Activities Of Key Glycolytic Enzymes Research Articles

Cortical glucose metabolism is altered in aged transgenic Tg2576 mice that demonstrate Alzheimer plaque pathology.

Alzheimer's disease is associated with markedly impaired cerebral glucose metabolism as detected by reduced cortical desoxyglucose utilization, by altered activities of key glycolytic enzymes or by reduced densities of cortical glucose transporter subtypes. To determine whether formation and/or deposition of beta-amyloid plays a role in the pathology of glucose metabolism, transgenic Tg2576 mice that overexpress the Swedish mutation of the human amyloid precursor protein and demonstrate a progressive, age-related cortical and hippocampal deposition of beta-amyloid plaques, were used to study expression and activity of key enzymes of brain glycolysis (phosphofructokinase, PFK) and glyconeogenesis (fructose1,6-bisphosphatase; FbPase). Quantitative RT-PCR revealed high expression levels of both C- and M-type PFK mRNA in non-transgenic mouse cerebral cortex, whilst there was little expression of the L-type. In 24-month-old transgenic Tg2576 mouse cortex, but not in 7-, 13-, and 17-month-old mice, the copy number of PFK-C mRNA was significantly reduced in comparison to non-transgenic littermates, while the mRNA level of the other PFK isoforms and FbPase did not differ between transgenic and non-transgenic tissue samples. In situ hybridization in brain sections from aged Tg2576 mice revealed reduced PFK-C mRNA expression in beta-amyloid plaque-associated neurons and upregulation in reactive astrocytes surrounding beta-amyloid deposits. The decreased PFK-C protein level detected by Western analysis in cerebral cortical tissue from 24-month-old transgenic Tg2576 mice was accompanied by reduced enzyme activity of PFK in comparison to non-transgenic littermates. Our data demonstrate that impairment of cerebral cortical glucose metabolism occurs only due to the long-lasting high beta-amyloid burden. This results from a reduction in glycolytic activity in beta-amyloid plaque-associated neurons and a concomitant upregulation in reactive, plaque-surrounding astrocytes.

Growth Factors Stimulate the Activity of Key Glycolytic Enzymes in Isolated Digestive Gland Cells from Mussels (Mytilus galloprovincialis Lam.) through Tyrosine Kinase Mediated Signal Transduction

Digestive gland cells isolated from mussels (Mytilus) have previously been demonstrated to respond to mammalian EGF with a cytosolic Ca2+ transient and stimulated DNA synthesis; both responses were mediated by activation of tyrosine kinase receptors. The present study examines the mechanisms involved in further signal progression and possible targets of phosphorylation/dephosphorylation processes. The effects of EGF, IGF-I, and insulin on the activity of two key glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase) were evaluated. All the peptides tested induced a transient and dose-dependent stimulation of the activity of both PFK and PK, which involved activation of MAPKs. Quantitative immunoelectron microscopy, utilizing monoclonal anti-phosphotyrosine antibodies, revealed that EGF induced a transient increase in tyrosine phosphorylation. The results demonstrate that, in marine invertebrate cells, activation of tyrosine kinase membrane receptors by growth factors triggers signal transduction pathways involving a phosphorylative cascade similar to that of mammalian cells. Moreover, these data suggest that, in mussel cells, growth factors may play a physiological role in the in vivo regulation of glucose metabolism by modulating, through reversible phosphorylation, the activity of key glycolytic enzymes.

Activities of key glycolytic enzymes in the brains of patients with Alzheimer's disease.

The activities of hexokinase, aldolase, pyruvate kinase, lactate dehydrogenase and glucose 6-phosphate dehydrogenase were determined in brains of patients with Alzheimer's disease (AD) and in age matched controls. For pyruvate kinase and lactate dehydrogenase a significant increase in specific activity was found in frontal and temporal cortex of AD brains, while the activities of aldolase and hexokinase are not changed. Glucose 6-phosphate dehydrogenase activity was significantly reduced in hippocampus. The increase of some glycolytic enzyme activities is correlated with increased contents of lactate dehydrogenase and glial fibrillary acidic protein (GFAP) in homogenates of frontal and temporal cortex and elevated phosphofructokinase (PFK) and GFAP in astrocytes from the same brain areas. The data extend previous findings on an increase in brain PFK specific activity in AD and suggest that the increased activity of some glycolytic enzymes may be, at least in part, the result of the reactive astrocytosis developing in the course of AD.

Dichlorvos induced alterations in glucose homeostasis: possible implications on the state of neuronal function in rats.

The present study was carried out to assess the effect of chronic dichlorvos exposure on various aspects of glucose homeostasis in different regions of rat brain. Dichlorvos administration caused a significant depletion in the brain glycogen content accompanied with an increase in the activity of glycogen phosphorylase. The activities of key glycolytic enzymes, hexokinase, phosphofructokinase and lactate dehydrogenase were decreased significantly following dichlorvos exposure. The decreased glycolytic flux was further reflected in terms of decreased regional glucose utilization, determined by measuring 14C-glucose influx. The altered neuronal glucose homeostasis had a significant impact on the neurobehavioural patterns of dichlorvos treated animals which was reflected in terms of severe deterioration in their memory and learning functions.

In vitro effect of extracellular AMP on MCF-7 breast cancer cells: inhibition of glycolysis and cell proliferation.

MCF-7 human breast cancer cells propagated in vitro were treated with adenosine derivatives added to the culture medium. The effects on cell proliferation, glycolysis, and glutaminolysis were investigated. Of all adenosine derivatives tested, AMP was the most efficient inhibitor of cell proliferation. In AMP-treated cells, DNA synthesis decreased, whereas RNA and protein syntheses rose normally with time. In terms of carbohydrate metabolism, lactate production from glucose was drastically reduced; therefore, most of lactate produced must have been derived from glutamine. Increases in the enzyme activities involved in glutamate degradation and in the malate-aspartate shuttle were observed. In contrast, actual glycolytic flux rates declined, whereas key glycolytic enzyme activities increased. Metabolites such as fructose 1,6-bisphosphate and pyruvate accumulated in AMP-arrested cells. Based on the lowered NAD level in the AMP-treated cells, lactate dehydrogenase, but not malate dehydrogenase, was impaired; thereby the whole of glycolysis was inhibited. In compensation, glutamine catabolism was increased. NAD concentrations fell drastically because of the known inhibition of P-ribose-PP synthesis through heightened intracellular AMP levels. A hypothetical metabolic scheme to explain these results and to show how extracellular AMP may influence carbohydrate metabolism and cell proliferation is presented.

Maximal activities of glutaminase, citrate synthase, hexokinase, phosphofructokinase and lactate dehydrogenase in skin of rats and mice at different ages

The activities of key glycolytic enzymes are, in general, similar at the three different ages of the animals used in this work (very young, adult and old). Glutaminase is present in skin of both mice and rats, but the activity was much lower in adult animals compared to the very young or the old. It is suggested that this activity is important for the provision of nitrogen for the de novo synthesis of purine and pyrimidine nucleotides during the growth of skin in the young animal and for DNA repair in the old animals; it might be important in the adult skin in response to wound healing.

Effect of ethanol on glucose transport, key glycolytic enzymes, and proton extrusion in Saccharomyces cerevisiae

The effect of ethanol on the activities of the key enzymes of the glycolytic pathway and on two membrane functions related with fermentation, the glucose uptake system, and proton extrusion rate are examined. The results indicate that ethanol, up to 2M, does not cause any change of the glucose uptake velocity nor any substantial change in the key glycolytic enzyme activities while the fermentation rate is reduced by about 50%. In a cell extract 3M ethanol as well as incubation of yeast cells with 4M ethanol caused a considerable decrease of pyruvate kinase and hexokinase activities. Phosphofructokinase remained unchanged even at higher ethanol concentrations. Transmembrane proton flow was found to be the most sensitive of the functions tested toward ethanol, and it could represent the first target of ethanol action on fermentation.

REGULATION OF GLUCOSE METABOLISM IN TISSUES OF THE SHEEP HIND-LIMB: RESPONSE TO LACTATION

Decreased rates of glucose utilization by peripheral tissues of sheep during lactation are probably due to a fall in the activity of key glycolytic enzymes of skeletal muscle, while the increase in lactate output by the hind-limb during lactation is probably due to a fall in pyruvate dehydrogenase activity in both muscle and adipose tissue. Key words: Sheep, glycolysis, pyruvate dehydrogenase, lactation

実験的エタノール・ピラゾール肝障害に関する研究

After administration of ethanol and pyrazole, a potent inhibitor of alcohol dehydrogenase, for 3 weeks to rats, enzyme activities of key glycolytic enzymes of the liver (glucokinase, hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase), a key gluconeogenic enzyme, (glucose-6-phosphatase), and alcohol dehydrogenase were invesgated. The resulting enzyme patterns are as follows: In rats treated with ethanol alone, glucose-6-phosphate dehydrogenase activity was decreased and glucose-6-phosphatase activity was increased. In the group treated with pyrazole, activities of hexokinase and glucose-6-phosphatase were decreased. In the group treated with pyrazole and ethanol, the activity of glucose-6-phosphate dehydrogenase was significantly increased, and those of pyruvate kinase and glucose-6-phosphatase were significantly decreased. The enzyme patterns appear to be different from those in liver injured by alcohol or hepatotoxins. This result suggests that liver damage caused by ethanol and pyrazole is due not only to high blood alcohol levels, but also to a combined effect of pyrazole and alcohol in regard to hepatic microsomes.

Insulin and epidermal growth factor stimulate glycolysis in quiescent 3T3 fibroblasts with no changes in key glycolytic enzyme activities.

In order to study the effect of insulin and epidermal growth factor (EGF) on glycolysis in quiescent 3T3 fibroblasts and their mechanisms of action, lactic acid produced by cells and activities of key glycolytic enzymes in cell extracts were determined. Insulin increased lactic acid production; the maximal stimulation occurred at the concentrations above 250 ng/ml and the half-maximal dose was 50 ng/ml. This effect of insulin appeared as early as one hour, and lactic acid production in the presence of insulin linearly increased up to 4 h. The 24-h pretreatment with insulin exhibited no significant effect on the production by cells afterward incubated either with or without insulin. Lactic acid production decreased as the concentration of phloridzin increased. However, insulin stimulation of the production still remained in the presence of phloridzin. Parahydroxymercuribenzoate reduced production only by the equivalent of the increase due to insulin. EGF also increased lactic acid production; this effect occurred at 1 ng/ml and was maximal at 100 ng/ml. The activities of hexokinase, phosphofructokinase and pyruvate kinase in quiescent cells were not increased by insulin, and the affinities for substrates of these enzymes remained unaltered. These findings suggest that glucose uptake is a rate-limiting step in glycolysis in quiescent 3T3 fibroblasts and that the stimulatory effect of insulin on glycolysis is mediated by enhanced glucose entry.

Glycogen content and activities of key glycolytic enzymes in muscle biopsies from control subjects and patients with chronic alcoholic skeletal myopathy.

The capacity for glycolysis in muscle biopsies obtained from long-term heavy alcohol drinking patients has been compared with tissue from control subjects by assay in vitro of the total activities of glycogen phosphorylase, phosphofructokinase and fructose 1,6-bisphosphatase, key regulatory enzymes in the anaerobic glycolytic pathway. Biopsies from 13 of 22 patients had type II fibre atrophy, and the activities of all three enzymes were reduced in these biopsies, when expressed in terms of DNA content, the most striking reduction being in phosphofructokinase activity. The amount of glycogen in the tissue correlated closely with these enzyme activities and was slightly lower in the most atrophic tissue, when expressed in terms of DNA content. The activities of acid and neutral alpha-glucosidases were similar in biopsies from control subjects and patients with various severities of alcohol myopathy. The reduced activities are consistent with a reduced proportion of type II fibre muscle mass in these patients, and suggest that there may be a reduced capacity for glycolysis with resultant reduced lactate production. Whether the changes in enzyme activities are primary to the selective atrophy remains to be established.

Evidence for a functional ATP-citrate lyase:NADP-malate dehydrogenase pathway in bovine adipose tissue: Enzyme and metabolite levels

Activities of key lipogenic and glycolytic enzymes were determined in extracts of crude homogenates to elucidate the rate-limiting step(s) for lipogenesis from lactate and glucose in bovine subcutaneous adipose tissue. The enzymes ATP-citrate lyase, NADP-malate dehydrogenase, and pyruvate carboxylase were shown to have enough activity to account for the rates of in vitro lipogenesis from 10 m m lactate with or without 2 m m glucose. Glucose utilization for fatty acid synthesis appears to be limited by the low activities of key glycolytic enzymes, especially hexokinase. Attempts were also made to estimate enzyme activities in bovine subcutaneous adipose tissue being incubated in vitro by relating primary substrate levels to kinetic characteristics for the enzymes. ATP-citrate lyase was estimated to be operating at levels equivalent to the rates of lactate incorporation into fatty acids in the absence or presence of 2 m m glucose in the incubation media. Additionally, metabolite levels were measured in rapidly frozen samples of bovine subcutaneous adipose tissue to estimate the relative importance of key lipogenic enzymes in vivo. At the citrate and malate levels measured in vivo, ATP-citrate lyase would be operating at levels that approximate those estimated in vitro.

Effect of endotoxin administration on heart glycolytic enzyme activities in normal and diabetic dogs.

AbstractThe effects of Escherichia coli endotoxin administration on the activities of key glycolytic enzymes in normal and alloxan-diabetic dog hearts were studied and the results were correlated with changes in plasma glucose concentrations. Normal dogs had a fasting plasma glucose concentration of 100 mg%, which was increased to 130 mg% 1 hr posten-dotoxin, then followed by a progressive decrease at 2 hr and thereafter to hypoglycemic levels (68 mg% at 4 hr). Diabetic dogs had a fasting plasma glucose concentration of 360 mg%, which was decreased to and maintained at 250 mg% at 2 and 4 hr following endotoxin injection. Hexokinase activity was not significantly different between the normal and diabetic dogs (P = 0.09). After endotoxin injection hexokinase activity tended to increase at 2 to 4 hr (P = 0.056) but it did not respond differently between the two groups, normal versus diabetic (P = 0.29). Phosphofructokinase activity was not different between normal and diabetic dogs (P = 0.49). After endotoxi...

The osteoglossid gill: correlations of structure, function, and metabolism with transition to air breathing

The gills of aruana and Arapiama, closely related water- and air-breathing osteoglossids commonly found living in similar environments in the Amazon, were compared in terms of cell composition, structure, and metabolic organization. Enzymes selected to represent different metabolic functions, including glycolysis, pentose shunt, gluconeogenesis, the Krebs cycle, hydrogen shuttling, and ammonia release, were found in the gills of both species. However, the aruana gill had higher activities of key glycolytic enzymes, such as pyruvate kinase and phosphofructokinase, but lower potential for gluconeogenesis (lower fructose diphosphatase: phosphofructokinase ratios), and lower activities of enzymes in oxidative metabolism (citrate synthase, malate dehydrogenase, aspartate aminotransferase, and glutamate dehydrogenase) and in the pentose shunt (glucose-6-phosphate dehydrogenase). Gill lactate dehydrogenase activities were quite high in both species, while hexokinase activities were low; coupled with other studies, these data were taken to mean that lactate is a major source of carbon and energy for the gill. In Arapaima adjustments in cell composition, ultrastructure, and structural organization develop the potential for close control of the regional distribution of cardiac output flowing through the gill. It was speculated that use of this potential control of blood flow would have a major effect (a) on the uptake of blood metabolites such as lactate, (b) on CO2 release, and (c) on ion regulation by the abundant chloride cell population of the Arapaima gill.

Studies on cellular toxicity of bilirubin: Effect on brain glycolysis in the young rat

(1) The levels of glycolytic intermediates in the brain tissue of 15-day-old homozygous and heterozygous Gunn rats were determined with or without novobiocin treatment in vivo, and the inhibitory effects of bilirubin on the glycolytic activity and the key glycolytic enzyme activities were investigated in vitro. (2) the pattern of glycolytic intermediate levels in the non-treated homozygotes compared with those in the non-treated heterozygotes showed a significant increase of G6P and F6P, and decrease of GAP plus DAP. Enhancement of bilirubin concentration in the brain tissue of homozygotes by novobiocin treatment brought about a significant decrease of FDP, 3PG, Pyr and Lac, and a slight increase of G6P compared with the respective values on the treated heterozygotes. (3) the overall glycolytic activity with 2mM glucose as a substrate was significantly inhibited in vitro by 100 muM bilirubin at 3-4, 20 and 600muM NAD concentration, whereas with 0.15 mM glucose the inhibition was observed only at a concentration of 3-4muM NAD. The inhibitory effect of bilirubin (120-140 muM) on lactate formation with FDP as a substrate was also found at three different NAD concentrations. And the marked inhibition of PFK activity by bilirubin was observed at physiological concentrations of ATP and F6P. (4) The present findings suggest that reduced rates of glycolysis might be involved in bilirubin encephalopathy.

Enzymatic pattern of glucose metabolic pathways in pyruvate kinase-deficient erythrocytes

It has been shown that in some cases of congenital non-spherocytic haemolytic anaemia (CNSHA) with pyruvate kinase deficiency, the primary defect may be related to diminished magnesium-stimulated ATPase activity, followed by elevation of the erythrocyte ATP level. ATP as the end product of glycolysis inhibits by negative feedback control the activities of key glycolytic enzymes involved in energy production, i.e. pyruvate kinase (PK) and phosphofructokinase (PFK). Erythrocyte-deficient PK, however, is insensitive to the stimulating effect of fructose 1,6-diphosphate (FDP), which is normally a positive effector of PK. Both competing effectors, i.e. ATP and FDP, seem to show specific interaction. PK, inactive in the presence of high concentrations of ATP, seems to lose its sensitivity to FDP. This effect persists until ATP molecules are present in excess. In vitro incubation of deficient PK with ATPase resulted in increased PK activity as well as in recovery of its sensitivity to the stimulating effect of FDP. The same effects were obtained in vivo by administering magnesium levulinate intravenously to CNSHA patients with PK deficiency. This may indicate that magnesium ions stimulate deficient ATPase activity and lead to diminution of ATP as a negative effector for other regulatory enzymes.

Glycolysis and gluconeogenesis in the giant scallop, Placopecten magellanicus (gmelin)

1.1. It would appear from the relative activities of key glycolytic enzymes that succinate is the main end product of glycolysis in both the mantle and the adductor muscle of the giant scallop.2.2. All but one of the enzymes involved in succinate formation were found to be cytoplasmic.3.3. The enzymes of the Bücher shuttle were found in the adductor muscle.4.4. The high activities of phosphoenolpyruvate carboxykinase and fructose diphosphatase are compatible with active glycogen synthesis.

Regulation of human and rat brain metabolism: Inhibitory action of phenylalanine and phenylpyruvate on glycolysis, protein, lipid, DNA and RNA metabolism

In human and rat brain the activity of key glycolytic enzymes and glycolysis were examined during development. The differential inhibitory effect of l-phenylalanine and phenylpyruvate on key glycolytic enzymes, on lactate production and on the incorporation of labeled glucose into lipid, protein, RNA and DNA was investigated in the brain of differentiating human and rat. 1. (1)|In the fetal human brain hexokinase, pyruvate kinase and glycolytic activities were approximately 10% of those observed in the adult. These parameters in the average brain cell gradually rose during differentiation. A similar pattern for these three glycolytic parameters was observed in the differentiating rat brain. 2. (2)|The sensitivity to inhibition by l-phenylalanine and phenylpyruvate was the same order of magnitude for the glycolytic enzymes and lactate production during differentiation. However, since the activities were very low in the fetal and the newborn brain, these parameters may be more vulnerable to inhibition by these compounds that accumulate in the untreated phenylketonuric patients. 3. (3)|The human and fetal brain has a very high cellularity (600–700 millions per g) during gestation and continues to decrease until it reaches a level of about 100 millions of cells per gram wet weight in the adult brain. This emphasizes the importance of expressing biochemical results on a per cell basis during differentiation. 4. (4)|Studies on the mechanism of effect of l-phenylalanine and phenylpyruvate in slices from differentiating human and adult brain were carried out. The kinetic results suggest that these compounds act through a competitive mechanism and the inhibitions were reversible by increasing the substrate level. 5. (5)|The effect of phenylpyruvate on lactate production from glucose, glucose-6-phosphate and fructose-1,6-diphosphate was also explored in the fortified supernatant fluid system from brains of differentiating human and rat. Phenylpyruvate resulted in a progressive inhibition of lactate production from all three substrates. Reciprocal plots, the Dixon plot and the reversibility of the inhibition exerted by phenylpyruvate suggested a competitive type of inhibition. 6. (6)|Since the metabolic inhibitions exerted by phenylalanine and phenylpyruvate on carbohydrate metabolism were reversible, it is possible that the high carbohydrate diet might be able to protect the developing brain from the potential damage of the high levels of these compounds in the phenylketonuric patients. 7. (7)|When increasing concentrations of phenylpyruvate were added to brain slices from differentiating rat, there was a steady, dose-dependent decrease in the incorporation of labeled glucose into lipid, RNA and DNA, which in all cases was more pronounced in slices from the fetal brain than from the adult. It is striking that there was a progressive decrease in the incorporation of label into protein with increasing concentrations of phenylpyruvate in the fetal brain but the adult brain was not affected at any concentration. These data are in agreement with the postulate that phenylpyruvate might interfere with the biosynthesis of strategic macromolecules in the differentiating brain. The results of this work also agree with clinical experience that points to the early stages of brain development as especially vulnerable to toxic effects, including metabolic disturbances. 8. (8)|The enzymatic attacking points of l-phenylalanine and phenylpyruvate involve the inhibition of key enzymes of glycolysis (hexokinase and pyruvate kinase) and of the direct oxidative pathway (6-phosphogluconate dehydrogenase). The inhibition of these enzymes might explain, at least in part, the inhibition of the biosynthesis of key macromolecules in the brain.