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PubMed1999

A method for the determination of glucose synthesis in isolated bovine hepatocytes.

A simple method for determining glucose synthesis from radiolabeled precursors in isolated bovine hepatocytes using ion exchange resins is presented. This method allows processing of multiple small volume samples using suspensions of anion and cation exchange resins rather than traditional stacked column separation methods. Hepatocytes were isolated from calf liver by collagenase perfusion of the caudate lobe and were incubated with (14)C-labeled lactate or propionate as gluconeogenic substrates. Glucose synthesis was determined in an aliquot of cell suspension that was vortexed with a slurry of anion exchange (acetate form) resin, followed by a slurry of cation exchange resin. Newly synthesized, labeled glucose was recovered in the supernatant after centrifugation and quantitated by scintillation counting. Using this procedure, more than 98% of the unused labeled precursor was bound to the ion exchange resin and essentially 100% of a labeled glucose tracer was recovered in the supernatant. Pretreatment of hepatocyte suspensions with glucose oxidase was shown to eliminate the accumulation of radioactivity in the supernatant, thus confirming the specificity of this technique for measurement of newly synthesized glucose. This method was sensitive to changes in the rate of hepatic gluconeogenesis that resulted from changes in substrate concentration or the addition of glucagon or fatty acids to the hepatocyte incubations.

Journal Article↗

PubMed1997

Cationic and secretory effects of 6-O-acetyl-D-glucose in rat pancreatic islets.

Selected esters of D-glucose were recently proposed as tools to supply the sugar to cells affected by a defect in the carrier-mediated process of hexose transport across the plasma membrane. The present study extends this knowledge to 6-O-acetyl-D-glucose. At a 2.0 mM concentration, the ester was indeed found to stimulate insulin release from perifused rat pancreatic islets. This coincided with stimulation of calcium influx into the islet cells, as judged from comparison of the ester effects on 45Ca outflow from prelabelled islets perifused in either the absence or presence of extracellular Ca2+. The facilitated inflow of Ca2+ did not appear attributable to a decrease in K+ conductance, 6-O-acetyl-D-glucose augmenting 86Rb efflux from islets prelabelled with this radioactive cation. Both the latter phenomenon and the insulinotropic action of 6-O-acetyl-D-glucose failed to be antagonized by D-mannoheptulose. These findings indicate that the cationic events coupling the recognition of D-glucose to the stimulation of insulin release are not identical in islets exposed to the hexose or its ester.

Animals↗

PubMed2004

Reactive oxygen species and nitric oxide affect cell wall metabolism in tobacco BY-2 cells.

The effects of hydrogen peroxide (H2O2), nitric oxide (NO), and a combination of both on the metabolism of cell wall polysaccharides were studied in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY-2) suspension cultured cells in the presence of D-[U-14C]glucose or D-[U-14C]galactose as radioactive tracers. We found that the radiolabelling of newly synthesised total cell wall polysaccharides (pectins, hemicelluloses and alpha-cellulose), buffer-soluble polysaccharides, and membrane-associated polysaccharides decreased under the influence of exogenous systems generating H2O2 and NO. However, when the total amount of newly synthesised cell wall polysaccharides was calculated as a percentage of the total cellular radioactivity (ethanol-soluble pool plus the homogenate of ethanol-insoluble material), all treatments showed negligible effects in the presence of D-[U-14C]glucose or D-[U-14C]galactose as tracers. This occurred because the treatments generating H2O2, NO and H2O2 plus NO caused a marked decrease in the concentration of the ethanol-soluble pool as well as in the total radioactivity found in the homogenate of the ethanol-insoluble material. Most of the radioactivity taken up by the cells was evolved as 14CO2 during the respiratory processes. A qualitative and quantitative characterisation of the ethanol-soluble pool showed that radioactive UDP-sugars in BY-2 suspension cultured cells were differentially reduced by all treatments. Therefore, the decrease of the newly synthesised cell wall polysaccharides seems to be strictly dependent on the reduction of the UDP-sugars pool.

Cell Wall↗

PubMedDate not supplied

Simultaneous, quantitative measurement of local blood flow and glucose utilization in tissue samples in normal and injured feline brain.

Cerebral blood flow (CBF) and local cerebral glucose utilization (LCGU) were measured using radioactive microspheres and [14C]2-deoxyglucose, respectively, in 26 brain regions in control animals (n = 8) and in animals (n = 4) sustaining low-level experimental brain injury. Examination of the initial (resting) CBF measurement in the uninjured cats revealed two subgroups with significantly (p less than 0.01) different CBF levels. In uninjured cats with normal CBF levels (33.4 +/- 1.8 ml/100 g/min) there was a close linear relationship between CBF and LCGU (n = 0.71, p less than 0.01). In contrast, the remainder of the uninjured cats exhibited abnormally high levels of CBF (72.6 +/- 9.9 ml/100 g/min) and the absence of a close relationship between CBF and LCGU (r = 0.27). One hour following low-level (2.0 atm) fluid percussion brain injury, CBF was increased and LCGU was decreased, though not significantly. The relationship between CBF and LCGU remained intact (r = 0.66, p less than 0.01) in most brain regions. However, the relationship between CBF and LCGU in the hippocampus differed significantly from the relationship between the two parameters in the rest of the brain. Thus, the use of the radioactive microsphere method for CBF measurements allows multiple measurements of CBF and permits the assessment of the status of the cerebral vasculature prior to experimental manipulations such as traumatic brain injury. In view of our current findings of an abnormal relationship between CBF and LCGU in cats with high resting CBF levels, this is an important advantage. In addition, the combination of the microsphere and 2-DG techniques within the same tissue samples allows for the investigation of the effects of traumatic injury on the important relationship between CBF and LCGU.

Animals↗

PubMed1970

Synthesis of cell constituents by methane-grown Methylococcus capsulatus and Methanomonas methanooxidans.

1. A study was made of the incorporation of carbon from [(14)C]methanol by cultures of Methylococcus capsulatus and Methanomonas methanooxidans growing on methane. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled substrate for periods of up to 3min, was analysed by chromatography and radioautography. 3. Over 80% of the radioactivity fixed by Methylococcus capsulatus at 30 degrees C at the earliest times of sampling appeared in phosphorylated compounds, of which glucose phosphate constituted 60%. 4. Most of the radioactivity fixed by Methanomonas methanooxidans at 30 degrees C at the earliest times of sampling appeared in serine, malate, aspartate and an unknown compound(s) tentatively suggested to be folate derivative(s). At 16 degrees C, [(14)C]methanol was fixed predominantly into serine and the unknown compound(s). 5. Extracts of Methylococcus capsulatus contain an enzyme system that catalyses the condensation of formaldehyde and ribose 5-phosphate to give a mixture consisting mainly of fructose phosphate and allulose phosphate. No similar activity was detected in extracts of Methanomonas methanooxidans. A convenient method was developed for assay of this enzyme system. 6. The enzyme system catalysing the condensation of formaldehyde with ribose 5-phosphate is particle-bound in both Methylococcus capsulatus and Pseudomonas methanica and is unstable in the absence of Mg(2+). 7. Extracts of Methanomonas methanooxidans contain high activities of d-glycerate-NAD oxidoreductase, whereas extracts of Methylococcus capsulatus and Pseudomonas methanica contain negligible activities of this enzyme. 8. These results indicate that during growth of Methylococcus capsulatus on methane, as with Pseudomonas methanica, cell constituents are made by the ribose phosphate cycle of formaldehyde fixation. This contrasts with Methanomonas methanooxidans, whose assimilation pathway resembles in some features that of Pseudomonas AM1 growing on methanol.

Aspartic Acid↗

PubMed1985

Stimulation by glucose and carbamylcholine of phospholipase A2 in pancreatic islets.

Glucose, in high concentrations (16.7-27.8 mM), caused a modest increase in effluent radioactivity from rat pancreatic islets prelabelled with [U-14C] arachidonate. The response to glucose was abolished in the absence of extracellular Ca2+. At a low glucose concentration (5.6 mM), carbamylcholine (1.0 mM) provoked a more marked and sustained increase in effluent radioactivity. The response to the cholinergic agent was abolished in the presence of atropine or absence of extracellular Ca2+. These results suggest that carbamylcholine and, to a lesser extent, glucose cause a Ca2+-dependent activation of phospholipase A2 in intact islet cells.

Animals↗

PubMed1987

Relationship between albumin and fructosamine concentration in diabetic and non-diabetic sera.

We investigated the contribution of albumin to serum fructosamine activity. On gel exclusion chromatography, pooled diabetic and pooled non-diabetic sera before and after incubation with [14C]glucose differed in fructosamine activity and radioactivity mainly in the albumin-containing fractions. We confirmed the major contribution of glycated albumin to serum fructosamine concentration using affinity chromatography on Blue Sepharose. The importance of high molecular mass species was further demonstrated by the recovery of reducing activity following dialysis and charcoal treatment. Fructosamine measurements in diabetic patients were not correlated with serum albumin concentration in individuals with urinary albumin less than 1 g/l. We conclude that fructosamine activity is a convenient measure of glycosylprotein concentration and that the index responds mainly to glycation of albumin. However, routine correction for serum albumin concentration is inappropriate.

Adolescent↗

PubMed1998

Can experimental conditions explain the discrepancy over glutamate stimulation of aerobic glycolysis?

Uncertainty reigns over whether or not glutamate uptake in astrocytes leads to strong stimulation of glucose utilization, measured as accumulation of radioactive deoxyglucose-6-phosphate. This is an important issue, not only because glutamate is the major excitatory transmitter, but also because it has been postulated that glutamate-induced stimulation of glycolysis links brain excitation with activation of energy production. The effect of glutamate on deoxyglucose utilization in cultured rat and mouse astrocytes grown in different media and incubated under various conditions during the deoxyglucose assay has, therefore, been studied. Under most conditions, no stimulation occurred but rather a decrease in deoxyglucose utilization during exposure to glutamate; under certain conditions, the contribution of non-metabolized deoxyglucose to the intracellular 14C signal was significant.

Aerobiosis↗

PubMed1983

Asymmetric distribution of decanoate in triacylglycerol synthesized in vitro by mammary glands of lactating mice.

Slices, prepared from the mammary glands of lactating mice, were incubated with either [1-14C]acetate, [U-14C]glucose, or [1-14C]decanoate. From all 3 substrates, radioactivity in the synthesized lipids was found mainly in triacylglycerols (TG). When acetate or glucose served as substrate, decanoate (C10) accounted for 24% of the fatty acids in TG. Hydrolysis of the TG by pancreatic lipase yielded [14C] fatty acids which had relatively more C10 (38%) than did either of the other hydrolysis products mono- or diacylglycerol (14-17%). However, when TG produced by slices from C10 were hydrolyzed, the acid was found to be esterified equally at the C-1, C-2 and C-3 of glycerol. Thus, when fatty acids are synthesized de novo and are converted to TG by gland slices, C10 is predominantly located in the C-3 position, a finding in accord with the situation in milk TG, although such preferential incorporation does not occur when the free acid is presented to the tissue slices.

Animals↗

PubMed1985

Activation of yeast plasma membrane ATPase by phorbol ester.

Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to yeast cells produces a 2-fold activation of the plasma membrane ATPase. The activation is reversible and time-and dose-dependent. The activated enzyme shows an increased affinity for its substrate, ATP, and its optimum pH is shifted to a more alkaline range. These changes are similar to those observed in the reported activation by glucose. Upon incubation of yeast cells with 32Pi incorporation of radioactivity in a membrane polypeptide of 105 kDa is observed after addition of either glucose or TPA.

Adenosine Triphosphatases↗

PubMed1992

Inhibition of intestinal glucose absorption in rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Male Sprague-Dawley rats were injected with either 125 micrograms 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg or vehicle (pair-fed and ad libitum-fed controls). Transfer of water, electrolytes and D-glucose as well as fats of a tracer dose of the non-metabolizable radioactive marker 3-O-methyl-D-[U-14C]glucose was studied in isolated perfused jejunal segments 1, 2, 7, and 21 d after treatment (TCDD-treated and pair-fed control rats) and after 26 d in ad libitum-fed controls. TCDD-treated rats demonstrated reduced feed consumption and loss of body weight. Active intestinal absorption of glucose was significantly inhibited 30 and 22% compared to pair-fed controls, respectively 2 and 7 d after TCDD treatment. After 21 d the inhibition (14%) was less significant. There were no differences in glucose transfer between severely starved pair-fed controls (body weights 370 +/- 26 g) and ad libitum-fed rats (body weights 512 +/- 15 g). Water absorption and transfer of sodium and calcium was not influenced by TCDD treatment. However, a significant increase of potassium transfer was observed in parallel with impaired glucose absorption. The uptake of 3-O-methylglucose into mucosal tissue was not impaired, whereas the transfer to the serosal side was significantly inhibited by 30-60% compared to pair-fed as well as ad libitum-fed animals from day 2 until the end of the experiment. These results suggest that TCDD is involved in an inhibition of glucose transport at the basolateral membrane.

Animals↗

PubMed1981

Subcellular compartmentation of glycolytic intermediates in Trypanosoma brucei.

In cell-fractionation experiments most of the glycolytic enzymes in bloodstream forms of Trypanosoma brucei are recovered in a microbody, called the glycosome [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360-364]. To see whether this compartmentation of glycolytic enzymes is accompanied by compartmentation of metabolites we have pulse-labelled intact T. brucei with [U-14C]glucose and followed the incorporation of radioactivity into glycolytic intermediates separated by anion-exchange chromatography. The kinetics of incorporation provide direct evidence for the existence of two pools of glycolytic intermediates. One pool is completely labelled within 15 s and represents 20-30% of total cellular metabolites. Radioactively labelled pyruvate is already produced after 15 s. Since this pool is directly involved in the glycolytic flux, we conclude that it is present in the glycosome. The second pool which represents 70-80% of the total appears not to be directly involved in glycolysis. Its content equilibrates relatively slowly with the glycosomal pool. It probably represents the cytosol. Incorporation of radioactivity into the total glycerol 3-phosphate pool is more rapid than for the other metabolites studied. This indicates rapid mixing of the glycosomal and cell-sap pools of glycerol 3-phosphate, as required for the extra-glycosomal oxidation of glycerol 3-phosphate by the mitochondrial oxidase. In the presence of 1 mM salicylhydroxamic acid, which mimics anaerobiosis, the trypanosome produces equimolar amounts of pyruvate and glycerol. Labelling of glycerol and glycerol 3-phosphate proceeds at identical rates but at all times the specific activity of glycerol is less than that of glycerol 3-phosphate. This is compatible with our earlier proposal that glycerol is made from glycerol 3-phosphate by glycerol kinase. We conclude that glycolysis in trypanosomes takes place in the glycosome and that the membrane of this organelle is poorly permeable to most glycolytic intermediates.

Animals↗

PubMed2000

Living on a high sugar diet: the fate of sucrose ingested by a phloem-feeding insect, the pea aphid Acyrthosiphon pisum.

The natural diet of aphids, plant phloem sap, generally contains high concentrations of sucrose. When pea aphids (Acyrthosiphon pisum) were fed on chemically defined diets containing sucrose radiolabelled in the glucose or fructose moiety, 2 to 12-fold and 87 to 110-fold more radioactivity was recovered from the tissues and honeydew, respectively, of aphids that ingested [U-(14)C-glucose]-sucrose than from those ingesting [U-(14)C-fructose]-sucrose. The total radioactivity recovered was 70% of the ingested [U-(14)C-glucose]-sucrose and <5% of ingested [U-(14)C-fructose]-sucrose. The dominant honeydew sugars produced by aphids feeding on 0.75 M sucrose diets were oligosaccharides comprising glucose. In vitro the guts of pea aphids had high sucrase activity, 1-5 U mg(-1) protein, generating equimolar glucose and fructose except at high sucrose concentrations where glucose production was inhibited (K(si)=0.1 M). These data suggest that the fructose moiety of ingested sucrose is assimilated very efficiently and may be preferentially respired by the aphid, and that the glucose moiety of sucrose is incorporated into oligosaccharides by the transglucosidase activity of the gut sucrase at high sucrose concentrations. These differences in the fate of sucrose-derived glucose and fructose are important elements in both the carbon nutrition and osmoregulation of aphids.

Journal Article↗

PubMed1975

Site of initial glycosylation of mannoproteins from Saccharomyces cerevisiae.

The cellular site of initial glycosylation of proteins from Saccharomyces cerevisiae has been studied. Short pulses of [U-14C]mannose label the ribosomal fraction of the yeast. Most of the label was associated with polysomes; monosomes contained only a small amount of radioactivity. All of the radioactivity present in the polysomal fraction was accounted by mannose and smaller amounts of glucose and glucosamine. Puromycin treatment detached more than 50% of the radioactivity from the polysomes; treatment of polysomes at pH 10.0 also caused the release of radioactivity. These results indicate that initial sugar binding occurs while the nascent polypeptide chains are still growing on the ribosomes. When the cells were preincubated with 2-deoxy-D-glucose, incorporation of [U-14C]mannose into the polysomes and the cell wall was inhibited, whereas its incorporation into membrane fractions was unimpaired. It was concluded that 2-deoxy-D-glucose inhibited the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level.

Carbon Radioisotopes↗

PubMed1992

Mechanisms regulating cardiac fuel selection in hyperthyroidism.

Starvation (48 h) decreases fructose 2,6-bisphosphate (Fru-2,6-P2) concentrations and the ratio of free to acylated carnitine in hearts of euthyroid rats. These decreases, which are indicative of increased lipid fuel oxidation, are accompanied by decreased rates of glucose uptake and phosphorylation, assessed by using radioactive 2-deoxyglucose. Cardiac concentrations of acylated carnitines were increased at the expense of free carnitine even in the fed state in response to experimental hyperthyroidism, but neither Fru-2,6-P2 concentrations nor rates of glucose utilization were suppressed. Starvation (48 h) did not further increase the proportion of acylated carnitine in the heart in hyperthyroidism, and suppression of Fru-2,6-P2 concentrations and glucose utilization rates by starvation was attenuated. Although glucose utilization rates were decreased, starvation did not decrease immunoreactive GLUT 4 protein concentrations. Furthermore, although hyperthyroidism was associated with a statistically significant (30-40%) increase in relative abundance of GLUT 4 mRNA, the amount of GLUT 4 protein was not increased by hyperthyroidism in either the fed or the starved state. The results demonstrate a significant effect of hyperthyroidism to enhance cardiac glucose utilization in starvation by a mechanism which does not involve changes in GLUT 4 expression but may be secondary to changes in glucose-lipid interactions at the tissue level.

Animals↗

PubMed1972

The turnover of protein in discrete areas of rat brain.

1. Rats were injected serially with [(14)C]glucose to obtain a constant specific radioactivity of brain amino acids. Measurements with this system for periods of up to 8h gave an apparent mean half-life for protein in whole brain of 85h (indicating the presence of a protein fraction with much more rapid turnover than this). 2. The half-lives of proteins in the granule-cell, molecular and white-matter layers of cerebellum were also determined. These had values of 33, 59 and 136h respectively. In addition, the incorporation into protein in six layers of the cerebral cortex, subjacent white matter and five layers of Ammon's horn was studied. All cell-body layers incorporated amino acids at about the same rate irrespective of location, and these rates were considerably higher than those for incorporation into proteins in areas rich in dendrites or fibre tracts. 3. A new method for measuring small amounts of glutamate with a cyclic enzyme system is presented.

Amino Acids↗

PubMed1975

Purification of cerebral glucose-6-phosphatase. An enzyme involved in sleep.

An insoluble phosphoprotein of rat brain acquires radioactivity from inorganic phosphate more rapidly during sleep than during wakefulness. It was purified in two ways. The first was solvent delipidation of brain tissue followed by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis. The second was sucrose gradient centrifugation of a brain homogenate to remove myelin, and gel filtration on Sephadex G-100 and adsorption chromatography on DEAE-Sephadex in the presence of sodium deoxycholate. The products were homogeneous within the limits of the analytical methods used. The apparent molecular weight of the phosphoprotein was 28,000 on sodium dodecyl sulfate polyacrylamide gels, but was much higher in the presence of sodium deoxycholate. The protein had a high content of aspartic and glutamic acids compared to basic amino acids. Analysis of a base hydrolysate, as well as studies of the kinetics of hydrolysis, showed that the radioactive phosphorus was attached to histidine. The NH2-terminal residue was identified as isoleucine. The phosphoprotein purified by the second method was enzymatically active. When it was incubated in vitro with a 32P-labeled supernatant fraction from rat brain (and later with glucose [6-32P]phosphate), a radioactive phosphorylated protein intermediate was formed. Exploration of the several enzymatic activities of the preparation indicated close correspondence to those reported for the glucose-6-phosphatases of liver and kidney. Glucose-6-phosphatase activity was found in all parts of the brain in the membranous subcellular fractions of neurons. It was shown to be co-purified with the sleep-related phosphoprotein. This report constitutes, we believe, the first complete purification of glucose-6-phosphatase from any tissue and an instance in which a change in the state of a cerebral enzyme has been linked to a normal change in the physiological state of the brain.

Amino Acids↗

PubMedDate not supplied

[Transformation of [5'-3H]riboflavin into 5,6-dimethylbenzimidazole].

The 5,6-dimethylbenzimidazole unit of vitamin B12 is formed from riboflavin via FMN in aerobic and some aerotolerant bacteria. Thereby C-1' of the ribityl side chain gets C-2 of 5,6-dimethylbenzimidazole. Experiments with homogenates of Propionibacterium shermanii on the fate of C-atoms 2' to 5' of the ribityl side chain of riboflavin in this transformation are reported. It was found that [5'-3H]riboflavin leads to radioactive sugar phosphates. These were isolated, dephosphorylated, and separated. Thus 3H-D-fructose and 3H-D-glucose were detected. The degradation of 3H-D-glucose revealed that 14 per cent of the total radioactivity was located in C-1, and 86 per cent in C-6. This indicates that during 5,6-dimethylbenzimidazole biosynthesis a three carbon unit is formed from the ribityl side chain of riboflavin.

Benzimidazoles↗