1.7 +/- 0.1 mIU/mg protein, while the same concentration of both fructose, mannose, and sorbitol induced total hexokinase activity from 0.3 +/- 0.1 mIU/mg protein to 0.60 +/- 1 mIU/mg protein. Kinetic analysis of the total pyruvate kinase activity indicated that this activity was greatly dependent on the presence of ADP and also showed a great affinity for PEP, with an estimated Km in supernatants of 0.15-0.20 mM. Immunological location of proteins closely related to glycolysis, like GLUT-3 hexose transporter and hexokinase-I, indicated that these proteins showed the trend to be distributed around or in the cellular membranes of both head and midpiece in a grouped manner. We conclude that glycolysis is regulated by both the specific availability of a concrete sugar and the internal equilibrium between ATP and ADP levels. Furthermore, localization of proteins involved in the control of monosaccharide uptake and phosphorylation suggests that glycolysis starts at concrete points in the boar-sperm surface.     

Regulation of the human-erythrocyte hexose-monophosphate shunt under conditions of oxidative stress. A study using NMR spectroscopy, a kinetic isotope effect, a reconstituted system and computer simulation

The regulation of the hexose monophosphate shunt of human erythrocytes under conditions of oxidative stress has been investigated by monitoring the reduction of oxidised glutathione (GSSG) to reduced glutathione (GSH) in erythrocytes containing high levels of GSSG; 1H NMR and a biochemical assay were used to measure the changes. A reconstituted metabolic system prepared with the purified erythrocyte enzymes was used in conjunction with studies of intact cells and haemolysates to determine the dependence of the rate of GSH production on the activities of hexokinase and glucose-6-phosphate dehydrogenase. Both of these enzymes have previously been claimed to be the rate-limiting step of oxidatively stimulated flux through the hexose monophosphate shunt. The absence of a kinetic isotope effect on the rate of GSH production in these systems, when [1-2H]glucose replaced glucose as the source of reducing equivalents, showed that glucose-6-phosphate dehydrogenase activity was not a strong determinant of the flux. The dependence of the rate of GSH production on the concentration of the hexokinase inhibitors glucose 1,6-bisphosphate and glycerate 2,3-bisphosphate showed that, under conditions of oxidative stress, hexokinase was the principal determinant of flux through the shunt. Glucose 1,6-bisphosphate at the concentration present in vivo appears to be more important in limiting hexokinase activity, and thus the rate of glucose utilisation, than was previously assumed. A detailed computer model of the system was developed based on the reported kinetic parameters of the enzymes involved. A sensitivity analysis of this model predicted that the hexokinase reaction would have a sensitivity coefficient of 0.995 with respect to the maximal rate of GSH production.     

“Carrier activation” and glucose transport in Chinese hamster fibroblasts

Incubation of chinese hamster fibroblasts in glucose free medium, resulted in a 4 to 8 fold increase in the rate of D-glucose uptake and in a 3 to 4 fold increase in the uptake rate of glucose analogs (D-glucosamine, 2-Deoxy-D-glucose, 3-O-Methylglucose). In contrast to what is known for chick embryo fibroblasts, this increased hexose uptake activity is not blocked by cycloheximide in chinese hamster cells. The stimulation of synthesis of the Glucose Regulated Protein, GRP 95 which preceeds by 4 hours the stimulation of GRP 75 cannot account for the increase in hexose uptake-activity. Kinetic data have shown that the activation of glucose uptake activity following sugar starvation resulted only in a V_max increase; K_m for glucose remained constant at 0.6–0.7 mM. However, only the “activated” form of glucose uptake (glucose starvation) was very sensitive to N-ethylmaleimide. A mechanism of hexose “carrier activation” by glucose or a close metabolite is discussed.     

Carbon dioxide production by red skeletal muscle of goldfish (Carassius auratus L.) aerobic and anaerobic metabolism of glucose and glutamate

Oxygen uptake and metabolic CO2 production by lateral red muscle of goldfish have been measured in vitro. Added glucose 6-phosphate depresses the rate of oxygen uptake by minced red muscle (Crabtree effect). Total CO2 production is stimulated resulting in a respiratory quotient which is considerably greater than one. 14CO2 release from [U-14C] glucose 6-phosphate and [U-14C] glutamate continues during anoxia. No activity of the hexose monophosphate shunt was observed. The results suggest that both aerobic and anaerobic CO2 production is of mitochondrial origin and, at least partially, derived from TCA cycle reactions.     

The effect of oxidant stress on diamide-treated human granulocytes

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xanthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.     

Evidence for an apical membrane effect in the regulation of the hexose monophosphate shunt pathway in toad bladder studies with amiloride

Aldosterone stimulates Na⁺ transport in toad bladder and, simultaneously with a coincident dose-response relationship, inhibits the hexose monophosphate shunt pathway. Amiloride, an acylguanidine diuretic, inhibits sodium transport when applied to the apical surface of the bladder. In this study amiloride was found to partially reverse the inhibitory effect of aldosterone on the hexose monophosphate shunt pathway. The amiloride effect upon glucose metabolism was detected when it was applied to both surfaces of the bladder simultaneously, in flask experiments, and when it was applied to the apical surface. No effect of amiloride on the shunt pathway was detected when it was applied to the serosal surface only, even at very high concentrations. It may be, but has not been proven, that the effects of aldosterone and amiloride on the hexose monophosphate shunt pathway are mediated by a common site at the apical membrane.     

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.     

Suppression of monocyte and neutrophil function by recombinant IL-2

Little IS known about the influence of IL-2 on phagocytes. We now describe the effects of human recombinant IL-2 on human neutrophil and monocyte functions related to mobility, phagocytosis, glucose uptake, respiration and degranulation. Neutrophil adherence and hexose monophosphate shunt activities were both suppressed after incubation with IL-2. IL-2 had no effect on neutrophil migration, phagocytosis, deoxyglucose uptake or degranulation, ionocytes demonstrated a greater sensitivity to IL-2 with suppression of monocyte adherence, random and stimulated migration, glucose uptake and hexose monophosphate shunt activity, even after addition of phorbol myristate acetate. Monocyte phagocytosis and degranulation were not affected. All of the effects observed were dose-dependent within a biologically active range for IL-2. These studies suggest that IL-2 may have an important down-regulatory role across a broad range of monocyte functions including movement, deoxyglucose uptake and respiration. However, its role in regulation of neutrophil function is limited to adherence and respiration. IL-2 may be a more versatile cytokine than has previously been appreciated.