scholarly journals The operation of the γ-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitro

1970 ◽  
Vol 116 (3) ◽  
pp. 445-461 ◽  
Author(s):  
B. J. Hammond ◽  
R. Balázs ◽  
Y. Machiyama ◽  
T. Julian ◽  
D. Richter
Keyword(s):  
Amino Acids ◽  
Brain Tissue ◽  
Time Course ◽  
Tricarboxylic Acid Cycle ◽  
Specific Radioactivity ◽  
Tricarboxylic Acid ◽  
Minor Importance ◽  
Acid Cycle ◽  
Metabolic Compartmentation ◽  
High Specific Radioactivity

1. Cerebral-cortex slices prelabelled with γ-amino[1-14C]butyrate (GABA) were incubated in a glucose–saline medium. After the initial rapid uptake there was no appreciable re-entry of 14C into the GABA pool, either from the medium or from labelled metabolites formed in the tissue. The kinetic constants of GABA metabolism were determined by computer simulation of the experimental results by using mathematical procedures. The GABA flux was estimated to be 0.03μmol per min/g, or about 8% of the total flux through the tricarboxylic acid cycle. It was found that the assumption of compartmentation did not greatly affect the estimates of the GABA flux. 2. The time-course of incorporation of 14C into amino acids associated with the tricarboxylic acid cycle was followed with [1-14C]GABA and [U-14C]-glucose as labelled substrates. The results were consistent with the utilization of GABA via succinate. This was confirmed by determining the position of 14C in the carbon skeletons of aspartate and glutamate formed after the oxidation of [1-14C]GABA. These results also indicated that under the experimental conditions the reversal of reactions catalysed by α-oxoglutarate dehydrogenase and glutamate decarboxylase respectively was negligible. The conversion of [14C]GABA into γ-hydroxybutyrate was probably also of minor importance, but decarboxylation of oxaloacetate did occur at a relatively slow rate. 3. When [1-14C]GABA was the labelled substrate there was evidence of a metabolic compartmentation of glutamate since, even before the peak of the incorporation of 14C into glutamate had been reached, the glutamine/glutamate specific-radioactivity ratio was greater than unity. When [U-14C]glucose was oxidized this ratio was less than unity. The heterogeneity of the glutamate pool was indicated also by the relatively high specific radioactivity of GABA, which was comparable with that of aspartate during the whole incubation time (40min). The rates of equilibration of labelled amino acids between slice and medium gave evidence that the permeability properties of the glutamate compartments labelled as a result of oxidation of [1-14C]GABA were different from those labelled by the metabolism of [14C]glucose. The results showed therefore that in brain tissue incubated under the conditions used, the organization underlying metabolic compartmentation was preserved. The observed concentration ratios of amino acids between tissue and medium were also similar to those obtaining in vivo. These ratios decreased in the order: GABA>acidic acids>neutral amino acids>glutamine. 4. The approximate pool sizes of the amino acids in the different metabolic compartments were calculated. The glutamate content of the pool responsible for most of the labelling of glutamine during oxidation of [1-14C]GABA was estimated to be not more than 30% of the total tissue glutamate. The GABA content of the ‘transmitter pool’ was estimated to be 25–30% of the total GABA in the tissue. The structural correlates of metabolic compartmentation were considered.

scholarly journals The metabolism of γ-aminobutyrate and glucose in potassium ion-stimulated brain tissue in vitro

1970 ◽  
Vol 116 (3) ◽  
pp. 469-481 ◽  
Author(s):  
B. J. Hammond ◽  
Y. Machiyama ◽  
R. Balázs ◽  
T. Julian ◽  
D. Richter
Keyword(s):  
Amino Acids ◽  
Brain Tissue ◽  
Tricarboxylic Acid Cycle ◽  
Specific Radioactivity ◽  
Tricarboxylic Acid ◽  
Mammalian Brain ◽  
Acid Cycle ◽  
Metabolic Compartmentation ◽  
Cortex Slices ◽  
Saline Medium

1. The metabolism of γ-aminobutyrate (GABA) was investigated in cerebral-cortex slices incubated in glucose–saline medium with [1-14C]GABA and [U-14C]-glucose as labelled substrates. 2. A rapid release of GABA from the tissue, amounting to 25–30% of the total, was observed on addition of 66m-equiv. of K+/1 to the medium; the liberation of other amino acids was relatively small. The effect was apparently specific for K+; GABA was not released on addition of equivalent amounts of Na+ or on increasing the respiration rate with 10mm-ammonium chloride. The results show that GABA behaves like the transmitter compounds (acetylcholine, catecholamines) on K+ stimulation, and therefore now satisfies certain of the criteria required for a transmitter in mammalian brain. 3. The release of GABA from the tissue on addition of K+ was followed by a slow re-uptake. The rate of uptake of GABA in a medium containing 5.9m-equiv. of K+/1 was more than four times that in a medium containing 66m-equiv. of K+/1. 4. The concentration of GABA in brain tissue incubated for 1h in a medium containing 66m-equiv. of K+/1 was about 50% higher than that observed under normal conditions. 5. There was evidence that exogenous [14C]GABA mixed with the endogenous pool(s), since the proportion of the total GABA released on K+ stimulation was the same, and the specific radioactivity of the liberated GABA was close to that remaining in the tissue, whether the GABA was labelled by [1-14C]GABA from the medium or generated in the tissue from [14C]glucose. 6. On the basis of these findings and the observations outlined in the preceding papers it was possible to calculate the kinetic constants of GABA metabolism by computer simulation of the results. K+ stimulation led to a 2.5-fold increase in the flux through the tricarboxylic acid cycle, whereas the flux in the GABA bypath was little affected; as a result the flux through the GABA bypath, which under normal conditions was 8% of that through the tricarboxylic acid cycle, decreased to 3–5%. 7. The metabolism of glutamine was greatly affected by K+-stimulation. The ratio of the concentration of glutamine in the slices to that in the medium, which under normal conditions was the smallest among the amino acids investigated, increased from about 17 to 63 in 1h. This effect was attributable partly to an uptake of glutamine from the medium (1.8μmol/h per g) and partly to a net increase in the total amount of glutamine (2.6μmol/h per g). At 1h after the addition of K+ the net gain of glutamine could be accounted for by the decrease of glutamate. 8. Metabolic compartmentation was evident when brain-cortex slices were incubated in glucose–saline medium and the labelled substrate was [14C]GABA, since the specific radioactivity of glutamine exceeded that of glutamate. On addition of K+ the signs of metabolic compartmentation promptly disappeared: this effect was apparently associated with an increase in the permeability of the compartments containing labelled metabolites derived from [14C]GABA. The change in the permeability, however, did not affect all the compartments; when the labelled substrate was [14C]glucose the equilibration of labelled amino acids between tissue and medium was similar under normal conditions and in the presence of high concentrations of K+. 9. The metabolism of [14C]glucose was followed by measuring oxygen uptake, respiratory 14CO2, and incorporation of 14C into amino acids. The results showed that K+ stimulation increased the flux of glucose carbon, both in the glycolytic pathway and in the tricarboxylic acid cycle.

scholarly journals Effect of thyroid hormone on metabolic compartmentation in the developing rat brain

1971 ◽  
Vol 121 (3) ◽  
pp. 469-481 ◽  
Author(s):  
A. J. Patel ◽  
R. Balázs
Keyword(s):  
Amino Acids ◽  
Thyroid Hormone ◽  
Tricarboxylic Acid Cycle ◽  
Specific Radioactivity ◽  
Tricarboxylic Acid ◽  
The Other ◽  
Developmental Changes ◽  
Acid Cycle ◽  
Metabolic Compartmentation ◽  
Age Dependent

1. The effects of treatment with thyroid hormone (tri-iodothyronine) and of neonatal thyroidectomy on the cerebral metabolism of [U-14C]leucine were investigated during the period of functional maturation of the rat brain extending from 9 to 25 days after birth. 2. Age-dependent changes in the labelling of brain constituents under normal conditions appear to depend on changes in the availability of blood-borne [14C]leucine resulting from differential rates of growth of body and brain; but developmental changes in the pool size of free leucine and in the rates of protein synthesis and oxidation of leucine are also involved. 3. Treatment with thyroid hormone had no significant effect on the conversion of leucine carbon into proteins and lipids; and the age-dependent changes in the concentration and specific radioactivity of leucine were similar to controls. On the other hand there was an acceleration in the conversion of leucine carbon into amino acids associated with the tricarboxylic acid cycle. These observations indicate that leucine oxidation was the process mainly affected. 4. The specific radioactivity of glutamine relative to that of glutamate was used as an index of metabolic compartmentation in brain tissue. Treatment with thyroid hormone advanced the development of metabolic compartmentation. 5. Neonatal thyroidectomy led to a marked decrease in the conversion of leucine carbon into proteins and lipids and to a significant increase in the amount of 14C combined in the amino acids associated with the tricarboxylic acid cycle. The age-dependent increase in the glutamate/glutamine specific-radioactivity ratio was strongly retarded. 6. The increased conversion of leucine carbon into cerebral amino acids applied to glutamate and aspartate, but not to glutamine and γ-aminobutyrate. This observation facilitated the understanding of the effects of thyroid deprivation on brain metabolism and provided new evidence for the allocation of morphological structures to the metabolic compartments in brain tissue. 7. In contrast with the marked effects of the thyroid state on metabolic compartmentation, it had relatively little effect on the developmental changes in the concentration of amino acids in the brain. 8. The rate of conversion of leucine carbon into the ‘cycle amino acids’ both under normal conditions and in thyroid deficiency indicated a special metabolic relationship between glutamate and aspartate on the one hand, and glutamine and γ-aminobutyrate on the other.

scholarly journals Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1969 ◽  
Vol 113 (2) ◽  
pp. 281-290 ◽  
Author(s):  
C. J. Van Den Berg ◽  
LJ. Kržalić ◽  
P. Mela ◽  
H. Waelsch
Keyword(s):  
Time Course ◽  
Close Contact ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Glutamate Metabolism ◽  
Acid Cycle ◽  
The Brain

1. 14C from [1−14C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than 14C from [2−14C]glucose. This difference for [1−14C]glucose and [2−14C]glucose increases with time. The amount of 14C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. 14C from [1−14C]acetate and [2−14C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of 14C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.

scholarly journals Neuronal-glial metabolism under depolarizing conditions. A 13C-n.m.r. study

1992 ◽  
Vol 282 (1) ◽  
pp. 225-230 ◽  
Author(s):  
R S Badar-Goffer ◽  
O Ben-Yoseph ◽  
H S Bachelard ◽  
P G Morris
Keyword(s):  
Amino Acids ◽  
Glial Cells ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acid Cycle ◽  
Maximum Percentage ◽  
Theoretical Maximum ◽  
Metabolic Pool ◽  
Time Courses ◽  
The Individual

Time courses of incorporation of 13C from 13C-labelled glucose and/or acetate into the individual carbon atoms of amino acids, citrate and lactate in depolarized cerebral tissues were monitored by using 13C-n.m.r. spectroscopy. There was no change in the maximum percentage of 13C enrichments of the amino acids on depolarization, but the maxima were reached more rapidly, indicating that rates of metabolism in both glycolysis and the tricarboxylic acid cycle were accelerated. Although labelling of lactate and of citrate approached the theoretical maximum of 50%, labelling of the amino acids was always below 20%, suggesting that there is a metabolic pool or compartment that is inaccessible to exogenous substrates. Under resting conditions labelling of citrate and of glutamine from [1-13C]glucose was not detected, whereas both were labelled from [2-13C]acetate, which is considered to reflect glial metabolism. In contrast, considerable labelling of these two metabolites from [1-13C]glucose was observed in depolarized tissues, suggesting that the increased metabolism may be due to increased consumption of glucose by glial cells. The labelling patterns on depolarization from [1-13C]glucose alone and from both precursors [( 1-13C]glucose plus [2-13C]acetate) were similar, which also indicates that the changes are due to increased consumption of glucose rather than acetate.

scholarly journals Effect of renal tubule-specific knockdown of the Na+/H+exchanger NHE3 in Akita diabetic mice

2019 ◽  
Vol 317 (2) ◽  
pp. F419-F434 ◽  
Author(s):  
Akira Onishi ◽  
Yiling Fu ◽  
Manjula Darshi ◽  
Maria Crespo-Masip ◽  
Winnie Huang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Tricarboxylic Acid Cycle ◽  
Branched Chain Amino Acids ◽  
Tricarboxylic Acid ◽  
Proximal Tubules ◽  
Wild Type ◽  
Kidney Weight ◽  
Acid Cycle ◽  
Branched Chain

Na+/H+exchanger isoform 3 (NHE3) contributes to Na+/bicarbonate reabsorption and ammonium secretion in early proximal tubules. To determine its role in the diabetic kidney, type 1 diabetic Akita mice with tubular NHE3 knockdown [Pax8-Cre; NHE3-knockout (KO) mice] were generated. NHE3-KO mice had higher urine pH, more bicarbonaturia, and compensating increases in renal mRNA expression for genes associated with generation of ammonium, bicarbonate, and glucose (phosphoenolpyruvate carboxykinase) in proximal tubules and H+and ammonia secretion and glycolysis in distal tubules. This left blood pH and bicarbonate unaffected in nondiabetic and diabetic NHE3-KO versus wild-type mice but was associated with renal upregulation of proinflammatory markers. Higher renal phosphoenolpyruvate carboxykinase expression in NHE3-KO mice was associated with lower Na+-glucose cotransporter (SGLT)2 and higher SGLT1 expression, indicating a downward tubular shift in Na+and glucose reabsorption. NHE3-KO was associated with lesser kidney weight and glomerular filtration rate (GFR) independent of diabetes and prevented diabetes-associated albuminuria. NHE3-KO, however, did not attenuate hyperglycemia or prevent diabetes from increasing kidney weight and GFR. Higher renal gluconeogenesis may explain similar hyperglycemia despite lower SGLT2 expression and higher glucosuria in diabetic NHE3-KO versus wild-type mice; stronger SGLT1 engagement could have affected kidney weight and GFR responses. Chronic kidney disease in humans is associated with reduced urinary excretion of metabolites of branched-chain amino acids and the tricarboxylic acid cycle, a pattern mimicked in diabetic wild-type mice. This pattern was reversed in nondiabetic NHE3-KO mice, possibly reflecting branched-chain amino acids use for ammoniagenesis and tricarboxylic acid cycle upregulation to support formation of ammonia, bicarbonate, and glucose in proximal tubule. NHE3-KO, however, did not prevent the diabetes-induced urinary downregulation in these metabolites.

scholarly journals Metabolism of Lactate in Cultured GABAergic Neurons Studied by 13C Nuclear Magnetic Resonance Spectroscopy

1998 ◽  
Vol 18 (1) ◽  
pp. 109-117 ◽  
Author(s):  
Helle S. Waagepetersen ◽  
Inger J. Bakken ◽  
Orla M. Larsson ◽  
Ursala Sonnewald ◽  
Arne Schousboe
Keyword(s):  
Amino Acids ◽  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Resonance Spectroscopy ◽  
Acid Cycle ◽  
Cell Extracts ◽  
Gaba Synthesis ◽  
Nuclear Magnetic

Primary cultures of mouse cerebral cortical neurons (GABAergic) were incubated for 4 hours in media without glucose containing 1.0 mmol/L [U-13C]lactate in the absence or presence of 0.5 mmol/L glutamine. Redissolved, lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance spectroscopy to investigate neuronal metabolism of lactate and by HPLC for determination of the total amounts of glutamate (Glu), γ-aminobutyric acid (GABA), and aspartate (Asp). The 13C nuclear magnetic resonance spectra of cell extracts exhibited multiplets for Glu, GABA, and Asp, indicating pronounced recycling of labeled tricarboxylic acid cycle constituents. There was extensive incorporation of 13C label into amino acids in neurons incubated without glutamine, with the percent enrichments being approximately 60% for Glu and Asp, and 27% for GABA. When 0.5 mmol/L glutamine was added to the incubation medium, the enrichments for Asp, Glu, and GABA were 25%, 35%, and 25%, respectively. This strongly suggests that glutamine is readily converted to Glu and Asp but that conversion to GABA may be complex. The observation that enrichment in GABA was identical in the absence and presence of glutamine whereas cycling was decreased in the presence of glutamine indicates that only C-2 units derived from glutamine are used for GABA synthesis, that is, that metabolism through the tricarboxylic acid cycle is a prerequisite for GABA synthesis from glutamine. The current study gives further support to the hypothesis that cellular metabolism is compartmentalized and that lactate is an important fuel for neurons in terms of energy metabolism and extensively labels amino acids synthesized from tricarboxylic acid cycle intermediates (Asp and Glu) as well as the neurotransmitter in these neurons (GABA).

scholarly journals Studies on the Metabolism of Locust Fat Body

1959 ◽  
Vol 36 (4) ◽  
pp. 665-675
Author(s):  
A. N. CLEMENTS
Keyword(s):  
Amino Acids ◽  
Sodium Acetate ◽  
Flight Muscle ◽  
Fat Body ◽  
Enzyme System ◽  
Tricarboxylic Acid Cycle ◽  
Succinic Dehydrogenase ◽  
Tricarboxylic Acid ◽  
Acid Cycle

1. The incorporation of glycine-14C (G), leucine-14C (G), sodium acetate-2-14C and glucose-14C (G) into Schistocerca fat body was studied under in vitro conditions, and the distribution of radioactivity in the various fat body fractions and the labelling of compounds within the fractions is described. 2. The overall picture was of high incorporation into fat and protein and of very low incorporation into glycogen. 3. Incubation with glycine-14C led to radioactivity appearing in the glycine and serine of the protein and of the amino acid pool. Incubation with sodium acetate-2-14C led to radioactivity appearing in glutamate, proline, aspartate and alanine, showing that the intermediates of the tricarboxylic acid cycle provide the carbon skeletons of certain amino acids. Glucose-14C was largely converted to trehalose. 4. Succinic dehydrogenase and the condensing enzyme system were shown to be present in fat body, contrary to previous reports. The succinic oxidase system was highly labile on homogenizing the tissue. 5. Fat body, unlike flight muscle, used glycine-14C and leucine-14C as respiratory substrates, and it is suggested that fat body acts like the vertebrate liver by transdeaminating amino acids and making them available for further metabolism by other tissues.

METABOLISM OF C14 AMINO ACIDS AND AMIDES IN DETACHED LEAVES

1956 ◽  
Vol 34 (4) ◽  
pp. 423-433 ◽  
Author(s):  
C. D. Nelson ◽  
G. Krotkov
Keyword(s):  
Amino Acids ◽  
Double Bond ◽  
Glutamic Acid ◽  
Broad Bean ◽  
Specific Activity ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Activity Data ◽  
Acid Cycle ◽  
Bean Leaves

Detached broad bean leaves were placed with their petioles in 0.01 M ammonium nitrate and allowed to carry on photosynthesis in C14O2 for various periods from 12 to 125 min. The radioactivities of the various amino acids formed from C14O2 were determined. In addition, these amino acids were degraded by decarboxylation with ninhydrin. From the specific activity data it was concluded that the amino acid closest to the site of carbon dioxide fixation in photosynthesis was alanine, followed by aspartic and glutamic acids, with the amides farthest removed. From the intramolecular distribution of label it was concluded that asparagine and glutamine were formed from their corresponding amino acids. The labelling in aspartic and glutamic acids was not consistent with the view that these two amino acids are formed from their corresponding α-keto acids produced by operation of the conventional tricarboxylic acid cycle. A C2 plus C2 condensation is postulated for the formation of aspartic acid. A shift in the double bond in the aconitase reaction of the tricarboxylic acid cycle would account for the observed labelling in glutamic acid. When acetate-1-C14 was fed to detached broad bean leaves in the light or dark, the distribution of label in glutamic acid supported the suggestion that there is such a. shift in the double bond in the aconitase reaction. Sodium arsenite, infiltrated into tobacco leaves, inhibited the biosynthesis of asparagine but not that of glutamine.

More Than Skin Deep

2018 ◽  
Author(s):  
David R. Dalton
Keyword(s):  
Amino Acids ◽  
Nucleic Acids ◽  
Carboxylic Acids ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Grape Berry ◽  
Acid Cycle ◽  
Solid Portion ◽  
Present Complex ◽  
The Individual

The grape berry is composed of skin, flesh (pulp) and seeds. After destemming (Chapter 13), the grapes are sent on for crushing. On crushing, the thick walls of the skin, including the waxy cuticle, are broken. Crushing the grapes (Figure 16.1) is a question of quantity. Small quantities are handled differently than large. The skins, including the contaminants thereon, as well as the majority of the materials discussed above for the individual grapes (i.e., phenols, anthocyanins, tanins, some acids, terpenes, pyrazines, and some carbohydrates including those attached to the anthocyanidins, forming anthocyanins) therein, are released. The cells of the pulp are also broken and released into the juice on crushing. This berry cell juice is mainly water (70–80% by weight) which contains the mixture of sugars (mostly glucose and fructose, but small concentrations of many other carbohydrates are also present), carboxylic acids (mostly tartaric and malic, but additional members of the tricarboxylic acid cycle, oxalic, glucuronic, etc. are also present), complex cross-linked polysaccharides from cell walls (pectins), some phenols and proteins (as well as the peptides and simple amino acids from which they are constructed), and minerals, including oxides of iron (Fe), phosphorus (P), and sulfur (S), as well as salts of potassium (K) and sodium (Na) brought up in the xylem to the growing berry. The seeds have their cellulose carbohydrate-based exterior coatings, which are also rich in complexed polyphenols (tannins). Additionally, amino acids, generally found as constituents of peptides, proteins, and enzymes, and their cofactors needed for all life, nucleic acids and their attached sugars needed for the next generation, are all present too. Thus, overall, the result of crushing the berries is a mixture consisting of skins, seeds, and fruit juice (the must = Latin vinum mustum = young wine). This mixture may, if the grapes were “white,” be cooled and the cap on the must—sometimes called the pomace (the solid portion of the must) removed early or late (usually between 12 and 24 hours) by the vintner. Most of the flavoring constituents are quickly extracted, and brightly colored phenols, tannins, anthocyanins, etc.

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