Hexose Monophosphate Shunt Pathway Research Articles

Rare red cell enzymopathies in the indian population: A comprehensive review

Red blood cell enzyme deficiencies are a rare category of hemolytic anaemia presented in children with varying degrees of hemolysis, indirect hyperbilirubinemia and splenomegaly. Glucose 6-phosphate dehydrogenase deficiency is the most common enzyme deficiency, followed by pyruvate kinase deficiency. This article aims to understand the pathophysiology of the rare enzymopathies due to deficiencies in the Embden-Meyerhoff pathway, glutathione metabolism, hexose monophosphate shunt and Nucleotide metabolism pathway, and to study clinical symptoms, diagnostic strategy, management and incidence of enzyme deficiencies in Indian patients. Most RBC enzyme deficiencies are inherited in an autosomal recessive fashion except for glucose 6- phosphate dehydrogenase, and phosphoglycerate kinase deficiency which has X-linked inheritance. Presentation depends on the representation of the enzyme on different tissues of the body, hence some enzyme deficiencies may present with neurological or muscular manifestations. In patients with suspected hemolytic anaemia, complete blood count and peripheral smear help in differentiating from hemoglobinopathies etc. To clinch the diagnosis enzyme levels and genetic testing may be required. These tests are important to guide antenatal diagnosis in subsequent pregnancies. Management of RBC enzymopathies will depend on the predominant symptoms and severity of hemolysis. Patients with marked hemolysis require regular blood transfusions and hence appropriate chelation therapy. Hematopoeitic stem cell transplant is attempted in patients with severe spectrum with variable results. Newer drugs like Mitapivat have proven to be beneficial in adults with PK deficiency and ongoing trials in children are showing promising results.

Continuous versus intermittent methylene blue administration: which spin will win?

Sir, The article by George et al. is indeed interesting.[1] However, a few aspects of their report require contemplation. The use of methylene blue continuous infusion in the management of methemoglobinemia due to insecticide poisoning is not congruent with current evidence. Methylene blue is a cationic thiazine dye and acts as an oxidant. It has assumed a significant role, with diverse applications in clinical practice, but is fraught with risks of side-effects.[2] Methylene blue is reduced to leukomethylene blue by erythrocyte methemoglobin reductase in the presence of nicotinamide adenine dinucleotide phosphate (NADPH). Further, leukomethylene blue reduces methemoglobin to oxyhemoglobin. Therefore, large doses of methylene blue may result in higher levels of methylene blue rather than the expected leukomethylene blue, which could potentially induce acute hemolytic anemia, independent of preexisting methemoglobinemia.[3] This is strengthened by reports of the paradoxical induction of methemoglobinemia by methylene blue. The possible mechanisms resulting in rebound methemoglobinemia include continued absorption of the inciting drug as well as prolonged half-life in the setting of renal or hepatic dysfunction.[4] For example, the hydroxylamine metabolites of dapsone responsible for the formation of methemoglobin have a half-life of over 30 h and may linger in circulation for up to 35 days. These agents are metabolized to reactive metabolites that oxidize hemoglobin. The redox reaction to form methemoglobin regenerates the parent compound, which can then be remetabolized to the oxidative metabolite. Therefore, to prevent the relapse of methemoglobinemia, adequate decontamination with activated charcoal is essential. In addition, dextrose infusion is necessary, to provide adequate substrate to form NADPH, through the hexose monophosphate shunt pathway, which is essential for reducing enzymes to be effective. The authors have used continuous infusion of methylene blue, to prevent rebound methemoglobinemia. Although there are reports[5] which advocate the continuous infusion of methylene blue to prevent rebound methemoglobinemia, it is prudent to avoid it as standard therapy.[6] The current recommended regimen is intermittent bolus dose of methylene blue for the treatment of methemoglobinemia.

Studies on the protective effect of flaxseed oil on cisplatin-induced hepatotoxicity

Cisplatin (CP) is known as one of the most potent chemotherapeutic antitumor drugs. The tissue-specific toxicity of CP in the kidneys is well documented. However, at higher doses less common toxic effects such as hepatotoxicity may arise. Since CP remains one of the most effective antineoplastic drug used in chemotherapy, strategies to protect tissues against CP toxicity are of clinical interest. Recently, ω-3 polyunsaturated fatty acids (PUFAs) from certain plants/seeds notably flaxseed have shown numerous health benefits. In view of this, the present study investigates the protective effect of flaxseed oil (FXO) on CP-induced damage in liver. Rats were pre-fed normal diet and the diet rich in FXO for 10 days and then a single dose of CP (6 mg/kg body weight) was administered intraperitoneally while still on diet. Serum/urine parameters, enzymes of carbohydrate metabolism and oxidative stress were analyzed. CP caused perturbation of the antioxidant defense as reflected by the decrease in the activities of catalase, superoxide dismutase and glutathione peroxidase. Further the activities of various enzymes involved in glycolysis, tricarboxylic acid cycle, gluconeogenesis and hexose monophosphate shunt pathways were determined and were found to be differentially altered by CP treatment. However, these alterations were ameliorated in CP-treated rats fed on FXO. Present results show that dietary supplementation of FXO in CP-treated rats ameliorated CP-induced hepatotoxic and other deleterious effects due to its intrinsic biochemical/antioxidant properties.

Oxidative and antioxidative defense system in testicular torsion/detorsion

Aim:The present study was aimed to assess the early effects of ischemia/reperfusion injury on the oxidants and anti-oxidant defense status in rat testicular tissue by measuring MDA, glucose-6-phosphte dehydrogenase activity and reduced glutathione levels in a designated time frame sequel to reperfusion. Animals were divided randomly into six groups (12 animals per group) in the following order: Group I: Sham-operated control group (Cso) without the application of the torsion. Group 2: Torsion-induced ischemia group (T30 m): Ischemia was induced through the torsion of spermatic cord for a period of 30 min. Group 3: One hour reperfusion group after detorsion (T30 mR1 h). Group 4: Twenty-four hour reperfusion group after detorsion (T30 mR24 h). Group 5: Forty-eight hours reperfusion group after detorsion (T30mR48h). Group 6: One week reperfusion group after detorsion (T30mR1wk). Results and Discussion:The oxidant-antioxidant system of the testicular tissue is altered during torsion as well as detorsion which results in the altered activities involved in the key enzyme of hexose monophosphate shunt pathway, glucose 6 phosphate dehydrogenase activity along with a reduction of glutathione (G.SH) content. The increase in G6PDH activity during torsion and followed by an increase in detorsion indicates the tissue's response to counter the oxidant stress caused by reduced blood supply. Continued exposure to such oxidant stressed physiological state of a tissue may lead to decreased capacity of the tissue to perform its physiological function such as testicular steroidogenesis and spermiogenesis shown in the present study.

Cell growth and cholesterol metabolism in human glucose-6-phosphate dehydrogenase deficient lymphomononuclear cells.

Atherosclerosis is an inflammatory-fibroproliferative response of the arterial wall involving a complex set of interconnected events where cell proliferation (lymphomonocytes, and endothelial and smooth-muscle cells) and substantial perturbations of intracellular cholesterol metabolism are considered to be among the main features. Glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the hexose-monophosphate shunt pathway, is an essential enzyme involved in both cell growth and cholesterol metabolism, raising the question as to whether G6PD deficiency may have metabolic and growth implications in a deficient population. In the present study, we investigated cell growth and cholesterol metabolism in peripheral blood lymphomononuclear cells (PBMC) from G6PD-normal (n = 5) and -deficient (n = 5) subjects stimulated with lectins (phytohaemoagglutinin and Concanavalin A). G6PD activity, DNA ([3H]-thymidine incorporation) cholesterol synthesis and esterification ([14C]-acetate and [14C]-oleate incorporation), and G6PD, HMGCoA reductase and low density lipoprotein (LDL) receptor mRNA levels (RT-PCR) all increased following lectin stimulation in both normal and G6PD-deficient cells. However, these parameters were significantly lower in G6PD-deficient cells (P < 0.05). It is of interest that G6PD-deficient PBMC, which showed lower expression of G6PD and higher expression of the LDL receptor gene than normal PBMC under basal conditions, exhibited an opposite pattern after stimulation: G6PD and HMGCoA reductase being expressed at significantly higher levels in deficient than in normal cells (P < 0.05). We conclude that the reduced capability of G6PD-deficient cells to respond to mitogenic stimuli and to synthesize cholesterol esters may represent favourable conditions for reducing the risk of cardiovascular diseases.

Determination of erythrocyte antioxidant capacity in haemodialysis patients using electron paramagnetic resonance.

The increased oxidative stress of uraemia is caused both by an increased generation of oxygen-free radicals and a decrease of antioxidative forces. There are, however, conflicting data concerning disturbances of the radical-scavenging power of red blood cells (RBCs) in uraemic patients. The antioxidant capacities of the RBCs of 10 haemodialysis (HD) patients and 10 controls were examined after treatment with 0.324 mM tert-butylhydroperoxide (t-BOOH) in phosphate-buffered saline at 37 degrees C using electron paramagnetic resonance (EPR) with 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap and glutathione (GSH) regeneration as an indicator of hexose monophosphate shunt (HMPS) activity. EPR investigations were also done after pre-incubation with N-ethylmaleimide (NEM) to inhibit the GSH system. Furthermore, we determined the RBC redox state in 15 HD patients and 15 controls. There was no difference between HD patients and controls in the elimination of t-BOOH-generated free radicals in the RBCs. A more than 20-fold increase in radical concentration was observed after GSH trapping with NEM. In this case, we found a delayed decrease of the relative radical concentration in HD patients compared with controls with a significant difference after 7 min (2.2+/-0.26 vs 1.60+/-0.21; P=0.005) and after 10 min (1.82+/-0.41 vs 0.83+/-0.44; P=0.001). GSH regeneration via HMPS did not differ between the RBCs of HD patients (99.5+/-13.5 nmol/min x ml RBC) and those of the controls (94.2+/-16.9 nmol/min x ml RBC). There were no differences in the RBC concentrations of GSH, GSSG, NADP, NADPH, and in the GSH/GSSG and NADP/NADPH ratios between HD patients and controls. These data suggest a strong antioxidant potential in the GSH system of erythrocytes without any evidence of a disturbance in HD patients. The HMPS pathway also appears not to be impaired in the RBCs of HD patients. However, the slower radical elimination in the RBCs of HD patients after inhibition of GSH-depending radical scavengers as compared with controls indicates a defect in the antioxidant forces outside the GSH system, and could be one reason for the reduced lifespan of RBCs in HD patients.

Effect of pyrethroids on carbohydrate metabolic pathways in common carp, Cyprinus carpio.

The activity levels of succinate dehydrogenase (SDH) and glucose-6-phosphate dehydrogenase (G6PD) were assessed in various tissues of Cyprinus carpio var communis which had been exposed to lethal concentrations of group-II pyrethroids (deltamethrin, cypermethrin, fenvalerate and fluvalinate) for a period of 72 h. The results indicated a steady decrease in SDH activity with a concomitant increase in G6PD activity. The decreased SDH activity indicated inhibition of SDH at mitochondrial level and the increased G6PD activity an enhancement of an alternative pathway of carbohydrate metabolism, viz the hexose monophosphate shunt (HMP) or pentose phosphate pathway as a biochemical adaptation to overcome the toxic stress.

High glucose-6-phosphate dehydrogenase activity contributes to the structural plasticity of periglomerular cells in the olfactory bulb of adult rats

Glucose-6-phosphate dehydrogenase (G6PD) activity, assayed spectrophotometrically, was found to be higher in the olfactory bulb (OB) than in other brain areas of adult rats [P. Ninfali, G. Aluigi, W. Balduini, A. Pompella, Glucose-6-phosphate dehydrogenase is higher in the olfactory bulb than into other brain areas, Brain Res. 744 (1997) 138–142]. Histochemical demonstration of G6PD activity in cryostat sections of OB, analyzed with optical microscopy, revealed a marked and well defined line of formazan deposition in the internal part of the glomerular layer (Glm), indicating that G6PD was much higher in cells distributed along the glomeruli. Electron microscope analysis showed that G6PD activity was mainly concentrated in cytoplasm and dendrites of periglomerular cells, the interneurons which span glomeruli and connect olfactory nerves with mitral/tufted cells. Since G6PD regulates the flux through the hexose monophosphate shunt (HMS) pathway, which provides NADPH for reductive biosynthesis and pentose phosphates for nucleic acid formation, it can be concluded that high G6PD activity in periglomerular neurons is functional to their differentiating capability. This result is consistent with the occurrence of structural plasticity events in the OB of adult rats.

Metabolic support of collecting duct transport

The present studies address the metabolic processes that support the reabsorption of sodium and the secretion of bicarbonate in the interspersed but distinct principal and intercalated cells of the cortical collecting duct (CCD). In microperfused rabbit CCD, sodium reabsorption was measured by lumen-to-bath 22Na flux, and bicarbonate transport was assayed by microcalorimetry. Flux measurements were made before and after metabolic substrate changes or application of metabolic inhibitors. Both sodium reabsorption and bicarbonate secretion were dependent on oxidative metabolism (inhibited by antimycin A) and appeared to have no special dependence on glycolysis or the hexose-monophosphate shunt pathways. Endogenous substrates (in the absence of exogenous metabolic substrates) supported a small component of sodium transport; in contrast, bicarbonate reabsorption in the outer medullary collecting duct, which was studied for comparison, was fully supported by endogenous substrates. In the CCD, sodium reabsorption was supported best by a mixture of basolateral metabolic substrates (glucose and acetate, as a fatty acid), whereas bicarbonate secretion was fully supported by either glucose or acetate. Alanine, as a representative amino acid, was not an effective metabolic substrate. Another contrasting feature of the two transport processes was that bicarbonate secretion, and not sodium transport, was supported to some extent by luminal glucose. In sum, principal cells and intercalated cells differ not only in their morphology and function, but also in their metabolism.

Fructose induced deactivation of glucose-6-phosphate dehydrogenase activity and its prevention by pyruvate: Implications in cataract prevention

Glucose-6-phosphate dehydrogenase (G6PDH) is an important lens enzyme diverting about 14% of the tissue glucose to the hexose monophosphate shunt pathway. The main function of such a pronounced activity of the enzyme is to support reductive biosyntheses, as well as to maintain a reducing environment in the tissue so as to prevent oxy-radical induced damage and consequent cataract formation. Sugars are one of the well-known cataractogenic agents. Several reports suggest that the cataractogenic effect of the sugars in diabetes as well as in normal aging is initiated by the glycation of the proteins including the enzymes and subsequent formation of more complex and biologically inactive or harmful structures. In a diabetic lens the concentration of fructose exceeds significantly the concentration of glucose, suggesting that the contribution of fructosylation may be greater than that of glucosylation. These studies were undertaken to examine further the possibility that in addition to glycation, generation of oxygen free radicals by fructose and consequent oxidative modifications in certain enzymes may be an important participant in the cataractogenic process. This hypothesis was tested by using G6PDH. The enzyme was incubated with various levels of fructose (0–20 mM) and its activity determined as a function of time. This led to a significant loss of its activity, which was prevented by superoxide dismutase, catalase, mannitol and myoinositol. Most interestingly, pyruvate at levels between 0.2 and 1.0 mM also offered substantial protection. Hence, the results, while elucidating further the mechanism of enzyme deactivation by sugars such as fructose, also demonstrate the possibility of therapeutic prevention of cataracts by pyruvate and other such keto acids, in diabetes and other disabilities involving oxygen free radicals in the pathogenetic process.

Effect of PGE2 and of agents that raise cAMP levels on macrophage activation induced by IFN-gamma and TNF-alpha.

The effect of prostaglandin (PG) E2 on macrophage activation by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) was evaluated. Murine macrophages infected with Leishmania enriettii or Leishmania major were activated by exposure to IFN-gamma (10-50 U/ml) and TNF-alpha (30-3000 U/ml), leading to intracellular parasite destruction within 24-48 h. Leishmanicidal activity was markedly increased when activation was performed in the presence of PGE2 (10(-9)-10(-7) M) or arachidonate (10(-5) M, a PG precursor), concomitant with enhanced nitrite release and glucose oxidation through the hexose monophosphate shunt pathway. Conversely, activation was reduced by indomethacin and hydrocortisone, two inhibitors of PG synthesis. Parasite killing and nitrite production were fully restored by exogenous PGE2, indicating that inhibition by these drugs was related to their ability to block PG production. PG can stimulate adenylate cyclase, thus raising intracellular cAMP levels. Accordingly, dibutyryl-cAMP, theophylline (which prevents cAMP breakdown), and forskolin (an activator of adenylate cyclase) all stimulated macrophage activation. Finally, PGE2 and cAMP enhanced expression of inducible nitric oxide synthase mRNA in response to IFN-gamma and TNF-alpha, and this effect was inhibited by the cAMP antagonist 2'-O-methyl adenosine. These findings are consistent with the hypothesis that PGE2 acts as a positive agonist in macrophage activation by IFN-gamma and TNF-alpha via its capacity to modulate intracellular cAMP levels.

31P–Nuclear Magnetic Resonance Evidence of an Activated Hexose-Monophosphate Shunt in Hyperglycemic Rat Lenses In Vivo

Using 31P-nuclear magnetic resonance spectroscopy, we have identified elevated concentrations of sedoheptulose-7-phosphate (S-7-P) in lenses from three animal models of hyperglycemia: streptozotocin-induced diabetic rats, galactose-fed rats, and xylose-fed rats. This observation provides a unique and independent confirmation of the activation of the hexose monophosphate shunt (HMPS) pathway in the hyperglycemic lens in vivo. While the elevation in concentration of S-7-P was very dramatic, the other HMPS metabolites in these tissues were below the threshold of detection, as expected for the HMPS pathway near equilibrium. In terms of nonenzymatic glycation, these results suggest that the only HMPS metabolite of importance in the hyperglycemic rat lens is S-7-P. Although in the diabetic lens its role appears to be relatively minor, in the galactosemic lens this compound may be an important contributor to the increased production of advanced glycosylation end products.

Alterations in Carbohydrate Metabolism in Selected Tissues of Crab,Oziotelphusa Senex Senex(Fabricius) Under Fenvalerate-Induced Stress

Abstract Changes in carbohydrate metabolism in hepatopancreas and muscle of the crab, Oziotelphusa senex senex exposed to a sublethal concentration (0.2 ppm) of fenvalerate, a pyrethroid insecticide, were studied. the glycogen and total carbohydrate levels decreased significantly in the tissues of crab exposed to fenvalerate. an increase in phosphorylase ‘a’ and decrease in aldolase activity levels suggested increased glycogenolysis, and decreased glycolysis during fenvalerate toxicity. Krebs cycle enzymes such as NAD-isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase were decreased, suggesting reduced mitochondrial oxidative metabolism. Glucose-6-phosphate dehydrogenase activity was increased significantly, indicating enhanced oxidation of glucose through the hexose monophosphate shunt pathway. Lactate dehydrogenase activity was elevated indicating the development of anaerobic conditions at tissue level in the stressed crab. Cytochrome C oxidase and Mg2+ ATPase activity levels were also decreased, indicating the impaired energy synthesis and prevalence of energy crisis. These results suggest that fenvalerate has a profound effect on the glucose metabolism of crab.

Uraemic toxic retention solutes depress polymorphonuclear response to phagocytosis

Previous studies from our laboratory have demonstrated that the activity of the hexose monophosphate shunt (HMS) pathway in phagocytosis-related respiratory burst is disturbed in end-stage renal disease. To determine whether uraemic solute retention is responsible for this defect the HMS-path was evaluated by measurements of glucose-1-C14 utilization and determination of 14CO2 production in polymorphonuclear cells (PMNLs), suspended in normal plasma or uraemic biological fluids. Normal PMNLs, while suspended in normal or uraemic plasma, were stimulated with either latex, zymosan or Staph. aureus; CO2 generation (measured as DPM/10(3) PMNL, normal versus uraemic plasma) was depressed in uraemic plasma in response to latex (from 43 +/- 5 to 20 +/- 3), zymosan (from 72 +/- 8 to 47 +/- 4) (P < 0.01), and Staph aureus (from 73 +/- 17 to 47 +/- 8 DPM/10(3) PMNL) (P < 0.05). The degree of inhibition was similar for each stimulus. To characterize the substances responsible for this defect we fractionated uraemic plasma ultrafiltrate by polarity-based semipreparative C18 reversed phase HPLC and found a decreased response to Staph. aureus in the presence of fraction 2 (from 102 +/- 13 to 23 +/- 10 DPM/10(3) PMNL, P < 0.05), and in fractions 8 and 11 (lowest value in fraction 8, 54 +/- 14 DPM/10(3) PMNL, P < 0.05 versus control). The pattern of HPLC elution on a gradient from 100% formiate (pH 4.0) to 100% methanol indicates that there are at least two chemically distinguishable groups of compounds, one hydrophilic (in fraction 2), and one lipophilic (in fractions 8 and 11). We conclude that uraemic biological fluids contain factors that inhibit HMS activity related to phagocytosis, and that at least two groups of components with different characteristics are involved.

Effect of propane-1,2-diol ingestion on carbohydrate metabolism in female rat erythrocytes.

This study was undertaken to assess the effect of propane-1,2-diol(PD) ingestion on carbohydrate metabolism in female rat erythrocytes. For this purpose, two different groups of adult albino female rats were treated orally with PD at two different dose levels of 73 and 294 mg 100 g-1 body wt. The blood samples drawn from the retro-orbital sinus prior to the treatment served as the controls, whereas the treated samples were collected at peak periods (1/2 and 2 h) 2 and 7 days after the treatment. A single dose of PD was found to elevate levels of blood glucose, lactate, pyruvate and the lactate/pyruvate ratio at the peak periods (P < 0.001) and after 2 days (P < 0.001) in both the groups. A significant (P < 0.05) increase in the contents of erythrocyte 2,3-diphosphoglycerate (2,3-DPG) was observed only at the peak periods. All these parameters returned to their base level after 7 days of treatment. The activities of hexokinase (HK), 2,3-diphosphoglycerate phosphatase (2,3-DPG Pase), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD) and aldehyde reductase II (AR II) declined markedly, whereas those of pyruvate kinase (PK) and aldose reductase increased as a result of PD ingestion. The changes in the activities of 2,3-DPG Pase and LDH were persistent up to 8 days post-treatment. The [14C]glucose flux through glycolysis and the hexose monophosphate shunt pathway in erythrocytes was found to be lowered (P < 0.001) in response to PD treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Some glucose metabolic enzymes in various fractions of sarcocysts of Sarcocystis fusiformis of buffalo ( Bubalus bubalis)

A comparative biochemical study on some enzymes of glycogenolysis, glycolysis and the hexose monophosphate shunt pathway in various fractions (cyst wall, cyst fluid and zoites) of the sarcocysts of Sarcocystis fusiformis from the oesophageal muscles of naturally infected Indian water buffalo ( Bubalus bubalis) was carried out. The pattern and the magnitude of enzymatic activity differed markedly in these fractions. Phosphorylase, hexokinase, aldolase and pyruvate kinase showed their highest levels of activity in the zoites fractions, whereas lactate dehydrogenase was the highest in cyst fluid. Alcohol dehydrogenases were non-detectable. Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were localized in the cyst wall only. Zoites were considered to be the most active metabolic sites for glucose breakdown.

Macrophage activation for intracellular killing as induced by a Ca2+ ionophore. Dependence on l-arginine-derived nitrogen oxidation products

Mouse macrophages activated by interferon-gamma kill intracellular Leishmania by a process that depends on the generation of L-arginine-derived nitrogen oxidation products. Interferon-induced intracellular killing can be mimicked by exposure of macrophages to the Ca2+ ionophore A23187 in the presence of lipopolysaccharide. The mechanisms of this effect were therefore investigated. Destruction of the parasite was accompanied by accumulation of nitrite in the macrophage culture fluids. Leishmanicidal activity and nitrite production in cultures stimulated with ionophore A23187 and lipopolysaccharide were abrogated when cells were activated in medium containing arginase or the L-arginine analogues L-canavanine, guanidine or NG-monomethyl-L-arginine. L-Arginine was required during the lipopolysaccharide-induced triggering phase only. Indeed, macrophage priming with ionophore A23187 in L-arginine-depleted medium led to full microbicidal activity and nitrite generation provided that L-arginine was present during subsequent triggering by lipopolysaccharide. Addition of NG-monomethyl-L-arginine to ionophore-activated macrophages increased O2- production on phorbol myristate stimulation, while inhibiting glucose oxidation through the hexose monophosphate shunt pathway. Leishmanicidal activity and nitrite production were also inhibited when ionophore-treated cultures were incubated with excess iron, implying a role for iron as a defence mechanism against the toxicity of nitrogen derivatives. These results indicate that the ionophore-induced leishmanicidal activity occurs through a process similar to that evoked by interferon-gamma, i.e. the production of L-arginine-derived nitrogen oxidation products.

Macrophage activation by OM-85 BV.

Peritoneal or bone-marrow-derived murine macrophages were exposed for 24 h in vitro to dilutions of the bacterial extract OM-85 BV, in the presence or absence of other added compounds [macrophage-activating factor (MAF), recombinant murine interferon-gamma (IFN-gamma)]. Various metabolic responses and functional activities were then measured. Glucose oxidation through the hexose monophosphate shunt pathway was markedly stimulated in OM-85 BV-treated macrophages compared to control macrophages. Similarly, OM-85 BV primed macrophages for superoxide production upon triggering by phorbol myristate acetate. Both effects were further enhanced by simultaneous treatment of the cells with MAF with OM-85 BV. The bacterial extract also induced macrophages to release large amounts of nitrite (a marker of the activated state). As regards functional responses, coincubation with MAF and OM-85 BV activated macrophages to destroy target cells as well as intracellular microorganisms; in the latter case, similar results were obtained when MAF was replaced by IFN-gamma. In all these tests, the possibility that the observed effects were due to contamination of the bacterial extracts by endotoxin could be excluded. The above results indicate that OM-85 BV induces metabolic and functional properties in macrophages that are characteristic of the activated state and are important for host defence.

Up-regulation of glucose metabolism in Kupffer cells following infusion of tumour necrosis factor

Alterations of glucose metabolism and the oxidation of glutamine and palmitate were studied, by using specifically labelled substrates, in freshly isolated Kupffer cells and hepatic endothelial cells after infusion in vivo of human recombinant tumour necrosis factor-alpha (TNF; 7.5 x 10(5) IU/30 min per kg body wt., intravenously). Cells were incubated in a medium containing 5 mM-glucose, 0.4 mM-palmitate, 1 mM-lactate and 0.5 mM-glutamine. Administration of TNF in vivo increased glucose use in Kupffer cells by 70%. Glucose oxidation in the tricarboxylic acid cycle and flux in the Embden-Meyerhof (EM) pathway were elevated by 40 and 80% respectively. Treatment in vitro with 1 microM-phorbol 12-myristate 13-acetate (PMA) resulted in a similar percentage increase in glucose use by Kupffer cells prepared from either saline- or TNF-treated rats. However, PMA increased the activity of the hexose monophosphate shunt (HMS) by 3- and 10-fold in cells isolated from saline- or TNF-infused animals respectively. A phagocyte stimulus in vitro, opsonized zymosan, increased glucose use by 30% and doubled the flux through the HMS in Kupffer cells from saline-infused animals. The activity of the HMS in response to zymosan was increased by 400% after TNF treatment. In endothelial cells, basal glucose utilization was not altered by TNF treatment. PMA increased HMS activity in endothelial cells to a similar degree after saline or TNF infusion. Zymosan, however, increased HMS activity only in endothelial cells from TNF-treated rats. Oxidation of palmitate or glutamine was not affected by TNF treatment either under basal conditions or after challenge in vitro. Our data indicate that, after phagocytosis in vitro or protein kinase C activation, glucose use and flux through the HMS increase in Kupffer cells. This is accompanied by increased glycolytic flux, with no changes in glucose oxidation in the tricarboxylic acid cycle. After TNF exposure, followed by a secondary stimulus, the enhanced glucose use by Kupffer cells is primarily channelled through the HMS pathway. These data suggest that the increased glucose use in vivo by Kupffer cells found after immune-stimulated conditions may subserve primarily the increased need for NADPH and HMS intermediates.

Effects of macrophage colony-stimulating factor and phorbol myristate acetate on 2-D-deoxyglucose transport and superoxide production in rat peritoneal macrophages.

2-D-Deoxyglucose (2-dGlc) uptake and accumulation into rat peritoneal macrophages was increased by colony-stimulating factor (mCSF) by stimulating the coupling between endofacial hexokinase activity and the sugar transporter. The evidence for this is as follows: (1) mCSF significantly decreased the Km for zero-trans uptake (P less than 0.05), without altering Vmax.; (2) the accumulation of free 2-dGlc was increased by mCSF (P less than 0.05); (3) mCSF retarded the rate of exit of accumulated free 2-dGlc. The mCSF-dependent increase in 2-dGlc uptake by macrophages was enhanced by preincubation of the cells in mCSF-free solution. The activity of the hexose monophosphate shunt (HMPS) measured by the differential uptake of 2-d[1-3H]Glc and 2-d[2,6-3H]Glc was not stimulated by mCSF. Also, in quiescent cells, superoxide production, as determined by cytochrome c reduction, was unaffected by mCSF. Phorbol myristate acetate (PMA; 40 nM) stimulated both the HMPS activity and superoxide production. Both these effects were dependent on the uptake of external sugar (2-dGlc). Incubation of the macrophages with mCSF enhanced the sugar transport and PMA-dependent stimulation of HMPS activity and superoxide production, indicating a role for mCSF in the 'priming' of macrophage functions. Both HMPS activity and superoxide production are entirely dependent on uptake of exogenous sugar, since the potent sugar-transport inhibitor cytochalasin B competitively inhibited 2-dGlc uptake, HMPS activity and superoxide generation in PMA-activated cells (Ki approximately 0.3 microM for all three processes). Over a wide range of 2-dGlc concentrations, 4 mol of superoxide were generated/mol of 2-dGlc metabolized in the HMPS pathway, indicating coupling between these processes. The Km of 2-d[2,6-3H]Glc uptake in PMA-treated cells was 0.45 +/- 0.07 mM, and Vmax. was 1.32 +/- 0.05 mumol.min-1.ml of cell water-1. It is evident that there is a large degree of slippage between HMPS activity and membrane-associated hexokinase activity, since the Km for HMPS activity was 0.06 +/- 0.02 mM and the Vmax. was 0.10 +/- 0.03 mumol.min-1.ml of cell water-1.