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PubMed1975

14C-glucose and 35S-metabolism of pig epiphysial cartilage and its variations after osteotomy.

On in vitro incubation of articular and epiphysial cartilage of the ulna of the domestic pig 70 to 80 per cent of [U-14C] glucose was metabolised to 14C-lactate, but cartilage of the epiphysial plate produced up to five times as much 14C-CO2 as articular cartilage, and the specific radioactivity of 14C(or 35S)-chondroitin 4(6)-sulphate isolated from epiphysial cartilage (following 35S-sulphate incorporation) was about twice as high as that of articular cartilage. Six weeks after an osteotomy on both sides of the proximal epiphysial plate of the lift ulna, the glucose uptake, lactate production, and the specific radioactivity of the glycosaminoglycans displayed no significant differences when compared with those of the corresponding epiphysial plate of the control right ulna, whereas a 50 per cent increase in the oxidation of 14C-glucose to 14C-CO2 was observed.

Animals↗

PubMedDate not supplied

[Oxidation in vivo of glucose, palmitate, alanine and leucine in the pig during the neonatal period].

Determination of 14CO2 content in expired air after the intravenous injection of energetic substrates marked by the radioactive carbon to the pigs showed that the oxidative intensity of these substrates decreases in the series: [6-14C]glucose greater than [1-14C] alanine greater than [1-14C]leucine greater than [1-14C]glucose. The oxidation intensity of all substrates under study except for [1-14C]palmitate in the organism of one-day satisfied pigs is considerably higher, than during the first two hours after their birth. The starvation of pigs during the first 24 hours increases the oxidation of both investigated amino acid and [1-14C]-palmitate in tissues of their organism with a decrease in the metabolic intensity of [6-14C] and [1-14C]glucose.

Alanine↗

PubMed1998

Cell-surface biotinylation of GLUT4 using bis-mannose photolabels.

New cell-impermeant bis-mannose photolabels have been developed with biotinyl groups attached to 4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1, 3-bis(d-mannos-4-yloxy)-2-propylamine (ATB-BMPA) by either a polyethoxy spacer (Bio-ATB-BMPA) or an additional hexanoic acid spacer (Bio-LC-ATB-BMPA). The half-maximal inhibition constants, Ki values, for inhibition of glucose transport activity in insulin-stimulated rat adipocytes were determined to be 359+/-10 and 273+/-28 microM for Bio-ATB-BMPA and Bio-LC-ATB-BMPA, respectively. These values are similar to those previously reported for the non-biotinylated compound ATB-BMPA. Following UV-irradiation-induced cross-linking of the biotinylated photolabels to rat adipocytes, the biotinylated glucose transporter isoform 4 (GLUT4) could be detected by non-radioactive and radioactive methods that utilized the interaction with streptavidin. Biotinylated GLUT4 from 1-2 microg of adipose cell membranes, precipitated onto magnetic streptavidin beads, could be sensitively and quantitatively detected using an electrochemiluminescent assay method. This utilized a ruthenium-tagged anti-GLUT4 antibody that on excitation at an electrode generated an electrochemiluminescent signal in an ORIGEN analyser. Alternatively, surface-biotinylated GLUT4 could be easily, but less sensitively, detected in streptavidin agarose precipitates which were analysed by conventional GLUT4 Western blotting. Data obtained using the non-radioactive methods compared favourably with those using tritiated versions of the biotinylated probes. Insulin treatment of adipocytes increased the levels of signals from surface biotinylated GLUT4 by approximately 10-fold or approximately 20-fold, respectively, when the electrochemiluminescent or the Western blot detection methods were used and these signals were blocked by cytochalasin B.

Adipocytes↗

PubMed1982

Degradation patterns and intermediates in the anaerobic digestion of glucose: experiments with 14C-labeled substrates.

A mineral salts medium containing 1% (w/v) glucose was subjected to anaerobic digestion in an upflow reactor. Performance with respect to utilization of glucose was monitored by collection of fermentation gases and calculation of carbon mass balances. Sub-samples of bacterial suspensions from the upflow reactor were incubated with (U-14C)-glucose, (U-14C)-acetate, (2-14C)-propionate, (1-14C)-butyrate or 14C-carbonate. Individual radioactive products in samples from incubation mixtures were analysed by radio gas chromatography. Quantitatively, acetate and propionate were the only important intermediates in glucose degradation by glucose-adapted sludge, with acetate accounting for the largest part of intermediary fatty acid flux.

Acetates↗

PubMed1982

Rates of flux through the pentose cycle in perfused rat liver. A procedure for the calculation of rates of substrate flux from 14CO2 production from [1-14C]glucose.

A method for the determination of substrate flux through the pentose cycle was developed employing [1-14C]glucose in experiments with perfused rat livers. The method consists first of a kinetic analysis which differentiates between the production of 14CO2 from [1-14C]glucose via the pentose cycle and via the citrate cycle and, second of a calculation of the specific radioactivity of the hexose monophosphate pool from measured rates of glycolysis and the specific radioactivity of lactate released into the perfusate. The method was validated by experiments comparing the results of tracer infusions with [1-14C]glucose, [6-14C]glucose and [3-14C]pyruvate. In livers from fed rats perfused with 10 mM glucose, the rate of substrate flux through the pentose cycle was around 0.2 mumol X min-1 X g-1; it was about 20% of the substrate flux via glycolysis. The kinetic data were inconsistent with the existence of an L-type pentose cycle in liver.

Animals↗

PubMed1990

A simple method for in-vitro studies of cancer tissue metabolism.

A simple laboratory method of investigating the metabolism of malignant tissues has been devised for liver cancer. The apparatus consists of a standard glass scintillation vial with an inner glass well suspended within it from a rubber lined cap. 14C-labelled glucose metabolised by the tumour tissue in the inner well into 14CO2 will be trapped by alkali in the outer vial, and the radioactivity quantified by liquid scintillation counting. The counts obtained will give a measure of the rate of tissue glucose oxidation. Application of this method to an animal liver tumour model has shown that the tumour tissue has a glucose oxidation rate of 2.8 times that of the host liver. The method is simple and versatile, can be adapted to various types of tissues, and may have potential clinical applications.

Animals↗

PubMed1994

Substitution of tyrosine 293 of GLUT1 locks the transporter into an outward facing conformation.

Tyrosines 292 and 293 in the mammalian glucose transporter GLUT1 have been substituted by either isoleucine or phenylalanine. Chinese hamster ovary clones that were transfected with Tyr-292-->Ile, Tyr-292-->Phe, Tyr-293-->Ile, and Tyr-293-->Phe constructs of GLUT1 were shown, by Western blotting and cell surface carbohydrate labeling, to have expression levels that were comparable with the wild type. The Vmax for 2-deoxy-D-glucose transport was markedly reduced only as a result of the Tyr-293-->Ile mutation. The ability of the Tyr-293-->Ile mutated GLUT1 to bind the exofacial ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) and the endofacial ligand cytochalasin B were assessed by photolabeling procedures. The ability to bind the bis-mannose compound was unimpaired, whereas the ability to bind cytochalasin B was totally abolished, and the level of labeling was lower than in the nontransfected clone. Affinities of the wild-type and Tyr-293-->Ile GLUT1 for D-glucose, the exofacial ligands (ATB-BMPA and 4,6-O-ethylidene-D-glucose), and the endofacial ligand (cytochalasin B) were assessed by the ability of these agents to displace the radioactive ATB-BMPA photolabel. These data indicated that the Tyr-293-->Ile substitution produced no change in the affinity for D-glucose, a relatively small enhancement in the affinity for exofacial ligands, but a large approximately 300-fold reduction in affinity for cytochalasin B, suggesting that the mutated GLUT1 is locked in an outward facing conformation. The observation that the Tyr-293-->Ile mutant transporter can bind nontransported C4 and C6 substituted hexose analogues but cannot catalyze transport is interpreted as indicating that Tyr-293 is involved in closing the exofacial site around C4 and C6 of D-glucose in the transport catalysis process.

Affinity Labels↗

PubMed1982

Changes in energy metabolism during the early development of Xenopus laevis.

Xenopus embryos at various development stages were incubated in the presence of labelled substrates and the 14CO2 production determined. From the rates of oxidation of glucose labelled in positions 1 and 6 and from that of radioactive acetate, pyruvate and glutamate, it was concluded that the Embden-Meyerhof pathway and the Krebs cycle are functional during early embryogenesis, but that their relative participation in the metabolic processes is limited and increases from gastrulation onwards. Early development is characterized by the predominance of the pentose cycle and the glutamate-aspartate cycle. Furthermore, it was shown that glutamate may be the main energy source up to gastrulation.

Acetates↗

PubMed1982

Nonenzymatic glycosylation of normal and abnormal haemoglobin alpha chains.

The total amount of haemoglobin-bound glucose is determined by the chemical method for quantification of glycosylated haemoglobin, used in the control of diabetic patients. About 40 per cent of the measured amount of glucose is bound by the alpha chains of haemoglobin. The effect of substitution of alpha chains on the glycosylation of the abnormal and normal chains was investigated in a J Buda and G Pest heterozygote. Intact red blood cells were incubated in the presence of labelled glucose of 0.20 MBq/mumol specific activity and 35 mM concentration, and nonenzymatic glucose incorporation into normal and abnormal alpha chains was followed. By the help of fingerprinting and autoradiography of separated chains the distribution of the incorporated radioactivity was investigated. Based on the results we may conclude that (i) 65 per cent of incorporated glucose is bound to five peptides; (ii) the abnormal chain does not affect the glycosylation of the normal alpha chain; (iii) the substitution of a potent glucose binding site (alpha 61) does not affect the glycosylation of the other residues in the abnormal chain; (iv) the presence of the abnormal chain does not interfere with the diagnostic use of the chemical method for quantification of glycosylated haemoglobin.

Autoradiography↗

PubMed1985

The effects of glucose and an aldose reductase inhibitor on the sorbitol content and collagen synthesis of bovine retinal capillary pericytes in culture.

The absolute rate of collagen synthesis by cultured bovine retinal capillary pericytes, determined using the specific radioactivity of proline in the cellular amino acid pool, was compared in media containing different concentrations of glucose (5, 10 or 40 mM) and Sorbinil (0.0 or 0.1 mM), an inhibitor of aldose reductase. The absolute rate of collagen synthesis, in proline molar terms, by pericytes in medium with 5 mM glucose was 3.3 +/- 1.9 (S.D.) pmol 10(-7) cells 24 hr-1, and increased significantly to 8.8 +/- 3.7 pmol 10(-7) cells 24 hr-1 when the glucose concentration was increased to 40 mM. Sorbinil (0.1 mM) reduced the elevated sorbitol contents of pericytes induced by high concentrations of glucose, but did not significantly change the absolute rate of collagen synthesis per cell.

Aldehyde Reductase↗

PubMed1975

In vitro incorporation of (3H)threonine and (3H)glucose by the mucous and serous cells of the human bronchial submucosal gland. A quantitative electron microscope study.

Incorporation of [3H]threonine and [3H]glucose by the mucous and serous cells of the human bronchial submucosal gland has been studied over 8 h using, for the first time in vitro pulse labeling and electron microscope autoradiography. In assessing the autoradiographs, two methods were compared, the circle analysis and the recently described hypothetical grain analysis. Preliminary studies showed formaldehyde to be the most suitable fixative. Chemical analysis of tissue revealed that [3H]threonine was incorporated into the polypeptide moiety of the bronchial gland product and that metabolites of [3H]-glucose were incorporated into the carbohydrate. Tritiated threonine was first localized in the endoplasmic reticulum of both mucous and serous cells and later migrated to the Golgi apparatus, while metabolites of [3H]glucose localized first mainly in the Golgi apparatus. From here, both radioactive precursors were next identified in vacuoles and, finally, in secretory granules. The mucous cell incorporated strikingly more of both radioactive precursors than the serous cell. Thus, it seems that oligosaccharides of mucous and serous cell glycoproteins are synthesized mainly in the Golgi apparatus and added there to the polypeptide core which is synthesized in the endoplasmic reticulum. The relationship of the mucous cell to the serous cell is discussed. It seems that under "normal" conditions each cell represents a different line but that injury may transform a serous cell into a mucous cell.

Autoradiography↗

PubMed1979

Carbohydrate transport in Moniliformis dubius (Acanthocephala). III. Post-absorptive fate of fructose, mannose, and galactose.

The initial metabolism of fructose, mannose, and galactose in Moniliformis dubius (M. moniliformis; Acanthocephala) was examined following brief aerobic incubations in 14C-labeled substrate. The pattern of incorporation of radioactivity from 14C-fructose and 14C-mannose into intermediates of hexose metabolism was indistinguishable from that reported elsewhere for the initial post-absorptive metabolism of 14C-glucose under comparable conditions; these hexoses were phosphorylated rapidly following their absorption, and much of the radioactivity absorbed as mannose or fructose was recovered in the nonreducing disaccharide trehalose [alpha-D-glucopyranosyl-(1 leads to 1)-alpha-D-glucopyranoside]. 14C-Galactose was phosphorylated less readily than the other hexoses. More than half of the radioactivity absorbed as galactose was incorporated into disaccharide; some of the galactose-derived disaccharide had properties suggesting the presence of a galactosyl moiety. Incorporation of radioactivity from any of the hexoses into glycogen was minimal. The extensive incorporation of hexose moieties into trehalose or a trehalose-like disaccharide rather than glycogen underscores the probable importance of trehalose metabolism to carbohydrate assimilation in Moniliformis. Physiological factors which might favor trehalose biosynthesis over glycogenesis are considered.

Absorption↗

PubMed1998

Characterization of the O-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1).

P-selectin glycoprotein ligand-1, PSGL-1, a specific ligand for P-, E-, and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin and platelet P-selectin- or E-selectin receptor globulin-agarose chromatography. The O-linked oligosaccharides on the ligand were released by mild alkaline sodium borohydride treatment and analyzed by a combination of ion-exchange, size exclusion, lectin, and paper chromatography, together with specific exoglycosidase treatments and chemical modifications. Approximately 91% of the radioactivity released from PSGL-1 was recovered in five O-linked glycans: GalNAc (approximately 4% of the total structures), Galp, 3GalNAc (36%), and Galbeta, 3GalNAc substituted with one (45%), two (6%), or three (3%) N-acetyllactosamine repeat units. None of these structures contained fucose, and the majority were substituted with at least one sialic acid. The N-acetyllactosmine-containing structures appeared to be core 2. The remaining 9% of the radioactivity recovered in O-linked oligosaccharides from PSGL-1, eluted in two peaks at 11.8 and 10.2 glucose units, on size-exclusion chromatography. Results from lectin chromatography and chemical and enzymatic degradation experiments suggest that the major portion of the radioactivity in these peaks is associated with sialylated N-acetyllactosamine-type oligosaccharides, substituted with fucose at the penultimate residue in the nonreducing end. Since both sialic acid and fucose reportedly are crucial requirements for selectin binding, these results suggest that only a minor portion, approximately 4.5%, of the O-linked oligosaccharides on PSGL-1 are involved in the interaction with the selectins.

Carbohydrate Conformation↗

PubMed2000

Fate of 2-deoxy-2-[18F]fluoro-D-glucose in hyperglycemic rats.

Sustained hyperglycemia allows the preferential labelling of pancreatic glycogen by D-[U-14C]glucose in control rats, as compared to animals previously injected with streptozotocin (STZ rats). The major aim of the present study was to investigate whether a sizeable difference between control and STZ rats could also be observed in terms of the radioactive content of the pancreatic gland 8 h after the intravenous injection of 2-deoxy-2-[18F]fluoro-D-glucose, both types of animals being examined at the same level of hyperglycemia. Although the radioactive content of muscle, liver and kidney was lower in STZ rats than in control animals, such a difference failed to achieve statistical significance in brain, hypophysis, pancreas and parotid gland. It is proposed, therefore, that 11C-labelled D-glucose, rather than 2-deoxy-2-[18F]fluoro-D-glucose should be used in the perspective of the non-invasive imaging of the endocrine pancreas.

Animals↗

PubMed1983

Ischemia-induced noradrenaline release in the isolated rat heart: influence of perfusion substrate and duration of ischemia.

To examine some of the characteristics of the local noradrenaline (NA) release in myocardial ischemia a study was made on Langendorff-perfused rat hearts, prelabelled with 3H-NA. The left coronary artery was ligated for 15, 30, 60 or 120 min, followed by 10 min of reperfusion. The coronary effluent was collected and analyzed for radioactivity to indicate release of 3H-NA. In some of the experiments the fraction of 3H-NA was determined. As substrate in the perfusion medium either glucose (11.1 mM) or sodium lactate (5.0 mM) was used. During the ischemic period there was a slight decrease in the outflow of radioactivity. However, reperfusion was associated with a rapid and marked outflow of radioactivity, including an increased fraction of 3H-NA, in the effluent. Compared to glucose as perfusion substrate, lactate caused a significantly higher (P less than 0.01) outflow of tritiated substances after 60 and 120 min of regional ischemia. With lactate there was an almost linear relationship between reperfusion efflux of 3H and duration of ischemia. With glucose, the reperfusion outflow increased less rapidly after a duration of ischemia longer than 15 min. It is concluded that the degree of local NA release in myocardial ischemia depends on both the duration of the ischemic period and the substrate used. Glucose attenuates the reperfusion outflow of NA, especially after longer periods of ischemia. The effect may be due to decreased myocardial cell damage with this substrate and/or a direct protection of the adrenergic nerve endings.

Animals↗

PubMed1991

Influence of raffinose on the relative synthesis rate of K88 fimbriae and the adhesive capacity of Escherichia coli K88.

The synthesis rate of K88 fimbriae relative to the rate of total protein synthesis was estimated during growth of a wild-type Escherichia coli Bd 1107/7508 (K88ac) in M9 minimal medium supplemented with glucose or raffinose as the carbon source. Rates of synthesis of fimbriae and total protein were analysed by immunoprecipitation and TCA precipitation of radioactive pulse labelled K88 fimbriae and E. coli K88 cells, respectively. In addition, the effect of raffinose on the in vitro adhesion of E. coli K88 cells to immobilized piglet ileal mucus was studied. The relative rate of K88 fimbriae synthesis increased gradually during log phase, reached maximum at late log phase, and thereafter decreased continuously. The use of raffinose as the carbon source, did not alter the relative rate of K88 fimbrial synthesis or the adhesive capacity of E. coli K88 cells, as compared to glucose.

Animals↗

PubMed1978

Pathway of succinate and propionate formation in Bacteroides fragilis.

Cell suspensions of Bacteroides fragilis were allowed to ferment glucose and lactate labeled with (14)C in different positions. The fermentation products, propionate and acetate, were isolated, and the distribution of radioactivity was determined. An analysis of key enzymes of possible pathways was also made. The results of the labeling experiments showed that: (i) B. fragilis ferments glucose via the Embden-Meyerhof pathway; and (ii) there was a randomization of carbons 1, 2, and 6 of glucose during conversion to propionate, which is in accordance with propionate formation via fumarate and succinate. The enzymes 6-phosphofrucktokinase (pyrophosphate-dependent), fructose-1,6-diphosphate aldolase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, and methylmalonyl-coenzyme A mutase could be demonstrated in cell extracts. Their presence supported the labeling results and suggested that propionate is formed from succinate via succinyl-, methylmalonyl-, and propionyl-coenzyme A. From the results it also is clear that CO(2) is necessary for growth because it is needed for the formation of C4 acids. There was also a randomization of carbons 1, 2, and 6 of glucose during conversion to acetate, which indicated that pyruvate kinase played a minor role in pyruvate formation from phosphoenolpyruvate. Phosphoenolpyruvate carboxykinase, oxaloacetate decarboxylase, and malic enzyme (nicotinamide adenine dinucleotide phosphate-dependent) were present in cell extracts of B. fragilis, and the results of the labeling experiments agreed with pyruvate synthesis via oxaloacetate and malate if these acids are in equilibrium with fumarate. The conversion of [2-(14)C]- and [3-(14)C]lactate to acetate was not associated with a randomization of radioactivity.

Acetates↗

PubMed1982

Regulation of glutamate biosynthesis and release in vitro by low levels of ammonium ions.

The content and release of endogenous amino acids from isolated rat hippocampal slices were measured. The tissue was perfused with control media and pulsed with high potassium media in order to induce synaptic release. Pathophysiological concentrations of ammonium ions (3--5 mM) were added to the control medium for 60 min prior to the induced release. Amino acids belonging to the putative transmitter group were released extensively during potassium perfusion and, except for glutamate, even after ammonium ion perfusion. The spontaneous secretion of glutamate increased, however, slowly after the addition of ammonia. The incorporation of 14C from radiolabelled glucose and acetate into the amino acid fraction was studied in the presence of ammonia-containing media. Glucose was utilized to a moderately increasing extent, but acetate-derived radioactivity was strikingly decreased in the amino acid fraction during ammonia perfusion. The decreased acetate incorporation into amino acids was mainly due to an inhibition by ammonia of the accumulation of acetate by the CNS tissue.

Acetates↗