Evidence for cell-mediated immunity to collagen in progressive systemic sclerosis

The activities of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, G6PD), 6-phosphogluconate dehydrogenase (6-phospho-d-gluconate: NADP oxidoreductase, 6PGD), hexokinase (ATP:D-hexose 6-phosphotransferase, HK), lactic dehydrogeanse (L-lactate: NAD oxidoreductase, LDH) and aspirate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, Asp.T) were determined in red blood cells of 11 healthy individuals. The determinations were carried out on samples drawn every 4 h over a 24 h period. The activities of G6PD, 6PGD, LDH and Asp.T exhibited a semi-circadian rhythm, namely, two peaks of activity during 24 h while HK activity demonstrated a true circadian rhythm. In addition a polymorphism of the G6PD and LDH activity patterns was observed. The implications of a biological clock in enucleated cells are discussed.     

Hexokinase isoenzymes in the rat placenta

The phosphorylation of glucose to glucose-6-phosphate, the first enzymatic step for glucose utilization is catalysed by a family of four hexokinase isoenzymes (HKI-IV) which display a tissue-specific distribution. The expression of HK isoenzymes was investigated in the rat placenta. High levels of HKI and HKII mRNA were found in the junctional and the labyrinthine zones. HKIII mRNA was present at low levels in the junctional zone and glucokinase (HKIV) mRNA was not detected, indicating that HKI and HKII are the two major placental HK isoenzymes. HKII activity was increased in placenta of insulinopenic diabetic rats. This regulation is likely to support the increase in glucose utilization and storage characteristics of the enlarged placentae of diabetic rats.     

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (= GGS1 = FDP1 = BYP1 = CIF1 = GLC6 = TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1 delta mutant and the wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total D-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, +/- 10 mM in a hxk1 delta hxk2 delta glk1 delta and 2-3 mM in a tps1 delta strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity.     

Action of growth hormone on erythropoiesis: changes in red blood cell enzyme activities in growth-retarded patients with and without growth hormone deficiency

Fifteen red cell enzyme activities of growth-retarded patients with and without growth hormone (GH) deficiency were investigated before and after GH administration. The 15 enzymes were Hexokinase, phosphoglucomutase, glucose phosphate, isomerase, phosphofructokinase, fructose diphosphate aldolase, glyceraldehyde-3-phosphae dehydrogenase, triosephosphate isomerase, 2,3-diphosphoglycerate mutase, 3-phosphoglycerate kinase, 3-phosphoglycerate mutase, enolase, pyruvate kinase, glycose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, glutathione reducase. Sixty-six subjects were studied: 30 normal control subjects (group N) and 36 patients (aged 5-23 years) with short stature. Complete endocrine evaluation showed 21 (group I) to have GH deficiency (10 patients with isolated GH deficiency) and 15 (group II) to have normal hypothalamic and pituitary function except for two patients with a moderate hypothyroidism. Both had been receiving thyroid hormone treatment for a long time before our studies. All 36 patients were treated with 2 mg human growth hormone intramuscularly for 7 days. Before GH treatment no significant difference was observed between hematologic data in group I (GH deficiency) and group II (no GH deficiency). After GH therapy there was a significant increase in reticulocyte count in both groups of patients with short stature. The mean pretreatment value in group I was 1.294% +/- 0.084 (SEM); the mean post-treatment value was 2.081% +/- 0.287 (SEM)< P less than 0.005. The mean pretreatment value in group II was 1.0% 0.184 (SEM); the mean post-treatment value was 1.407% +/- 0.193 (SEM), P less than 0.01. In group II (no GH deficiency) mean pretreatment erythrocyte enzyme activities were not significantly different from those activities observed in normal control subjects (group N). However, in patients who lacked GH, the pretreatment activities of five red cell enzymes (glucose phosphate isomerase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, 2,3-diphosphoglycerate mutase, 3-phosphoglycerate kinase) were significantly decreased before GH administration compared with the values in normal control subjects...     

Rifampin, a useful drug for nonmycobacterial infections

Hexokinase I, the pacemaker of glycolysis in brain tissue and red blood cells, is comprised of two similar domains fused into a single polypeptide chain. The C-terminal half of hexokinase I is catalytically active, whereas the N-terminal half is necessary for the relief of product inhibition by phosphate. A crystalline complex of recombinant human hexokinase I with glucose and phosphate (2.8 A resolution) reveals a single binding site for phosphate and glucose at the N-terminal half of the enzyme. Glucose and phosphate stabilize the N-terminal half in a closed conformation. Unexpectedly, glucose binds weakly to the C-terminal half of the enzyme and does not by itself stabilize a closed conformation. Evidently a stable, closed C-terminal half requires either ATP or glucose 6-phosphate along with glucose. The crystal structure here, in conjunction with other studies in crystallography and directed mutation, puts the phosphate regulatory site at the N-terminal half, the site of potent product inhibition at the C-terminal half, and a secondary site for the weak interaction of glucose 6-phosphate at the N-terminal half of the enzyme. The relevance of crystal structures of hexokinase I to the properties of monomeric hexokinase I and oligomers of hexokinase I bound to the surface of mitochondria is discussed.     

Peculiarities of the action of protein positively reacting in the sedimentation test for cancer on the activity of glycolytic enzymes

Blood serum of oncologic patients due to immunoglobulin involved in its composition, activates glycolysis in the soluble fraction of muscles when using starch, glycogen and glucose as substrates. The activation is registered under both aerobic and anaerobic conditions. When elucidating the immunoglobulin effect in a glycolytic chain under aerobic conditions it is shown that its activating effect in the incomplete incubation system is manifested with such glycolysis substrates as fructose-6-phosphate and 2-phosphoglyceric acid. Glycolysis activation with serum is insignificant or absent at all with the presence of glucose-6-phosphate, fructose-1,6-diphosphate, 3-phosphoglyceric aldehide, 3-phosphoglyceric acid, phosphoenolpyruvic acid, sodium pyruvate. Immunoglobulin isolated from the blood serum of oncologic patients does not affect the activity of purified preparations of hexokinase, glycerinaldehydephosphate dehydrogenase, lactate dehydrogenase under aerobic and anaerobic conditions. When using the air as a gas medium lactate dehydrogenase is activated by immunoglobulin. Lactate dehydrogenase activity under aerobic and anaerobic conditions is essentially lower than in the case when the air serves as a gas medium.     

Effect of low altitude on glycolytic key enzymes in red blood cells

METHOD: The ATP level and the activities of hexokinase (HK), phosphofructokinase-1 (PFK-1) and pyruvate kinase (PK) in red blood cells (RBC) were measured in 50 school students 6-12 yr of age in the Jordan Valley (JV) which is the lowest region below sea level in the world (low altitude: 390 m below sea level, hyperoxic and with an average daily value of 796 mmHg barometric pressure) and in 50 school students of the same age group in Irbid region (normal altitude: 600 m above sea level and with an average daily value of 600 mmHg barometric pressure). The same parameters were measured also in 40 school students at sea level. RESULTS: A significant decrease in HK and PFK-1 activities and an increase in ATP level in the low altitude region, while no significant change in PK activity in JV-group when compared to Irbid and to the sea level control groups. Possible explanations will be discussed to interpret these observations.     

The effects of insulin on transport and metabolism of glucose in skeletal muscle from hyperthyroid and hypothyroid rats

The effects of insulin on the rates of glucose disposal were studied in soleus muscles isolated from hyper- or hypothyroid rats. Treatment with triiodothyronine for 5 or 10 days decreased the sensitivity of glycogen synthesis but increased the sensitivity of lactate formation to insulin. The sensitivity of 3-O methylglucose to insulin was increased only after 10 days of treatment and was accompanied by an increase in the sensitivity of 2-deoxyglucose phosphorylation; however, 2-deoxyglucose and glucose 6-phosphate in response to insulin remained unaltered. In hypothyroidism, insulin-stimulated rates of 3-O-methylglucose transport and 2-deoxyglucose phosphorylation were decreased; however, at basal levels of insulin, 3-O-methylglucose transport was increased, while 2-deoxyglucose phosphorylation was normal. In these muscles, the sensitivity of lactate formation to insulin was decreased; this defect was improved after incubation of the muscles with prostaglandin E2. The results suggest: (a) in hyperthyroidism, insulin-stimulated rates of glucose utilization in muscle to form lactate are increased mainly because of a decrease in glycogen synthesis; when hyperthyroidism progresses in severity, increases in the sensitivity of glucose transport to insulin and in the activity of hexokinase may also be involved; (b) in hypothyroidism, the decrease in insulin-stimulated rates of glucose utilization is caused by decreased rates of glycolysis; (c) prostaglandins may be involved in the changes in sensitivity of glucose utilization to insulin observed in muscle in altered thyroid states.     

The effect of 5-methylpyrazole-3-carboxylate and nicotinic acid on abnormalities of carbohydrate metabolism in alloxan-diabetic rat muscle

The administration of the antilipolytic agents sodium nicotinate (1 mmole/kg i.p.) or sodium 5-methylpyrazole-3-carboxylate (0.5 or 1.0 mmole/kg i.p.) to alloxan-diabetic rats produced a significant reduction in the plasma concentration of free fatty acids and a slight reduction in blood glucose concentration. The concentrations in the freeze-clamped heart of citrate , acetyl CoA, Glucose-6-phosphate and fructose-6-phosphate were increased in untreated alloxan-diabetic rats relative to normoglycaemic controls. Treatment of alloxan-diabetic rats with the antilipolytic agents or insulin (60 U/kg i.p.) lowered these increased concentrations of metabolites in the heart. Treatment of the diabetic rats with the antilipolytic agents also produced an increase in the activity of pyruvate dehydrogenase in heart, but only treatment with 5-methylpyrazole-3-carboxylate had a significant effect on the activity of the enzyme in freeze-clamped soleus muscle.     

Decreased cerebral glucose utilization in rats during the ebb phase of thermal injury

OBJECTIVE: Thermal injury is associated with the development of encephalopathy. The mechanism(s) for the development of this condition have not been established. In the present study, the effects of thermal injury were determined on rat brain glucose utilization (Rg), using 2-[18F]fluoro-2-deoxy-D-glucose (18FDG). DESIGN: Four types of studies were performed. In one group of rats, the effect of thermal injury on total rat brain glucose utilization (Rg) was determined at 6 hours, 24 hours, and 3 weeks after injury. The brains of thermally injured rats were also assayed for hexokinase and glucose-6-phosphatase activities, since these enzyme activities are responsible for the phosphorylation and dephosphorylation of the 18FDG. We also measured total body oxygen consumption in the thermally injured rats. We wanted to compare the changes produced by thermal injury on rat brain glucose utilization (Rg) with the effects produced by compounds known to modify energy metabolism and/or rat brain glucose utilization (Rg). For that reason, in a second group of rats, an inflammatory state was produced by lipopolysaccharide injection, and rat brain glucose utilization (Rg) was determined. In the third group of rats, overall metabolism in rats was reduced by pentobarbital injection, followed by hypothermia, and rat brain glucose utilization (Rg) was determined. In the fourth group of rats, overall metabolism in rats was stimulated by 2,4-dinitrophenol injection, and rat brain glucose utilization (Rg) was determined. MATERIALS AND METHODS: Glucose utilization (Rg) by the brains of these treated rats was determined using 18FDG. Oxygen consumption in vivo, as well as glucose-6-phosphatase and hexokinase activity in vitro, were measured by standard procedures. MEASUREMENTS AND MAIN RESULTS: Glucose utilization (Rg) by rat brain was significantly reduced (p < 0.01) at 6 and 24 hours after injury, but returned to normal values 3 weeks after injury. These reductions were associated with decreases in rat brain hexokinase activity, increases in rat brain glucose-6-phosphatase activity, and decreased oxygen consumption by rats in vivo. Pentobarbital injection followed by hypothermia reduced rat brain glucose utilization (Rg) (p < 0.01), while 2,4-dinitrophenol treatment elevated rat brain glucose utilization (Rg) (p < 0.01). In contrast, LPS treatment had no effect on rat brain glucose utilization (Rg). CONCLUSIONS: These data indicate that thermal injury decreases glucose utilization (Rg) in rat brain during the hypometabolic phase. This effect can be explained, at least in part, by alterations in hexokinase and glucose-6-phosphatase activities, as well as reductions in oxygen consumption. Thus, the changes in brain glucose utilization (Rg) appear to be associated with the ebb phase of the thermal injury. The present results observed in burned rats may provide evidence to explain the encephalopathy observed in burned patients.     

Glucose utilization rates in single neurons and neuropil determined by injecting nontracer amounts of 2-deoxyglucose

A nontracer amount of 2-deoxyglucose (DG) was intravenously injected into rats, which were frozen 2 and 4 min later in liquid nitrogen. The freeze-dried samples of cell bodies of anterior horn cells, dorsal root ganglion cells, and cerebellar Purkinje cells, as well as the neuropil adjacent to anterior horn cell bodies, were prepared. Their contents of glucose, glucose 6-phosphate, DG, and 2-deoxyglucose 6-phosphate were microassayed using an enzymatic amplification reaction. NADP cycling. Based on the resulting data and theoretical equations previously described, glucose utilization rate (GUR) and apparent distribution volumes (DVs) of glucose and DG were determined. Anterior horn cell bodies had the highest GUR and their neuropil the lowest, although apparent DVs of glucose and DG were similar in both. This indicates that the glucose supply was equally balanced in all, but that the cell bodies had higher functional activity supported by hexokinase (and other enzymes) related to their energy demands. Dorsal root ganglion cells showed the lowest 2-deoxyglucose 6-phosphate formation rate, but their GUR was slightly higher than that of neuropil because of their markedly large DV of glucose, thus demonstrating that the abundant glucose supply supports the neuronal function. Purkinje cells indicated GUR and apparent DVs similar to molecular and granular layers.     

Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation

We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH130 by disruption of the cell membrane by nitrogen cavitation. These isolated mitochondria were found to have essentially the same functional properties as rat liver mitochondria, but unlike the latter, hexokinase (HK) was bound to their membrane. Using the tumor mitochondrial preparation, we examined the source of ATP for phosphorylation of glucose by HK under conditions in which intra- and extramitochondrial ATP-generation systems operated separately or together. Results showed that the membrane-bound HK utilized ATP derived from the most efficiently operating ATP generation system, i.e., oxidative phosphorylation. However, when the rate of extramitochondrial ATP generation was much greater than that of oxidative phosphorylation, HK used ATP from the extramitochondrial ATP-generation system.     

Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 mumol/g wet weight and in muscle led to its increase from 3.64 to 25.1 mumol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK) by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.     

Biochemical polymorphism and the 2,3-diphosphoglycerate in the sheep red blood cells

There was no significant difference in the level of 2,3-DPG in the red blood cells of sheep of different haemoglobin types (Hb A and Hb B) or potassium types (HK and LK). However, low glutathione (GSHL) sheep had significantly higher (p less than 0.01) level of 2,3-DPG in their red blood cells than high glutathione (GSHH) sheep. There was also significant effect of interactions between glutathione, haemoglobin and potassium types (p less than 0.05) and glutathione and haemoglobin types (p less than 0.01) on red cell 2,3-DPG levels.     

Frequency analysis of the contralateral suppression of evoked otoacoustic emissions by narrow-band noise

Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1 delta, hxk2 delta grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1 delta, hxk2 delta double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucose-induced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the deletion of HXK2. However, both the ggs1 delta and the ggs1 delta, hk2 delta mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1 delta strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fructose metabolism and cell survival in freshly isolated rat hepatocytes incubated under hypoxic conditions: proposals for potential clinical use

The protective effect of fructose with regard to hypoxia-induced cell injury was investigated. The addition of fructose (2 to 20 mmol/L) protected hepatocytes against hypoxia-mediated cell lysis in a concentration-dependent way. The intracellular ATP content was initially decreased as a result of fructose-1-phosphate formation, but it remained constant during the hypoxic incubation. Conversely, high initial ATP values observed at low fructose concentrations progressively declined. Cellular protection was observed only when fructose was added before (and not after) the start of hypoxia. In addition, a sufficient amount of fructose-1-phosphate rapidly accumulated before the induction of hypoxia, and the linear production of lactate, during hypoxic incubation, indicated that cells synthesized ATP continuously. The lack of cell protection by fructose added after the onset of the hypoxia may be explained by a lesser fructose-1-phosphate formation and a subsequently low accumulation leading to insufficient glycolytic ATP production. Under aerobic conditions, both glycolysis (lactate formation) and gluconeogenesis (glucose formation) were carried out in fructose-1-phosphate-loaded cells with the same initial rates, whereas under hypoxic conditions glycolysis was the main metabolic event. The fact that protein synthesis activity recovered faster during reoxygenation of previously hypoxic fructose-treated cells than in glucose-treated cells led us to hypothesize that in situ perfusion of liver with fructose, before its removal, would improve its metabolic capacity during the hypoxic cold preservation and subsequent transplantation.     

Urinary sugar phosphates and related organic acids in fructose-1,6-diphosphatase deficiency

Two sisters with fructose-1,6-diphosphatase deficiency are reported. They presented with ketonuria, elevated plasma transaminase activity and severe metabolic acidosis during hypoglycaemic crises, which resembled Reye syndrome. Intravenous fructose tolerance tests provoked severe hypoglycaemia and metabolic acidosis. Fructose-1,6-diphosphatase activities in both peripheral leukocytes and cultured lymphocytes were below the limit of detection. Urinary organic acid analysis during crises revealed markedly increased excretion of lactate, ketone bodies, glycerol and glycerol-3-phosphate. We newly identified other glycolytic intermediates, glyceraldehyde, 3-phosphoglycerate and fructose-1,6-diphosphate, in the urine during hypoglycaemic attacks or after fructose tolerance tests. Identification of such compounds may be useful in the early diagnosis of this disease.     

Triosephosphate metabolism by mature boar spermatozoa

Boar sperm rapidly interconverted dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, produced fructose-1,6-bisphosphate, approximately equilibrium concentrations of fructose 6-phosphate and glucose 6-phosphate but not glycerol or glycerol 3-phosphate. In the presence of 3-chloro-1-hydroxypropanone, an inhibitor of stage 2 of the glycolytic pathway, the triosephosphates were metabolized faster, produced less fructose-1,6-bisphosphate, fructose 6-phosphate and glucose 6-phosphate, but not glycerol or glycerol 3-phosphate. This suggests that these cells may have the capacity to convert glycolytic intermediates into a storage metabolite to conserve carbon atoms for the eventual synthesis of lactate.