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PubMed1980

Uptake and decomposition of chlorozotocin in L5178Y lymphoblasts in vitro.

Uptake and metabolism of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin) by L5178Y lymphoblasts in vitro was investigated, using both glucose- and chloroethyl-labeled chlorozotocin. A time course of uptake of total radioactivity revealed a greater cell/medium distribution ratio of activity in cells treated with chloroethyl-labeled chlorozotocin compared to cells treated with the glucose-labeled compound. Thin-layer chromatographic analysis showed that uptake of intact chlorozotocin was identical in cells treated with either glucose- or chloroethyl-labeled drug and that the cell/medium distribution ratio never exceeded unity. Accumulation of 14C-chlorozotocin was not inhibited by an excess of unlabeled chlorozotocin, the structural analogs glucose and glucosamine, or several metabolic inhibitors or by sodium ion depletion. These observations, together with the relatively low temperature quotient for the uptake process, suggested that chlorozotocin uptake occurs by passive diffusion. In cells treated with glucose-labeled chlorozotocin, a bicyclic urethan derivative and polar metabolites soluble in trichloroacetic acid were formed. In cells exposed to chloroethyl-labeled drug, nonpolar as well as polar metabolites were noted. Formation of metabolites from the glucose moiety was impeded by the presence of an excess of unlabeled chlorozotocin, the structural analogs glucose and glucosamine, the glucose transport inhibitors phlorizin and phloretin, the metabolic inhibitor m-chlorophenyl carbonyl cyanide hydrazone and by sodium depletion. Appearance of metabolites arising from the chloroethyl moiety was also blocked by the presence of m-chlorophenyl carbonyl cyanide hydrazone and by sodium ion depletion. These results suggested that metabolism of chlorozotocin in L51789Y lymphoblasts appears to be enzyme mediated.

Animals↗

PubMed1976

Regulation of L-phenylalanine ammonia-lyase from Rhizoctonia solani.

Maximal levels of L-henylalanine ammonia-lyase activity were observed when the mycelial felts of Rhizoctonia solani were grown for 4.5 days on Byrde synthetic medium containing 3.5% glucose and 0.3% L-phenylalanine, Differential centrifugation studies have indicated that the enzyme is localized in the soluble fraction. The time course of induction of L-phenylalanine ammonia-lyase activity by L-phenylalanine showed a lag period of 1 to 1.5 h and reached a maximum around 4 to 6 h after the addition of the inducer to the medium. L-Phenylalanine, L-tyrosine, and L-tryptophan were nearly equally efficient inducers of the enzyme. D-Phenylalanine was as efficient as the L-isomer, whereas D-tyrosine was a poor inducer. Light, gibberellic acid, indole 3-acetic acid, and kinetin had no effect on the induction of L-phenylalanine ammonia-lyase activity. Cycloheximide did not inhibit the uptake of amino acids by the mycelia but completely blocked the incorporation of radioactive amino acids into soluble proteins and the development of L-phenylalanine ammonia-lyase activity. Actinomycin D inhibited both the incorporation of 32P into ribonucleic acid and the enzyme activity. Conclusive evidence for de novo synthesis of L-phenylalanine ammonia-lyase was obtained by the incorporation of radioactive amino acids into the enzyme. Electrophoretic analysis of the purified preparation showed a single protein band that coincided with radioactivity and L-phenylalanine ammonia-lyase activity. Glucose and intermediates of the tricarboxylic acid cycle, like citric acid, alpha-ketoglutaric acid, and succinic acid, and the metabolites of L-phenylalanine, like o-coumaric acid, o-hydroxyphenylacetic acid, and protocatechuic acid, significantly repressed L-phenylalanine ammonia-lyase activity. The observed repression was not relieved by cyclic adenosine 5'-triphosphate.

Ammonia-Lyases↗

PubMed1988

Validation of the triple-tracer autoradiographic method in rats.

We have developed a quantitative autoradiographic technique to measure simultaneously local cerebral blood flow, tissue pH, and local cerebral glucose utilization using 4-[18F]fluoroantipyrine, [14C]dimethyloxazolidine-2,5-dione, and 2-[3H]deoxyglucose as radioactive tracers. Our technique was validated by measurements of local cerebral blood flow, tissue pH, and local cerebral glucose utilization in the brains of sham-operated rats. Sham-operated rats showed a 14% reduction in local cerebral blood flow and a 10% reduction in local cerebral glucose utilization in the most affected piriform and entorhinal cortical regions compared with the contralateral homologous regions. There was no difference in tissue pH measured in the sham-operated side compared with normal rats or with the contralateral region.

Animals↗

PubMed1991

Fructose administration stimulates glucose phosphorylation in the livers of anesthetized rats.

A method allowing one to measure the rate of glucose phosphorylation in the livers of anesthetized rats is described. Upon injection of [2-3H]glucose into the portal vein, about 90% of the radioactivity remained in the liver for approximately 30 s. The proportion of radioactivity accounted for by tritiated water increased linearly at a rate of about 5%/min in control animals. Fructose injected into the penile vein stimulated this rate up to 2.2- and 2.7-fold in fed and overnight starved rats respectively, a maximal effect being observed at a dose of 50 mg/kg under both conditions. Fructose was also active when administered by intragastric infusion. The ketose caused increases in the concentration of fructose 1-phosphate, which reached values known to relieve the inhibition exerted on glucokinase by its regulatory protein.

Anesthesia↗

PubMed1993

Autonomic modulation of insulin levels in foetal sheep.

The autonomic nervous system plays an important part in metabolic and circulatory adaptation of the foetus to changes in its intrauterine environment and after delivery. Foetal and neonatal glucose metabolism and insulin secretion are influenced by changes in humoral catecholamine levels as they may occur during asphyxia. To assess the role of neuronal and humoral sympathetic activity in foetal endocrine pancreatic regulation, chronically catheterized foetal sheep near term were chemically sympathectomized with 6-hydroxydopamine. Experiments were carried out in unanaesthetized foetal sheep in utero in the absence of uterine contractions. Insulin and glucose levels, blood gases, acid-base status and catecholamines were measured before, during and after a 2 min occlusion of uterine blood flow caused by mechanical constriction of the maternal aorta. Pancreatic blood flow was determined using radioactive labelled microspheres. During normoxaemia, insulin levels, pancreatic blood flow and glucose transport to the organ in sympathectomized foetuses were elevated compared with intact animals, whereas glucose concentrations did not show any significant differences. After the onset of asphyxia humoral catecholamine levels rose significantly in both groups. Insulin concentrations in the plasma of both intact and sympathectomized foetuses were no longer different indicating both indirect (blood flow and humoral catecholamine related) and direct (neurally mediated) sympathetic effects on pancreatic beta cells.

Animals↗

PubMed1975

Glucose utilization by the salivary glands of the marmoset Saguinus oedipus.

Glucose utilization by the marmoset salivary glands was studied radioisotopically, both in vitro and in vivo. Surgically excised, minced parotid and submandibular glands from 7 adult animals were incubated in vitro in a medium containing uniformly labeled 14C glucose (glucose-UL-14C), then subjected to respirometry measurements and to extraction and separation procedures for the radioautographic isolation and identification of amino acids, organic acids, and glucose. Glucose utilization in vivo by the marmoset salivary glands was determined in 3 animals by analysis for 14C-containing amino acids, organic acids and glucose in pilocarpine-stimulated whole saliva, following intraperitoneal injection of glucose-UL-14C. The whole saliva was fractionated, separated and processed for localization of radioactivity by the same methods that were used in the in vitro studies. The isolated and in situ salivary glands converted glucose-UL-14C into 14C-containing carbon dioxide, glycine, alanine, glutamic acid, aspartic acid, and lactate. In no instance did the amount of 14C-glucose secreted in saliva exceed 0.52 mg/100 ml..

Amino Acids↗

PubMed1993

UDP-sugar metabolism in Swarm rat chondrosarcoma chondrocytes.

UDP-sugars and adenine nucleotides were extracted from freshly isolated chondrocytes and primary cell cultures and analysed by anion-exchange h.p.l.c. The pool sizes of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-glucose-galactose, UDP-glucuronate and UDP-xylose were 2.9, 1.2, 2.5, 0.6 and 0.03 nmol/10(6) freshly isolated chondrocytes. When chondrocytes were maintained in Dulbecco's modified Eagle medium supplemented with 15% foetal-bovine serum, synthesis of [35S]proteoglycan and [3H]protein decreased over the first 48 h in culture, as did the pools of UDP-glucuronate and ATP. In contrast, the size of the UDP-N-acetylhexosamine pools underwent little change during culture. [35S]Proteoglycan and [3H]protein syntheses were stimulated in cultures supplemented with serum or insulin compared with those maintained in medium alone, in agreement with previous results. However, the UDP-sugar pool sizes were the same in both supplemented and non-supplemented cultures. In cultures maintained in the presence of [1-3H]glucose, the UDP-sugars were labelled to a constant 3H specific radioactivity which was very similar to that of the labelling medium. UDP-N-acetylhexosamines were labelled to constant 3H specific radioactivity with [6-3H]glucosamine as a precursor, but only about 1 in 375 of these UDP-sugars was derived from the amino sugar in the presence of glucose. The half-life (t1/2) for UDP-hexoses, UDP-glucuronate and UDP-N-acetylhexosamines was about 12, 12 and 50 min respectively.

Animals↗

PubMed1985

Effect of antibody preparations on glucose uptake by a cariogenic Streptococcus.

The effect of antisera to whole cells or cell wall components on glucose uptake by S. mutans 6715 was examined. Early stationary phase 6715 cells were treated with test serum and incubated at 37 degrees C in the presence of 14C-glucose. Samples were removed at timed intervals and measured in a liquid scintillation counter for radioactive uptake. Antisera to both whole cells and components known to be present on the surface of the cells reduced glucose uptake relative to normal serum. It is suggested that inhibition of glucose uptake may be one mechanism by which a caries vaccine may operate.

Animals↗

PubMed1978

The energy metabolism of adult Haemonchus contortus, in vitro.

A comparison was made of the major excretory products when adult Haemonchus contortus worms were incubated with D-[U-14C]glucose under aerobic and anaerobic conditions. Catabolites measured were propan-1-ol, acetate, n-propionate and CO2 and the only major difference was that nearly twice as much CO2 both in terms of quantity and radioactivity was excreted under aerobic than anaerobic conditions. The worms were also much more physically active under aerobic conditions. When worms were incubated under aerobic conditions with increasing amounts of fluoroacetate their CO2 production was progressively reduced to the anaerobic level. Their movement and their ability to clump together was also progressively reduced. After aerobic incubation with fluoroacetate and D-[U-14C]glucose the quantity and radioactivity of citrate within worms increased greatly. When worms were similarly incubated anaerobically no increase in citrate occurred, no radioactivity was associated with the citrate and the worms appeared physically unaffected. When worms were incubated aerobically with fluoro[1(-14)C]acetate they produced radioactive fluorocitrate.

Acetates↗

PubMed1994

Acidic polysaccharides of the ascidian Styela plicata. Biosynthetic studies on the sulfated L-galactans of the tunic, and preliminary characterization of a dermatan sulfate-like polymer in body tissues.

Histochemical and chemical analyses reveal important differences between tunic and body of the ascidian Styela plicata. The body contains hydroxyproline, nucleic acid and hexuronic acid. Analyses of the hexuronic acid-containing molecules indicates the presence of dermatan sulfate and heparan sulfate. The tunic, on the other hand, contains no hydroxyproline and small amounts of nucleic acid and hexuronic acid. Large amounts of sulfated polysaccharides, identified by agarose gel electrophoresis, are also present in the tunic. In vitro, incorporation of 35S-sulfate and 14C-glucose and 35S-sulfate pulse-chase experiments show that sulfate and glucose are incorporated into the sulfated polysaccharides of the tunic. The radioactivity is associated mainly with the region of the tunic containing the epidermal cells; however, a small amount of radioactivity is also detected in other regions of the tunic, suggesting that the sulfated polysaccharides are synthesized mainly by the epidermal cells and, to a small extent, by other cell types present in the tunic. Conversion of D-glucose to L-galactose, previously observed in the tunic of Styela plicata (Biochemistry 30, 3458-3464, 1990) is more intense in the region of the tunic containing the epidermal cells.

Animals↗

PubMedDate not supplied

[Incorporation of 1-6-14C-glucose into lipid fractions of the liver and brain of rats with different alcohol motivation].

Specific radioactivity of total phospholipids, cholesterol and lysophosphatidyl choline was decreased in liver tissue of rats consuming ethanol as compared with the animals preferring water. In brain of rats with alcohol motivation incorporation of 1-6-14C-glucose into cardiolipin was decreased, while the label incorporated into triacylglycerols, sulfatides II and cerebrosides I at the increased rate.

Alcohol Drinking↗

PubMed1989

A simple and rapid microplate assay for glycoprotein-processing glycosidases.

A simple and convenient microplate assay for glycosidases involved in the glycoprotein-processing reactions is described. The assay is based on specific binding of high-mannose-type oligosaccharide substrates to concanavalin A-Sepharose, while monosaccharides liberated by enzymatic hydrolysis do not bind to concanavalin A-Sepharose. By the use of radiolabeled substrates [( 3H]glucose for glucosidases and [3H]mannose for mannosidases), the radioactivity in the liberated monosaccharides can be determined as a measure of the enzymatic activity. This principle was employed earlier for developing assays for glycosidases previously reported (B. Saunier et al. (1982) J. Biol. Chem. 257, 14155-14161; T. Szumilo and A. D. Elbein (1985) Anal. Biochem. 151, 32-40). These authors have reported the separation of substrate from the product by concanavalin A-Sepharose column chromatography. This procedure is handicapped by the fact that it cannot be used for a large number of samples and is time consuming. We have simplified this procedure and adapted it to the use of a microplate (96-well plate). This would help in processing a large number of samples in a short time. In this report we show that the assay is comparable to the column assay previously reported. It is linear with time and enzyme concentration and shows expected kinetics with castanospermine, a known inhibitor of alpha-glucosidase I.

Animals↗

PubMed1987

Biosynthesis of proteins by human gastric mucosa in vitro.

Incorporation of L-[U-14C]leucine and of D[U-14C]glucose into proteins of fresh human gastric mucosa in vitro was studied after incubation of homogenized tissue and of intact mucosal pieces. CsCl centrifugation was used to separate high-density mucus glycoproteins from other mucosal proteins, and the macromolecular nature of radioactive mucosal glycoprotein fractions was confirmed by SDS/polyacrylamide-gel electrophoresis and autoradiography of the polyacrylamide gels. In all experiments a substantial proportion of total incorporated radioactivity was associated with gastric-mucosal glycoprotein fractions (CsCl fraction L3), indicating their biosynthesis. Radioactivity of these fractions was shown to co-chromatograph with carbohydrates when fractionated either directly or after reduction and alkylation (1) Sephadex G-200 chromatography in the excluded fractions and (2) by DEAE-cellulose ion-exchange chromatography. On incubation of intact mucosa, the major portion of radioactivity associated with the glycoprotein fractions of both leucine- and glucose-labelled specimens was secreted into the mucosal media during the course of the experiment. It is suggested that biosynthesis of mucus in vivo by gastric mucosa may be associated with rapid secretion of the synthesized macromolecules into the lumen of the stomach and that investigations of the metabolic processes within the mucosa should consider the products of secretion of the tissue. Incorporation of L-[U-14C]leucine implies biosynthesis of the polypeptide components of the macromolecules.

Autoradiography↗

PubMed1986

Effect of glucose on systemic hemodynamics and blood flow rate in normal and tumor tissues in rats.

The effect of hyperglycemia on systemic hemodynamics and blood flow rate of Walker 256 carcinoma and several normal tissues of unanesthetized, unrestrained female Sprague-Dawley rats was measured, using the radioactive microsphere technique prior to and at 30 and 60 min after glucose administration (6 g/kg body weight i.v.). Whereas the mean arterial pressure and heart rate remained unchanged following glucose injection, cardiac output (CO), cardiac index, and stroke volume decreased by approximately 25% (P less than 0.05), and the total peripheral resistance increased by more than 25% (P less than 0.05). Redistribution of blood flow, expressed as a percentage of CO, among normal tissues was traced to the brain, kidneys, spleen, and liver and away from the skin, pancreas, and stomach. Changes in percentage of CO to the jejunum, colon, peritumor muscle, hindlimb, and forelimb muscles were not significant. Reduction in blood flow in large tumors (greater than 0.6 g) was proportional to the reduction in CO, and in small tumors (less than 0.6 g) was more than the reduction in CO. These results suggest that the reduction in blood flow due to hyperglycemia in large tumors is primarily due to reduction in CO and in small tumors due to both systemic and local effect; and changes in the blood flow to normal tissues should not be disregarded when using hyperglycemia in combination with hyperthermia and/or chemotherapy for cancer treatment.

Animals↗

PubMed1994

Determination of 13C enrichment at specific positions of plasma glucose by gas chromatographic-mass spectrometric analysis of the 1,2:5,6-diisopropylidene-3-O-acetyl-alpha-furanosyl derivative.

To determine 13C enrichment at specific positions of glucose, plasma samples (50-100 microliters) were deproteinized using ethyl alcohol, centrifuged, and evaporated to dryness, and the residues were derivatized with acetone/H2SO4 (100/1, v/v). After acetylation, the 1,2:5,6-diisopropylidene-3-O-acetyl-alpha-furanosyl derivative was analyzed by gas chromatography-mass spectrometry. The method was tested using mixtures of natural and 13C-labeled glucose molecules. Ions at m/z 113, 114, 143, 144, 287, and 288, corresponding to the C2-C4, C1-C4, and C1-C6 parts of natural or 13C-labeled glucose molecule, respectively, were selectively monitored to determine the 13C content of these fragments. This method was applied to the study of plasma glucose-labeling pattern following the infusion of [3-13C]lactic acid at tracer doses. Assuming equilibration of label through triose-phosphate isomerization in vivo, we determined the 13C enrichment of carbons 1, 2, and 3 of glucose; the values for E1/E3 and E2/E1 ratios of 8.1 and 0.77, respectively, are consistent with those obtained in studies using radioactive tracers.

Blood Glucose↗

PubMed1986

The fate of ultra-low viscosity 14C-hydroxypropyl methylcellulose in rats following gavage administration.

The disposition of 14C-Hydroxypropyl methylcellulose (HPMC) with a viscosity of 2.25 centipoise was studied in male and female Sprague-Dawley rats following a single 500 mg/kg body weight gavage dose, or five consecutive daily doses. Recoveries for the single dose were: feces, greater than 99%; urine, approximately 1%; carcass and tissues, approximately 0.2%; expired air, 0.07%; and bile, 0.05%. Plasma radioactivity had a monophasic excretion half-life of approximately 2 hours for either sex. The majority of the residual radioactivity in the tissues was found in the gastrointestinal tract. The absorbed radioactivity in the urine, based on thin layer chromatography (TLC) analyses, represented methyl ethers of glucose and oligomers; this amounted to 0.56% recovered in a study in which urine samples were isolated from possible contamination by radioactivity in the feces. The 0.56% correlated well with the 0.53% portion of the original dosing solution which consisted of cellulose units with an average molecular weight of less than 1000. Recovery of radioactivity in the feces of rats on the 5-day dosing regimen was 97% and 102% for males and females, respectively, without any evidence for accumulation in tissues. Approximately 1% was recovered in the urine. Thus, the results of this work show that ultra-low viscosity 2.25 centipoise HPMC was only minimally absorbed with essentially all of a single 500 mg/kg gavage dose, or 5 daily consecutive doses, being excreted unabsorbed in the feces.

Administration, Oral↗

PubMed2005

Positron-emission tomography imaging of early events after transplantation of islets of Langerhans.

The aim of our study was to assess cell trafficking and early events after intraportal islet transplantation. Sprague-Dawley rat islets were incubated for various times, with various concentrations of 2-[F]fluoro-2deoxy-D-glucose (FDG), and in presence of various glucose concentrations. FDG-labeled syngeneic islets or FDG alone were injected in rats. Radioactivity was measured in the liver and in various organs by positron-emission tomography for 6 hours. FDG uptake increased with incubation time or FDG concentration and decreased in presence of glucose. In vivo, all islets implanted in the liver, with an uptake 4.4 times higher than controls (44.2% vs. 10.1%, P=0.02). Radioactivity in the liver decreased at the same rate after injection of labeled-islets and FDG alone. Ex vivo labeling of islets and imaging of posttransplant early events were feasible. Islets engrafted exclusively in the liver. No islet loss could be demonstrated 6 hours after transplantation.

Animals↗

PubMed1995

Insulin and glucose modulate glucose transporter messenger ribonucleic acid expression and glucose uptake in trophoblasts isolated from first-trimester chorionic villi.

OBJECTIVE: Our purpose was to determine the effects of insulin and glucose on glucose transport and expression of GLUT1 glucose transporter messenger ribonucleic acid in first-trimester human trophoblast-like cells. STUDY DESIGN: First-trimester human trophoblast-like cells were maintained as a continuous cell line. For 2[3H]deoxy-D-glucose uptake and messenger ribonucleic acid studies the cells were incubated in the presence or absence of insulin (10(-7) to 10(-11) mol/L) or D-glucose (0 to 50 mmol/L) for 0 to 24 hours. Glucose transport was measured by incubating cells with 0.1 mmol/L 2[3H]deoxy-D-glucose for 5 minutes. Specific uptake was determined by incubating companion cultures with 10 mumol/L cytochalasin B. The cells were then solubilized with sodium hydroxide and the radioactivity counted. Data were expressed as nanomoles of 2[3H]deoxy-D-glucose transported per milligram of protein per 5 minutes and analyzed by one-way analysis of variance with post hoc testing by the method of Tukey. GLUT1 messenger ribonucleic acid was measured by Northern blotting of total ribonucleic acid samples hybridized to a phosphorus 32-labeled complementary deoxyribonucleic encoding the rat GLUT1 glucose transporter. As a control for loading efficiency, blots were stripped and rehybridized to a 40-mer phosphorus 32-labeled beta-actin oligonucleotide probe. RESULTS: Insulin treatment resulted in a dose-dependent increase in the transport of 2[3H]deoxy-D-glucose at 24 hours (p < 0.001 at 10(-7) mol/L). This change was first detected at 12 hours of incubation. These data closely paralleled the insulin-induced increase in GLUT1 messenger ribonucleic acid seen in Northern blots. In contrast to insulin, increasing concentrations of D-glucose did not change the transport of 2[3H]deoxy-D-glucose. However, when cells were incubated in low concentrations of D-glucose (0 or 1 mmol/L), an enhancement in the uptake of 2[3H]deoxy-D-glucose (p < 0.001) was observed. Kinetic studies indicated that D-glucose augmentation of 2[3H]eoxy-D-glucose uptake was significant at 9 hours (p < 0.05). The effects of D-glucose on GLUT1 messenger ribonucleic acid expression paralleled the uptake of 2[3H]deoxy-D-glucose, although the modulation of GLUT1 messenger ribonucleic acid levels by glucose was much less pronounced than in insulin-treated cells. CONCLUSION: Although it has been assumed that the placenta has a limited role in influencing glucose transport to the fetus, our in vitro data demonstrate that both insulin and glucose can modulate glucose transport at the cellular level of the placental trophoblast. Thus maternal insulin and glycemic status may influence the expression of GLUT1, the major trophoblast glucose transporter protein, therefore directly affecting first-trimester placental glucose transport. These in vitro data may help explain the association between maternal glucose abnormalities and impaired fetal development during the first trimester when placental GLUT1 messenger ribonucleic acid expression is at its peak.

Analysis of Variance↗