A simplified radioactive assay for the enzyme UDP-glucose pyrophosphorylase.
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Carbon Radioisotopes↗
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Results for “GLUCOSE, RADIOACTIVE”
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[Studies on the corneal metabolism by radioactive isotope. 3. Incorporation of 14C-glucose into the mucopolysaccharide of rabbit cornea and the effects of metabolic accelerators and inhibitors for 14C incorporation].
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Animals↗
Distribution of radioactive carbon from [U]-14C-glucose in adult mice brains in vivo under normal conditions.
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Animals↗
Distribution of Cellular Carbohydrates during Development of the Mycelium and Fruitbodies of Flammulina velutipes.
Flammulina velutipes (Curt. ex Fr.) Sing. was grown on potato-glucose solution freed of most starch. Glucose uptake and dry weight changes in the colony indicated that the large fruitbodies derived their substrates partly from glucose remaining in the medium and partly from cellular constituents stored in the mycelium and small fruitbodies. Changes in the amounts of low molecular weight carbohydrates, glycogen, and four cell wall polysaccharide fractions were followed in the mycelium and fruitbodies. Trehalose, arabitol, and smaller amounts of mannitol were the main stored low molecular weight carbohydrates. A large net loss of these compounds occurred in the mycelium and small fruitbodies after their growth ceased. The carbohydrates accumulated in the large fruitbodies, but were also partly metabolized in the colony. Reducing sugars were minor components, and included about 30 to 50% glucose and a small undetermined quantity of fructose. Glycogen was the main storage carbohydrate in the mycelium, and was also stored in the small fruitbodies. It was broken down in both structures during growth of the large fruitbodies which accumulated only small amounts. During the same period, almost 45% of the maximum amount of cell wall polysaccharides were degraded in the small fruitbodies, but not in the mycelium.By feeding (14)C-glucose in replacement medium, incorporation of radioactivity into carbohydrates was followed in the colony during fruit-body development. Total incorporation was highest in trehalose, next highest in glycogen, and the rest was found in polyols and cell wall polysaccharides except for a few per cent which remained in endogenous glucose. In the large fruitbodies, specific radioactivity in glucose was much lower than in trehalose and mannitol. The labeling patterns in the mycelium and large fruitbodies suggested that trehalose, mannitol, and possibly arabitol were translocated into the stipes and pilei.
Journal Article↗
Evidence against necessary phosphorylation during hexose transport in Aspergillus nidulans.
The transport of 2-deoxy-d-glucose, a nonmetabolizable glucose analogue, into Aspergillus nidulans against a concentration gradient does not appear to require phosphorylation, despite the high levels of sugar phosphates accumulated rapidly within the cell. Two other deoxy analogues of d-glucose, 6-deoxy-d-glucose and 1,5-anhydro-d-glucitol (1-deoxy-d-glucose), although they lack the C-6 and the C-1 hydroxyl groups, respectively, and thus cannot be phosphorylated in those positions, still competitively inhibit the entry of 2-deoxy-d-glucose. Moreover, 6-deoxy-d-glucose can be concentrated against a gradient within the cell without the accumulation of 6-deoxy-d-glucose-phosphate. d-Galactose shows an intracellular ratio of free to phosphorylated sugar similar to that found for 2-deoxy-d-glucose in cells that have galactokinase, but no sugar phosphates are found in a galactokinaseless mutant strain. These data suggest that intracellular kinases are responsible for the sugar phosphate pool; and indeed, a kinase capable of phosphorylating 2-deoxy-d-glucose has been demonstrated. Finally, experiments on the kinetics of labeling of intracellular free sugar and sugar phosphate pools with (14)C-2-deoxy-d-glucose show that radioactivity appears first in the free sugar pool and after a delay enters the sugar phosphate pool.
Aspergillus↗
Pulmonary surfactant lipid synthesis from ketone bodies, lactate and glucose in newborn rats.
The contribution of acetoacetate (AcAc), beta-hydroxybutyrate (beta OHB), lactate and glucose to pulmonary surfactant lipid synthesis in three- to five-day-old rats was measured. Minced lung tissue was incubated with 3H2O and [3-14C]AcAc, [3-14C]beta OHB, [U-14C]lactate or [U-14C]glucose, and the radioactivity incorporated into surfactant lipids was measured. When expressed as nmol of substrate incorporated/g lung tissue per four hr, lactate was incorporated more rapidly than other substrates into total surfactant lipids and phosphatidylcholine (PC). There was no difference in the rates of incorporation of lactate, AcAc or glucose into disaturated PC (DSPC). Substrates other than glucose were incorporated almost exclusively into fatty acids, whereas 60-80% of glucose incorporated into surfactant phospholipids was found in fatty acids, with the remaining in glyceride-glycerol. When expressed as nmol acetyl units incorporated/g lung tissue per four hr, the rates of AcAc, lactate and glucose incorporation into total surfactant fatty acids were comparable. Glucose incorporation into DSPC and PC was greater than that of AcAc and lactate. When glucose was the only exogenous substrate added to the incubation medium, it contributed 37% of total surfactant fatty acids synthesized de novo. In the presence of other substrates, the contribution of glucose to de novo fatty acid synthesis dropped to 14-20%. In the presence of unlabeled glucose, 14C-labeled AcAc, lactate and beta OHB contributed 52%, 40% and 19%, respectively, of the total fatty acids synthesized de novo. The rate of beta OHB incorporation into surfactant lipids was only about 50% that of other substrates and was accompanied by low activity of beta-hydroxybutyrate dehydrogenase measured for newborn lung.(ABSTRACT TRUNCATED AT 250 WORDS)
3-Hydroxybutyric Acid↗
Hepatic redox state and gluconeogenesis from lactate in vivo in the rat.
1. To examine the role of the hepatic redox state on the rate of gluconeogenesis the effects of sodium crotonate injection (6mmol/kg body wt.) on rat liver metabolite concentrations and gluconeogenesis from lactate were studied in vivo. 2. Crotonate caused a marked oxidation of cytoplasmic and mitochondrial redox couples; decreases were observed in the ratios of [lactate]/[pyruvate], [glycerol 3-phosphate]/[dihydroxyacetone phosphate], [hydroxybutyrate]/[acetoacetate] and measured [NAD(+)]/[NADH]. 3. Increases occurred in the liver concentrations of all gluconeogenic intermediates from pyruvate through to glucose 6-phosphate, but there was no change in lactate concentration. 4. To determine whether gluconeogenesis from lactate was altered by the more-oxidized hepatic redox state l-[2-(14)C]lactic acid was infused into the inferior vena cava (50mumol/min per kg body wt.) and the incorporation of radioactivity into blood glucose was measured. 5. Administration of crotonate transiently decreased the rate of lactate incorporation into glucose but within a few minutes the rate of incorporation returned to that of the controls. 6. The results indicate that in these experiments alteration of the NAD(+)-NADH systems of cytoplasm and mitochondria to a more-oxidized state did not change the rate of gluconeogenesis.
Acetoacetates↗
Stimulation of glucose catabolism through the pentose pathway by the absence of the two pyruvate kinase isoenzymes in Escherichia coli.
Escherichia coli strains devoid of one or both of the two pyruvate kinase isoenzymes (PKA and PKF), were grown on minimal media in batch fermentations. The strain lacking both PKs showed a 28% decrease on its specific growth rate when compared to the wild type. However, protein and CO2 yields did not change. Using radioactive 1-C14 glucose and collecting the CO2 produced by the cultures, it was found that the mutant lacking both pyruvate kinases, metabolized glucose mainly through the pentose pathway (PP). The increased participation of the PP in glucose metabolism in this strain, was also reflected on the levels of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases.
Escherichia coli↗
Metabolic effects and fate of succinic acid methyl esters in rat hepatocytes.
The metabolic fate and metabolic effects of succinic acid methyl esters were examined in rat isolated hepatocytes. Both the monomethyl ester (SAM) and dimethyl ester (SAD) of succinic acid inhibited D-glucose metabolism. Such an inhibition affected, in order of increasing severity, the direct incorporation of D-glucose into glycogen and futile cycling between the hexose and its 6-phosphate ester, the phosphorylation of D-glucose, the generation of triose phosphates from the hexose, and the production of 14C-labeled lactate, pyruvate, and amino acids from D-[U-14C]glucose and its oxidation. The dimethyl ester of [1,4-14C]succinic acid ([1,4-14C]SAD) or [2,3-14C]-succinic acid ([2,3-14C]SAD) was efficiently converted to acidic metabolites. The oxidation of [1,4-14C]SAD largely exceeded that of [2,3-14C]SAD. Inversely the generation of newly formed radioactive D-glucose and glycogen appeared higher in cells exposed to [2,3-14C]SAD, rather than [1,4-14C]SAD. It is proposed that SAM and SAD are suitable nutrients both to cover the energy need of hepatocytes and to act as gluconeogenic precursors.
Amino Acids↗
Glucose kinetics during prolonged exercise in euglycaemic and hyperglycaemic subjects.
To determine the limits to oxidation of exogenous glucose by skeletal muscle, the effects of euglycaemia (plasma glucose 5 mM, ET) and hyperglycaemia (plasma glucose 10 mM, HT) on fuel substrate kinetics were evaluated in 12 trained subjects cycling at 70% of maximal oxygen uptake (VO2, max) for 2 h. During exercise, subjects ingested water labelled with traces of U-14C-glucose so that the rates of plasma glucose oxidation (Rox) could be determined from plasma 14C-glucose and expired 14CO2 radioactivities, and respiratory gas exchange. Simultaneously, 2-3H-glucose was infused at a constant rate to estimate rates of endogenous glucose turnover (Ra), while unlabelled glucose (25% dextrose) was infused to maintain plasma glucose concentration at either 5 or 10 mM. During ET, endogenous liver glucose Ra (total Ra minus the rate of infusion) declined from 22.4 +/- 4.9 to 6.5 +/- 1.4 mumol/min per kg fat-free mass [FFM] (P < 0.05) and during HT it was completely suppressed. In contrast, Rox increased to 152 +/- 21 and 61 +/- 10 mumol/min per kg FFM at the end of HT and ET respectively (P < 0.05). HT (i.e., plasma glucose 10 mM) and hyperinsulinaemia (24.5 +/- 0.9 microU/ml) also increased total carbohydrate oxidation from 203 +/- 7 (ET) to 310 +/- 3 mumol/min per kg FFM (P < 0.0001) and suppressed fat oxidation from 51 +/- 3 (ET) to 18 +/- 2 mumol/min per kg FFM (P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Effects of the novel oral antidiabetic agent HQL-975 on glucose and lipid metabolism in diabetic db/db mice.
The antidiabetic effects of 3-¿4-[2-(5-methyl-2-phenyl-oxazol-4- yl)ethoxy]phenyl¿-2S-propylamino-propionic acid (CAS 185679-16-7, HQL-975), a novel oral agent, on a genetically obese non-insulin-dependent diabetes mellitus (NIDDM) model (db/db mice) were examined. HQL-975 administration (3.7-34.1 mg/kg/d for 7 days) decreased the levels of plasma glucose, triglyceride, total cholesterol, non-esterified fatty acid and insulin in the mice. In an intraperitoneal glucose tolerance test (IPGTT), HQL-975 administration decreased the fasting plasma glucose level and improved the glucose tolerance in the mice. The HQL-975 administration also significantly increased the glycogenesis and lipogenesis from 14C-glucose in liver, but did not alter the glycogenesis in the diaphragm or the lipogenesis in adipose tissues at 2 h after the glucose loading. In the HQL-975-treated db/db mice, the radioactivity of 14C-glucose incorporated into hepatic glycogen was higher than that incorporated into hepatic total lipids. After the administration of HQL-975 (34.1 mg/kg/d for 7 days) to db/db mice, the hepatic hexokinase and fatty acid synthetase activities were significantly increased, the glycogen synthase I activity was increased but not significantly, and the glucose-6-phosphatase and the phosphoenolpyruvate carboxykinase activities were decreased. These results suggest that HQL-975 increases the hepatic glucose utilization and decreases the hepatic glucose production. Since hepatic glycogenesis is regulated by glucose itself but not by insulin in normoglycemic ICR mice, HQL-975 is thought to enhance the effect of glucose on the stimulation of hepatic glycogenesis. It is concluded that the enhancement of the hepatic glucose utilization played an important role in the hypoglycemic action of HQL-975.
Animals↗
Evidence for the involvement of Na/Ca exchange in glucose-induced insulin release from rat pancreatic islets.
Glucose-induced inhibition of Ca(++) extrusion from the beta-cell may contribute to the rise in cytosol Ca(++) that leads to insulin release. To study whether interference with Na/Ca exchange is involved in this inhibition the effects of glucose were compared to those of ouabain. This substance inhibits Na/K ATPase, decreases the transmembrane Na(+) gradient in islets, and thus interferes with Na/Ca exchange. Collagenase isolated rat islets were maintained for 2 d in tissue culture with a trace amount of (45)Ca(++). Insulin release and (45)Ca(++) efflux were then measured during perifusion. In Ca(++)-deprived medium (to avoid changes in tissue specific radioactivity) 16.7 mM glucose inhibited (45)Ca(++) efflux. Initially 1 mM ouabain inhibited (45)Ca(++) efflux in a similar fashion, the onset being even faster than that of glucose. The effects of 16.7 mM glucose and ouabain were not additive, indicating that both substances may interfere with Na/Ca exchange. In the presence of Ca(++), 16.7 mM glucose induced biphasic insulin release. Ouabain alone caused a gradual increase of insulin release. Again, the effects of ouabain and 16.7 mM glucose were not additive. In contrast, at a submaximal glucose concentration (7 mM) ouabain enhanced both phases of release. An important role for Na/Ca exchange is suggested from experiments in which Ca(++) was removed at the time of glucose-stimulation (16.7 mM). The resulting marked inhibition of insulin release was completely overcome during first phase by ouabain added at the time of Ca(++) removal; second phase was restored to 60%. This could be due to the rapid inhibitory action of ouabain on Ca(++) efflux thereby preventing loss of cellular calcium critical for glucose to induce insulin release. It appears, therefore, that interference with Na/Ca exchange is an important event in the stimulation of insulin release by glucose.
Animals↗
Effect of grafted PEG-2000 on the size and permeability of vesicles.
The permeability of PEG-derivatized vesicles produced by the extrusion technique (VETs) to encapsulated D-glucose has been studied. Vesicles were prepared from mixtures of dipalmitoylphosphatidylcholine (DPPC), phosphatidylinositol (PI) and dipalmitoylphosphatidylethanolamine with covalently attached poly(ethylene glycol) of molecular weight 2000 (DPPE-PEG2000). The occurrence of an irreversible temperature-induced size transition of the PEG-derivatized VETs was detected close to the temperature of the main phase transition of the predominant phospholipid component by photon correlation spectroscopy. The permeability of the vesicles towards radioactive D-glucose was studied by a method similar to the one proposed by Johnson and Bangham at 25, 37 and 50 degrees C for VETs with DPPE-PEG content from 0 to 9 mol%. A complex effect of the PEG-coverage was found with a maximum leakage in the range of the transition of the polymer from the mushroom to the brush conformation.
1,2-Dipalmitoylphosphatidylcholine↗
Rat liver and lung mitochondria do not incorporate radioactivity from glycerol-3-phosphate or CDP-choline into glycero-3-phosphocholine.
The proposed formation of glycero-3-phosphocholine (GPC) from glycerol-3-phosphate (GP) and CDP-choline catalysed by the enzyme GPC-synthetase has been examined in liver and lung subcellular fractions. Previous observations on the incorporation of radioactive GP into the GPC-spot on paper chromatograms have been interpreted as evidence for the GPC-dependent synthesis of phosphatidylcholine. Although we could reproduce this incorporation of GP, we could not detect any incorporation of radioactive CDP-choline into the GPC-spot using the same paper chromatographic system. TLC separation of the substrate and products showed no detectable formation of GPC with either radioactive substrate. These results strongly suggest that the previously reported formation of GPC in liver and lung was due to an inaccurate identification of the true radioactive products. We demonstrate that the major radioactive product formed in liver mitochondria is glucose. A small amount of radioactive glycerol was also detected. Lung mitochondria incorporate radioactive GP into glycerol and into another unidentified compound or compounds. It is concluded that the occurrence of the GPC dependent formation of phosphatidylcholine is unlikely.
Animals↗
Studies on insulin degradation inside the pancreatic B-cell: evidence for a regulating role of glucose.
The present study aimed at testing whether insulin degradation inside the pancreatic B-cell is regulated by insulin release or by glucose per se. Radioactively prelabelled isolated rat islets were cultured in unlabelled medium for 24 h. During that chase period the media contained (a) 50 or 300 mg glucose/dl alone or (b) together with 100 micrograms diazoxide/ml and (c) 50 mg glucose/dl without or with 10 mM L-leucine or 300 mg glucose/dl. The content of labelled (pro-)insulin in islets and media was measured and related to degradation. No degradation was found at the high glucose concentration, not even when insulin release was blocked by diazoxide. Degradation was also inhibited in the presence of leucine. The results suggest that glucose per se or its metabolism prevents insulin from being degraded inside the B-cell.
Animals↗
The effect of pH on incorporation of galactose by a normal human cell line and cell lines from patients with defective galactose metabolism.
Incorporation of radioactive galactose into TCA-insoluble material of galactosemic fibroblasts is more sensitive to low pH than is the incorporation by normal human fibroblasts. This study was undertaken to determine (1) whether there was any pH which could correct or counteract the galactosemic defect relative to galactose incorporation, and (2) whether the low pH effect was specific for galactose metabolism or whether general cellular metabolism in galactosemic cells was more sensitive to low pH than that in normal cells. The pH dependencies of incorporation of radioactive galactose and glucose into cellular macromolecules were investigated in galactosemic and normal cells. Normal cells have a biphasic curve with respect to galactose incorporation with peaks at pH 7.0 and 8.5. Galactosemic cells have only the high pH peak. The maximum incorporation by galactosemic cells was never more than about 30% that seen by normal cells under the conditions of these experiments. Thus manipulation of the pH alone cannot correct the galactosemic defect. The rate of incorporation of radioactive galactose was studied in normal, galactosemic and galactokinase deficient cells, at pH 7.2 and at pH 6.3. At pH 7.2, galactosemic cells incorporate galactose at a linear rate which is 30 to 40% that of normal cells while incorporation by kinase-deficient cells is between 5 and 10% of normal. At pH 6.3, the incorporation is also linear. However, galactosemic cells now exhibit the same rate as kinase-deficient cells in which the low level of incorporation is unaffected by pH. These results suggest that incorporation of galactose by galactosemic cells at low pH is not due to metabolic death of the cells, but may be due to the inhibition of some specific step or steps along a metabolic route of galactose metabolism other than the Leloir pathway.
Cell Line↗
The endo-1,4-beta-glucanase I from Trichoderma reesei. Action on beta-1,4-oligomers and polymers derived from D-glucose and D-xylose.
The reaction mechanism of the non-specific endo-1,4-beta-glucanase from Trichoderma reesei QM 9414 (endoglucanase I) was investigated using both reducing-end3H-labelled and universally 14C-labelled cellooligosaccharides, as well as reducing-end3H-labelled xylooligosaccharides. The bond cleavage frequencies of cellooligosaccharides proved to be dependent upon the substrate concentration, especially in the case of cellotriose. In addition to simple hydrolytic cleavage, the enzyme catalyzes reactions along alternative pathways, including transglycosylations leading to products larger than the substrate. Some of these pathways were shown to be reversible. During cellotriose or cellopentaose degradation, substrate resynthesis was demonstrated by incorporation of added radioactive D-glucose or cellobiose. The endoglucanase I is active on xylan and xylooligosaccharides, but less than on soluble cellulose derivatives (e.g. hydroxyethylcellulose) and cellooligosaccharides. The fact that for these different types of substrates the same active site is operative is proven by the ability of the enzyme to utilize cellooligosaccharides and xylooligosaccharides as both glycosyl donors and acceptors. The mixed substrate reactions lead to products composed of D-glucosyl and D-xylosyl residues. The kinetic parameters for cellooligosaccharide degradation can be used for the description of an extended substrate binding site. Of the four putative glycosyl subsites, -II and +II show the highest affinities, 16.7 kJ.mol-1 and 7.1 kJ.mol-1, respectively.
Cellulase↗
Invertase, maltase, lactase, and peroxidase activities in duodenum of BB rats.
The development of immune-mediated diabetes in BB rats may involve a defect of the gastrointestinal tract (GI), as suggested by increased gut permeability. This study aimed at measuring invertase, maltase, lactase, and peroxidase activities in the duodenum of diabetesprone BioBreeding (BBdp) rats and control BioBreeding rats (BBc) given free access to NIH-07 diet up to the time of killing at 60 66 d of age. After washing the entire small intestine, the duodenal mucosa was scraped off in the first 5-cm segment from the pylorus and frozen in distilled water. Invertase, maltase, and lactase activities were measured by monitoring the conversion of [U-(14)C]sucrose, [U-(14)C]maltose, and [D-[1-(14)C]glucose] lactose to radioactive hexoses, which were phosphorylated in the presence of adenosine triphosphatase and yeast hexokinase and then separated from their precursor by ion-exchange chromatography. Peroxidase activity was measured by a spectrophotometric procedure. In the BBdp rats, the activity of invertase, maltase, and lactase averaged, respectively, 70.2 +/- 4.4, 81.2 +/- 4.3, and 75.7 +/- 4.1% (n = 16 and p < 0.001 in all cases) of the control values found in BBc rats of the same sex. Inversely, after exclusion of two female BBc rats with abnormally high plasma D-glucose concentration, the activity of peroxidase in the BBdp rats averaged 157.4 +/- 20.0% (n = 16; p < 0.02) of the mean control value recorded in BBc rats of the same sex (100.0 +/- 9.3%; n = 14). These findings are compatible with the view that a proinflammatory state of the GI associated with compromise function may precede the occurrence of pancreatic insulitis in BBdp rats and, possibly, human subjects with type 1 diabetes.
Animals↗