Raffaella Catanzaro

Free amino acids in fish brain: normal levels and changes upon exposure to high ammonia concentrations in vivo, and upon incubation of brain slices.

1. 1. When fish (Carassius auratus) were kept in water containing controlled concentrations of ammonia, the cerebral concentration of glutamine increased up to ten times, that of several other amino acids increased to a lesser extent. 2. 2. The changes observed were much more pronounced in brain than in other organs. 3. 3. Fish brain slices incubated aerobically in a medium containing glucose or glutamate with or without NH4C1 showed an increase in the level of most amino acids. Glutamine and histidine increased the most, in absolute terms. 4. 4. The amino acid changes of incubated rat brain slices differed, in several aspects, from those observed in fish.

The Influence of Insulin on Carbohydrate Metabolism in the Isolated Diaphragm Muscle of Normal and Alloxan Diabetic Rats

The fate of uniformly labelled 14C glucose in the isolated diaphragm muscle of normal and alloxan diabetic rats has been studied by a quantitative application of the radio paper-chromatographic technique. No significant differences were observed in the metabolism of glucose by muscle from normal and diabetic rats. About 80% of the glucose metabolized by the muscle in the absence and presence of insulin has been accounted for. The extra glucose disappearing from the incubation medium in the presence of insulin was found to be incorporated in oligo- and polysaccharides, the percentage of glucose converted into the other main metabolites (i.e. lactic acid, hexose-phosphate esters and CO2) was shown to be unchanged or decreased by insulin. Insulin markedly accelerated the synthesis of oligo- and polysaccharides. The amount of free glucose in the tissues, which reached a maximum after 15 min of incubation, was uninfluenced by insulin. When the muscle was first incubated with 14C glucose and then in the absence of substrate, there was a sharp decrease in the radioactivity of all the intermediates with the exception of the hexose-phosphate esters which were found to accumulate. Under anaerobic conditions the total glucose metabolism was very reduced, and the greater part of the glucose disappearing from the medium was recovered as free glucose in the tissues. Very little radioactive lactic acid was formed under anaerobic conditions, except when the muscle was first incubated with 14C glucose aerobically and then anaerobically; under these conditions there was a considerable accumulation of radioactive lactic acid. Current theories on the mode of action of insulin have been discussed in reference to the findings reported in the present work.

Fate of uniformly labelled 14C glucose in brain slices

The fate of uniformly labelled 14C glucose in rat-brain slices has been followed by a quantitative application of the radio paper-chromatography technique. After 60 min incubation with brain tissue approximately 60% of the glucose disappearing from the medium was accounted for as lactic acid, about 20% as CO2 and most of the remainder as free amino-acids. Of the total glucose metabolized approximately 9% was converted into glutamic acid, 1.5% alanine, 3% γ-amino-butyric acid and 2.4% aspartic acid. Glutamic acid was the first of the amino-acids to be formed from glucose, and was detectable after 3 min incubation of brain tissue with 14C glucose. Insulin had no effect on glucose metabolism in brain slices. Under anaerobic conditions the total glucose metabolized by brain slices was only about 10% of that found under aerobic conditions; of the total glucose disappearing from the medium anaerobically about 80% was accounted for as lactic acid and the remainder as free unchanged glucose in the tissue cells.

Influence of iron on oxidation of reduced nicotinamide- adenine-dinucleotide phosphate in rat-liver microsomes

Abstract 1. 1. When Fe2+ was added to a microsome suspension prepared from rat liver, there was an uptake of O2 and a concomitant lipid peroxidation. These reactions were uninfluenced by the addition of ADP or NADPH2. 2. 2. The addition of Fe3+ to a liver-microsomal suspension increased O2 uptake and lipid peroxidation in the presence of ADP and NADPH2. 3. 3. Ethylenediaminetetraacetate and antimycin A inhibited O2 uptake by rat- liver microsomes in the presence of NADPH2 and ADP, and this inhibition was reversed by the addition of Fe3+. 4. 4. The supernatant obtained after separating the microsomes by centrifugation at 105000 × g, inhibited O2 uptake by the microsomes in the presence of NADPH2 and low concentrations of ADP (2 μmoles/ml), and this inhibition was reversed by the addition of ADP (a further 2 μmoles/ml). 5. 5. Cytochrome b5 was reduced by addition of NADPH2 in the presence of antimycin A which blocked O2 uptake. The cytochrome was re-oxidized on addition of Fe3+ and ADP together. 6. 6. A tentative scheme for the participation of iron in the oxidation of NADPH2 in rat-liver microsomes is discussed.

The metabolism of glucose and pyruvate in rat retina

The metabolism of [U-14C]glucose and [3-14C]pyruvate in the adult rat retina is described. In vitro under aerobic conditions, in either phosphate or bicarbonate medium, glucose was converted into lactate, carbon dioxide, glutamate, γ-aminobutyrate, aspartate, glutamine and alanine. Under anaerobic conditions, total glucose metabolized was reduced to 60 to 70% of that under aerobic conditions, lactic acid being the only metabolic product detected. Under aerobic conditions [3-14C]pyruvate was converted by the retina into the same metabolites as was glucose. The quantitative data for oxygen uptake and 14CO2 formation were similar to those obtained with glucose as substrate; lactate production was lower and amino acid formation higher.

The Metabolism of Glutamate and Aspartate in Rat Cerebral Cortical Slices

The fate of [14C]glutamate and [14C]aspartate in rat cerebral cortical slices was followed quantitatively by means of the previously described automatic scanning technique of paper radio-chromatograms, in the presence and absence of non-radiocative glucose, and in the case of [14C]glutamate, also of the following non-radioactive tricarboxylic cycle acids: pyruvate, citrate, α-oxoglutarate, malate, oxalo-acetate and succinate. Both amino-acids were readily oxidized to CO2, glutamate increasing, aspartate not affecting the rate of endogenous oxygen uptake of the tissue. In addition the following metabolites accumulated: (a) From glutamate: aspartate, glutamine and γ-aminobutyrate, in order of decreasmg amounts. (b) From aspartate: glutamate, malate and citrate, in order of decreasmg amounts, with traces of glutamine, γ aminobutyrate and lactate. The presence of non-radioactive glucose as co-substrate caused the following effects. 1. On glutamate-.The total production of 14CO2aswell as the oxygen uptake increased. This indicated that glucose oxidation had not replaced glutamate oxidation, but actually caused its stimulation. The conversion into glutamine was increased by a factor of 3, an effect similar to that caused by K+ ions. The conversion into aspartate was decreased by a factor of 4. The accumulation of intracellular glutamate was much enhanced. 2. On aspartate: both 14CO2 production and oxygen uptake were stimulated, indicating that, as in the case of glutamate, glucose oxidation not only had not replaced the oxidation of amino-acid, but stimulated it. The production of glutamate, glutamine, γ-aminobutyrate and lactate was considerably increased while that of malate and citrate was unaffected. The following effects on glutamate metabolism were noted in the presence of non-radioactive tricarboxylic cycle acids: None of the acids affected 14CO2 production except α-oxoglutarate which caused a decrease of over half. This decrease was shown to be due to a dilution of [14C]glutamate through equilibration with non-radioactive α-oxoglutarate; the latter acquired about half of the radioactivity of the [14C]glutarnate added during the incubation. Pyruvate, like glucose, decreased the conversion to aspartate and increased glutamine formation, the latter, however, to a much lower degree than glucose. It also enhanced the accumulation of intracellular glutamate. Citrate, succinate, malate and oxaloacetate, particularly the latter two co-substrates, repressed the formation of [14C]glutamine, but had no other effect on the pattern of distiibution of the metabolites formed from glutamate and on the permeability of this amino-acid into the cell.

The Influence of Glucose on Acetate, Alanine and Pyruvate Metabolism in Rat Cerebral Cortical Slices

The effect of glucose as co-substrate has been studied on the metabolic fate of [U-14C]alanine [2-14C]acetate and [3-14C]pyruvate in brain cortical slices, using the automatic paper radio-chromatographic scanning technique previously described. In comparison to glucose and pyruvate, acetate and alanine were metabolized only to a very small extent by brain cortical tissue. The addition of glucose strongly stimulated the 14CO2 production from acetate, but did not affect 14CO2 production from pyruvate and alanine. The oxygen uptake was stimulated by glucose in presence of all three substances. In addition to CO2 the metabolites formed from pyruvate were aspartate, glutamate, alanine, GABA and lactate. The same metabolites were formed from alanine. The only metabolites, detected with the technique used, formed from acetate, were aspartate and glutamate. The effect of glucose on the metabolites formed from acetate was a marked increase of glutamate, glutamine and GABA and a decrease of aspartate. The effect of glucose on the metabolites formed from alanine was an increase of glutamate, GABA, and lactate, and a decrease of aspartate. The effect of glucose, on the metabolites formed from pyruvate was a very large increase of lactate, and a decrease of all the amino-acids formed except glutamine, the formation of which was stimulated.

Influence of Anti-insulin Serum on Glucose Metabolism: I. In Isolated Adipose Tissue

1. A consistent in vitro effect of anti-insulin serum on glucose metabolism in isolated rat adipose tissue has been demonstrated. 2. Anti-insulin serum added in vitro to epididymal fat pads caused an average decrease in the C-14-O2 formation from glucose-1-C-14 of over 50 per cent, whereas it reduced the incorporation of glucose-U-C-14 into lipids by about 20 per cent. 3. Epididymal fat pads pretreated in vitro with antiinsulin serum oxidized glucose-1-C-14 at a reduced rate compared to control fat pads preincubated in buffer; in the presence of added insulin, however, glucose oxidation was the same in the control and anti-insulin treated fat pads.

INFLUENCE OF INTERFERON ON GLUCOSE METABOLISM IN CHICK-EMBRYO CELLS.

Abstract The influence of interferon on the metabolism of glucose in tissue culture has been studied. It was shown that glucose incubated with chick-embryo cells was converted into lactate, glycogen, iligosaccharides, glutamate, aspartate and CO 2 . Approx. 85% of the glucose metabolized was accounted for as lactate. A slight increase in the lactate formation from glucose which was observed with interferon-treated cells was shown to be due probably to some substrate formed in the preparation of interfereon but not to interferon itself. Interferon had no influence on the oxidation of glucose.

The influence of insulin on the specific activities of the hexose phosphate esters and lactate formed from glucose in the isolated rat diaphragm muscle

A microchromatographic technique using ion exchange columns for the separation of glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-diphosphate in extracts of rat diaphragm muscle has been described. Applying this technique it has been shown that when diaphragm muscle was incubated in the presence of uniformly labelled C14-glucose for 2 hr in the absence of insulin the specific activity of the hexose phosphate esters was about half that of the added glucose whereas in the presence of insulin the specific activity of the hexose phosphate esters was equal or approaching to that of the added glucose substrate. Insulin had no influence on the specific activity of lactate formed from glucose. Insulin did not increase the total concentration of glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-diphosphate present in the muscle at the end of the incubation period.