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Effects of dichloroacetate on myocardial substrate extraction, epicardial ST-segment elevation, and ventricular blood flow following coronary occlusion in dogs.

Glucose metabolism in the healthy heart is stimulated by dichloroacetate (DCA). The possibility has been examined in dogs that DCA, by increasing glucose utilization, might limit the severity of acute myocardial ischaemic injury. Intravenous administration of DCA reduced the degree of epicardial ST-segment elevation induced by subsequent coronary occlusion, both under basal conditions and during isoprenaline infusion. A similar result was obtained when DCA was given during an established coronary occlusion. This effect could not be explained by changes in mean aortic blood pressure, heart rate, or regional myocardial blood flow as measured by radioactive microspheres. Measurements in arterial and coronary sinus blood demonstrated an increase in the extraction of glucose and a decrease in that of FFA by the heart. Glucose extraction also tended to be increased in the ischaemic zone, as shown by the differences in the concentrations of these substrates between arterial blood and blood obtained from the local vein draining that zone. Lactate release by the ischaemic zone was markedly reduced.

Acetates↗

Carbon dioxide fixation in Helminthosporium cynodontis.

A mycelial suspension of Helminthosporium cynodontis (ATCC24938), grown on glucose-peptone-yeast extract broth and exposed to NaH14CO3 for 5 h, fixed significant quantities of 14C into the following fractions (%): small molecular weight components, 7-4; lipid and lipoproteins, 3-9; nucleic acids, 59; the residual protein and cell wall fragments, 29-2. The labelled protein components were (%): aspartate, 39; glutamate, 18; cystine, 15; threonine, 9. Radioactive nucleic acid components were (%): adenine, 18; guanine, 18; cytidylate, 34; uridylate, 30. When the mycelium was grown in Czapek-Dox glucose medium and incubated in this medium plus NaH14CO3, the nucleic acid fraction contained 29-9% and the residual protein 49-5% of the cellular radioactivity. The removal of CO2 from the atmosphere did not reduce growth. Pyruvate carboxylase (PC) and phosphoenolypyruvate carboxykinase (PEPCK) activities were demonstrated in extracts of H. cynodontis. Synthesis of PEPCK was stimulated under conditions promoting gluconeogenesis and was reduced under conditions promoting glycolysis, while PC synthesis was similar under both conditions.

Carbon Dioxide↗

Mathematical modelling of stimulus-secretion coupling in the pancreatic B-cell. II. Calcium-stimulated calcium release.

An attempt was made to simulate in a mathematical model one of the two major effects of glucose upon 45Ca fractional outflow rate from prelabelled pancreatic islets, namely the increase in effluent radioactivity which is currently ascribed to the displacement of 45Ca from intracellular sites, as resulting from a facilitated influx of unlabelled 40Ca into the islet cells. The occurrence of such a rise in effluent radioactivity and its suppression in the absence of extracellular Ca2+ could only be simulated if the release of Ca by the vacuolar system was assumed to be stimulated by a rise in the cytosolic Ca concentration. It is proposed therefore that, in islets like in muscle, a process of Ca-stimulated Ca release may participate in the regulation of intracellular Ca distribution.

Animals↗

[Utilization of 1-14C- glucose, 1-14C-acetate and 1-14C-palmitate during lipid synthesis in vitro in the liver and skeletal muscles of pigs at different ontogenic stages].

The specific radioactivity of lipids synthesized in the liver and quadriceps muscle of 110-day embryos, and 1-, 30-, 60-, 90-day and 12-month pigs in vitro from [1-14C]acetate, [1-14C] glucose and [1-14C] palmitate was investigated. The lipid synthesis from most of the above compounds in the fetal liver developed at a greater rate and in the fetal skeletal muscles at a smaller rate than in livers and skeletal muscles of newborn piglets. During the first months after birth the lipid synthesis from [1-14C] acetate, [1-14C] glucose and [1-14C] palmitate in the liver increased gradually. At the same time the lipid synthesis in skeletal muscles from [1-14C]glucose and [1-14C]palmitate decreased and from [1-14C] acetate increased. The utilization of the above compounds in the lipid synthesis of the liver and quadriceps muscle of pigs follows the decreasing order: [1-14C] palmitate greater than [1-14C]acetate greater than [1-14C]glucose.

Acetates↗

Characterization of cytochalasin B binding to adult rat liver parenchymal cells in primary culture.

The characterization of cytochalasin B binding and the resulting effect on hexose transport in rat liver parenchymal cells in primary culture were studied. The cells were isolated from adult rats by perfusing the liver in situ with collagenase and separating the hepatocytes from the other cell types by differential centrifugation. The cells were established in primary culture on collagen-coated dishes. The binding of [4-3H]cytochalasin B and transport of 3-O-methyl-D-[14C]glucose into cells were investigated in monolayer culture followed by digestion of cells and scintillation counting of radioactivity. The binding of cytochalasin B to cells was rapid and reversible with association and dissociation being essentially complete within 2 min. Analysis of the kinetics of cytochalasin B binding by Scatchard plots revealed that binding was biphasic, with the parenchymal cell being extremely rich in high-affinity binding sites. The high-affinity site, thought to be the glucose-transport carrier, exhibited a KD of 2.86 x 10(-7) M, while the low-affinity site had a KD of 1.13 x 10(-5) M. Sugar transport was monitored by 3-O-methyl-D-glucose uptake and it was found that cytochalasin B (10(-5) M) drastically inhibited transport. However, D-glucose (10(-5) M) did not displace cytochalasin B, and cytochalasin E, which does not inhibit transport, was competitive for cytochalasin B at only the low-affinity site, demonstrating that the cytochalasin B inhibition of sugar transport occurs at the high-affinity site but that the inhibition is non-competitive in nature. Therefore, the liver parenchymal cells may represent an unusually rich source of glucose-transport system which may be useful in the isolation of this important membrane carrier.

Animals↗

Uptake of U-14C-glucose by Streptococcus mutans in the presence of saccharin.

The uptake of U-14C-glucose by resting cells of Streptococcus mutans OMZ-176 was studied in the presence of the artificial sweetener saccharin as well as sodium chloride. Glucose grown cells were resuspended in phosphate buffer (0.05 M, pH 7.8), and the uptake of U-14C-glucose was observed for 150 min in time intervals of 30 min, in the presence of 0.02 and 2.00 mg/ml of sodium saccharin as well as sodium chloride. As compared to the control and the sodium chloride treatments, sodium saccharin at the highest concentration range more than doubled the accumulation of radioactive labelled carbon within the cells.

Glucose↗

Studies on the respiratory metabolism of isolated human adipose cells.

1. Some metabolic characteristics of fat cells isolated from 50 patients were examined. 2. The respiratory activity of human fat cells was of the same order of magnitude as cells obtained from the rat, whether the comparison was based on the molar triglyceride content of cells or the nitrogen content of tissue. 3. There was no difference in the respiratory metabolism of fat cells isolated from three separate age groups: 20-40, 40-60 and 60-80 years. 4. The respiratory activity of human adipose cells was partially inhibited by malonate (0.1m), arsenite (1mm) and iodoacetate (1mm). 5. Oxygen consumption by human adipose cells could not be stimulated by adding glucose or glucose plus palmitic acid to the medium. However, stimulation of oxygen uptake with these substrates was observed after the tissue had been preincubated for 5hr. in a bicarbonate buffer. 6. After the tissue had been preincubated for 5hr., there was a linear relationship between the oxygen uptake by isolated cells and the amount of oleic acid added to the incubation medium. Stimulation of oxygen uptake by oleic acid (0.95mumole/ml.) was dependent on the presence of glucose (5.6mm) in the incubation medium. 7. Of the total [1-(14)C]palmitic acid metabolized by human adipose cells, over 99.8% of the radioactivity was recovered in the neutral-lipid fraction and less than 0.2% in carbon dioxide. Under conditions where oxygen uptake is stimulated by [(14)C]-palmitic acid, the radioactivity of the 1,2-diglyceride pool increased 20-fold.

Adipose Tissue↗

The involvement of lysophosphoglycerides in neurotransmitter release; the composition and turnover of phospholipids of synaptic vesicles of guinea-pig cerebral cortex and Torpedo electric organ and the effect of stimulation.

(1) Crude synaptosomal fractions (P2) derived from guinea-pig cerebral cortex were incubated in the presence of 50 mM KCl in a Krebs-glucose medium. Torpedo marmorata electric organs were stimulated electrically in vivo at 5 pulses/sec for 30 min by electrodes placed on the electric lobe. Synaptic vesicles were isolated from each source and the phospholipid compositions analysed and compared with vesicles from unstimulated controls. (2) Lysophosphatidylcholine was the only lysophosphoglyceride demonstrable in the synaptic vesicles from either source and its low levels did not increase as a result of chemical or electircal stimulation. In each case there was a close similarity of the phospholipid distributions in the vesicles taken from control and stimulated samples. (3) Control experiments indicated extensive decreases in the acetylcholine content of the vesicles from the stimulated electric organ and smaller decreases in the acetylcholine content of the synaptic vesicles from stimulated crude synaptosomal fractions. These fractions were found to respire linearly in the presence of 10 mM glucose and the vesicle fractions were shown to have low levels of contaiminating membranes as judged by marker enzyme analyses. (4) Crude synaptosomal fractions from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium with labelled fatty acids and [3H]glucose in the presence or absence of 50 mM KCl. Subsynaptosomal fractionation was carried out and specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomes) and H (disrupted synaptosomes). The release of neurotransmitter did not significantly enhance the labelling of phospholipids in any of the fractions studied as compared with phospholipids from unstimulated fractions. This was found after two incubation times and using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose.

Acetylcholine↗

Stimulated secretion of esterified lipids and VLDL by perifused hepatocytes.

This report describes a new method for investigation of hepatic metabolism by perifusion of medium through a batch of hepatocytes isolated from fed rats. Oxygenated medium flows by gravity through a hepatocyte-containing glass column that is immersed in 37 degrees C water bath. The effluent medium is then collected in consecutive aliquots in test tubes on ice. The pattern of export of esterified lipids, glucose, and VLDL by isolated liver cells into the perifusate was examined under both basal conditions and response to the infusion of certain metabolic stimulants. Perifusion of medium containing sodium clofibrate (1 mM and 10 mM levels) through lipid-prelabeled cells augmented the secretion of radioactive triacylglycerols that reached a maximal rate by about 30 min after exposure to this agent. Measurement of effluent glucose levels after perifusion of hepatocytes with media lacking glucose but containing a gluconeogenic precursor revealed steadily declining concentrations despite the addition of glucagon or epinephrine. Concomitantly, glycogen granules disappeared from the cytoplasm, but the cells retained intact ultrastructure after the course of perifusion. Protein-prelabeled hepatocytes released labeled VLDL into the perifusate, and this release was enhanced by prolonged exposure of the cells to medium containing palmitate (0.80 mM). The hepatocyte perifusion system thus offers a simple, reproducible method whereby hepatocellular secretory processes can be sequentially examined under carefully controlled basal and stimulated conditions.

Animals↗

Mannitol-specific carrier protein from the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system can be extracted as a dimer from the membrane.

The association state of the mannitol-specific enzyme II (EIIMtl) has been studied both in the purified form and embedded in the cytoplasmic membrane. Membrane fragments obtained from mannitol-grown Escherichia coli catalyze the phosphoenolpyruvate- (PEP) dependent phosphorylation of both glucose and mannitol; thus they contain both the glucose- and mannitol-specific enzymes II. The autoradiogram of an electrophoresed mixture of [32P]PEP, EI, HPr, and membrane fragments shows bands at 58 and 116 kilodaltons, in addition to the bands of P-EI and P-HPr. In an analogous experiment with purified EIIMtl, suspended in detergent micelles, only a 58 000-dalton band and the P-HPr and P-EI bands were found. Treatment of the phosphorylated membranes with mannitol results in an immediate substantial decrease in the radioactivity in the 58- and 116-kilodalton bands. A similar treatment of the phosphorylated membranes with glucose had no direct effect on the autoradiogram. We conclude therefore that the 58- and 116-kilodalton bands originate from enzyme IIMtl monomers and dimers, respectively. The interaction between the subunits of the dimer is not abolished by the addition of up to 5% sodium dodecyl sulfate. However, the nonionic detergent Lubrol PX, which is present during the purification of EIIMtl, is capable of transforming the enzyme IIMtl dimers into monomers.

Cell Membrane↗

GLUCOSE METABOLISM OF TWO STRAINS OF MYCOPLASMA LAIDLAWII.

Castrejon-Diez, Jaime (Tulane University School of Medicine, New Orleans, La.), Thelma N. Fisher, and Earl Fisher, Jr. Glucose metabolism of two strains of Mycoplasma laidlawii. J. Bacteriol. 86:627-636. 1963.-Two strains of Mycoplasma laidlawii were incubated in systems containing d-glucose-C(14); carbon dioxide, acetate, pyruvate, and lactate were isolated from appropriate fluids after resting-cell and growth experiments. In resting-cell experiments, radioactivity recoveries were shown to be 95% for M. laidlawii A and 89% for M. laidlawii (Adler). By growth studies, the radioactivity recovery for M. laidlawii A was 83% and for M. laidlawii (Adler) was 90.5%. Low specific activities of the products as compared with the specific activity of glucose suggested cellular pools, or that the dissimilation of other substances present in the complex growth medium yielded products which contributed to the dilution factors. Enzyme studies added support to the hypothesis that glycolysis is operative in these organisms. Experiments with d-glucose-1-C(14) or d-glucose-6-C(14) as substrate suggested that the hexose monophosphate shunt may be functional in M. laidlawii (Adler), particularly since glucose-6-phosphate dehydrogenase, ribose-5-phosphate isomerase, and transketolase were demonstrated. This pathway is absent in M. laidlawii A.

Acetates↗

Metabolic fates of U-14C-labelled monosaccharides and an enzyme-treated cell-wall substrate in the fowl.

A major benefit of supplementing non-ruminant feedstuffs with exogenous enzymes is presumed to be the degradation of plant cell-wall polysaccharides to metabolizable monosaccharide residues. In the present study, metabolic fates of (U-14C-labelled, 10 mM) glucose, galactose, mannose, xylose and arabinose were compared in the fowl, by measuring recoveries of 14C radioactivity in exhaled carbon dioxide excreta and body tissues after administration either by wing vein (iv) or into the crop (ic). A further comparison was made with a tube-fed, enzyme-treated, U-14C-labelled cell-wall substrate, Festuca arundinacea, and a final experiment tested possible competition for absorption between different cell-wall residues. Delays between iv and ic treatments in recovery of 14C in CO2, which were assumed to reflect intestinal absorption, indicated that xylose was absorbed more slowly than glucose and galactose, but faster than mannose and arabinose. Total recoveries of 14C in CO2 and excreta over the whole test period indicated that metabolizabilities were highest with glucose, galactose and mannose, and lowest with arabinose. After testing, 14C recovery in caecal contents was highest with ic arabinose, and recoveries in body tissues, with all sugar treatments, were in the order liver greater than breast and leg muscle greater than abdominal fat greater than plasma. Results with the Festuca substrate showed similar patterns of recovery in body tissues and confirmed an increase in metabolizability with addition of enzyme. The timing of the 14CO2 response with Festuca and a wet enzyme pretreatment was broadly similar to a 'predicted Festuca' response, based on the composition of the substrate and the measured responses with individual (ic) monosaccharides. There was no evidence of any competition for absorption or metabolism among cell-wall residues. It was concluded that glucose release from cellulose is potentially the most important product of cell-wall degradation to contribute to enzyme enhancement of metabolizable energy.

Animals↗

Glucose transport in Streptococcus salivarius. Evidence for the presence of a distinct phosphoenolpyruvate: glucose phosphotransferase system which catalyses the phosphorylation of alpha-methyl glucoside.

A spontaneous mutant of Streptococcus salivarius ATCC 25975 was isolated by inoculating an agar medium containing 11 mM lactose and 0.5 mM 2-deoxyglucose. This mutant grew poorly on 5 mM glucose but almost as well as the parental strain on 110 mM glucose. Uptake of 2-deoxyglucose was abolished by the mutation, and phosphoenolpyruvate: glucose phosphotransferase activity could not be detected with toluenized cells under normal conditions when the glucose concentration was below 5 mM. Data from growth experiments, glycolysis, and uptake studies indicated the presence of a second phosphoenolpyruvate: glucose phosphotransferase system that could catalyze the phosphorylation of alpha-methyl glucoside. The activity of this system was detected by a spectrophotometric assay coupled with lactate dehydrogenase and by a radioactive isotope method using methyl alpha-D-[U-14C] glucoside. The phosphorylation was phosphoenolpyruvate dependent. The apparent Km of the system for glucose and alpha-methyl glucoside was approximately 20 mM. Studies with energy poisons ruled out the possibility of an active transport system, and accumulation of alpha-methyl glucoside argued against facilitated diffusion. It was concluded that the other glucose transport system which allowed growth of the mutant strain of S. salivarius was a second phosphoenolpyruvate: glucose phosphotransferase system.

Biological Transport↗

Metabolism and distribution of cyclohexanecarboxylic Acid, a plant growth stimulant, in bush bean.

A foliar spray of 10 mm cyclohexanecarboxylic acid (CHC), a component of the growth stimulant naphthenic acid, to primary leaves of 14-day-old plants of bush bean, Phaseolus vulgaris L., cv Top Crop, resulted in increased vegetative growth and pod production. One minute after the application of 0.5 mm CHC-7-(14)C (CHC(*)) to a primary leaf, CHC(*) was present within it. The chief conversion of the CHC(*) in the leaf during the interval 15 minutes to 4 hours after the acid had been applied appeared to be CHC(*) to its glucose conjugate (CHC(*)-G), and during 4 to 48 hours, CHC(*)-G to CHC(*)-aspartate and an unknown metabolite. Radioactivity was confined to the leaf for at least 1 hour, but by the 12th hour was detected throughout the plant. In the interval 1 to 4 weeks after CHC(*) application, the mean percentage distribution of radioactivity was: treated leaf, 65.3; roots, 18.8; stem, 7.7; trifoliate leaves, 5.9; flower buds-flowers-pods, 2.3. During this period CHC(*)-G was the most prominent metabolite in all organs; no free CHC(*) was detected. Light favored the movement of CHC(*) conjugates out of the leaf; glucose fed to dark-grown plants substituted for light to some extent but aspartate was relatively ineffective, suggesting the dependence of outward movement on ATP. The presence of the glucose and aspartate conjugates of the acid in all organs of CHC-treated plants and the absence of free CHC from them suggest that one or both conjugates, rather than the acid itself, are involved in growth stimulation.

Journal Article↗

Sensitivity of myocardial fluorodeoxyglucose lumped constant to glucose and insulin.

The lumped constant (LC) that relates the steady-state phosphorylation rate of 2-[18F]-fluoro-2-deoxy-D-glucose (2-FDG) to that of glucose was determined in an isolated working rat heart model by direct assay of phosphorylation product formation. Five conditions were tested: 5 and 30 mM glucose without insulin, and 2, 3.5, and 5 mM glucose + 10 mU/ml insulin, all at high external work load. Hearts were continuously perfused with 2-FDG and tritiated glucose without recirculation. The steady-state production of tritiated water was used to monitor the glucose phosphorylation rate. Perfused hearts were freeze-clamped and extracted in perchloric acid, and 2-FDG-6-phosphate was separated from 2-FDG with a formate column. The accumulation of 2-FDG phosphorylation products in tissue was also determined from the slopes of the total tissue radioactivity time courses measured by external gamma-ray detection. Without insulin, the LC value decreased 18% as perfusate glucose concentration was increased sixfold (0.94 +/- 0.06 at 5 mM vs. 0.77 +/- 0.17 at 30 mM). With insulin, the LC rose from 0.33 +/- 0.03 at 5 mM to 1.19 +/- 0.05 at 2 mM glucose concentration. The trends can be interpreted in terms of the concept of control strength; the LC value rises as glycolysis becomes rate limited by transport into cells. This potential variability of the LC must be addressed in the quantitative interpretation of myocardial deoxyglucose studies.

Animals↗

Effects of oxygen saturation and pCO2 on brain uptake of glucose analogues in rabbits.

The effect of oxygen saturation and PCO2 on brain uptake of glucose analogues was studied in rabbits. Using a modified Oldendorf technique, 14C-labeled glucose analogues with a 3H2O reference standard were introduced into the cerebral circulation via the common carotid artery, and the radioactivity of the ipsilateral cerebral cortex was counted and expressed in terms of a brain uptake index (BUI). Severe hypoxia (oxygen saturation less than or equal to 18%) resulted in approximately a 40% decrease in the BUI of 2-deoxy-D-glucose and a 45% decrease in the BUI of 3-0-methyl-D-glucose. Severe hypercapnia (PCO2 = 100 mm Hg) caused a 45% decrease in the BUI of both of these glucose analogues. Hypercapnia superimposed on severe hypoxia had no additional effect. Hypocapnia (PCO2 = 15 mm Hg) increased the BUI of 3-0-methyl-D-glucose by 35% of the control value, and this increase was extremely sensitive to competitive inhibition. When BUI values were plotted against pH rather than PCO2 for the same experiments, there was a good correlation with the calculated linear regression. These results are compared with previous findings on pathologically induced changes in brain uptake of glucose analogues, and the possible role of blood flow is considered in detail.

Animals↗

Enzymatic lactate-specific radioactivity determination in biological samples.

A method for the measurement of specific lactate radioactivity in biological samples is presented. It is based on the following steps: (a) enzymatic conversion of lactate to pyruvate, (b) pyruvate conversion to 2,4-dinitrophenylhydrazone, (c) concentration-separation of the latter in reusable Amberlite XAD-7 polymeric adsorbent columns, and finally (d) estimation of the radioactivity thus retained compared with that of enzymatically untreated aliquots of the same samples. Specificity was ensured by the use of lactate dehydrogenase as specific recognizing agent for lactic acid. No interference from glucose, lactate, or amino acids was observed. The method presented is simple and can be applied in routine multiple estimations of lactic acid radioactivity in conjunction with the enzymatic measurement of lactate in biological samples in tracer metabolic studies.

Animals↗